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European Biopharmaceutical Review

Novel Platforms

RNA-targeting therapeutics have reached a pivotal moment in their development, with positive clinical data paving the way for imminent FDA approvals. Those in development today have built upon the lessons learned from previous generations' chemistries, and have incorporated modifications that potentially improve the safety and efficacy of these therapeutics to overcome the limitations faced by earlier approaches.

Unmodified RNA oligonucleotides are unstable in cells and must be chemically modified in order to be used as a therapeutic; however, historical approaches resulted in off-target effects and less-than-optimal efficacy profiles (1,2). Initially, RNA-targeted therapeutics were modified by replacing the non-bridging oxygen in the phosphate backbone with a sulphur atom, replacing the phosphodiester bond of the backbone with a phosphorothioate bond (1,3). Modifying the backbone in this way improves the stability of a compound by increasing resistance to nucleases, while still allowing RNase H − the enzyme that destroys the RNA in a DNA/RNA duplex − to be engaged (2,4). However, it was soon discovered that this increased stability was accompanied by off-target effects, caused when the therapeutic would bind to sequences similar to the intended target or directly bind to proteins (1). This observed effect was attributed to the negative charge of phosphorothioate oligonucleotides, which created a tendency to bind proteins found in the serum (2).

It was then discovered that phosphorothioate oligonucleotides activate the innate immune system in a non-specific manner, including components of the complement cascade, coagulation factors and Toll-like receptor (TLR), and that therapeutics based on this chemistry also tended to accumulate in the liver, leading to liver toxicity as indicated by elevated levels of transaminases (2,5).

With an understanding of these developmental challenges, the nextgeneration of RNA-targeted chemistries currently under development seek to address these limitations to improve their potential for clinical, and ultimately commercial, success.

Approaches Under Development

Several approaches using nextgeneration chemistries to develop RNA-targeting therapeutics are currently under development, and incorporate knowledge gained from past research.

2'-O-methoxyethyl (2'MOE) Gapmer Chemistries

Isis, the current leader in the antisense space, is developing a pipeline of RNA-based therapeutics based on the phosphorothioate 2'-O-methoxyethyl (2'MOE) gapmer chemistries. Isis brought the first antisense drug to market, fomivirsen, a 21-mer phosphorothioate oligodeoxynucleotide, and has since focused on developing its gapmer technology. This technology consists of a chimeric oligonucleotide with a ‘gap’ region of approximately 10 phosphorothioate-modified 2’ deoxynucleotides flanked on both sides by 2’-MOE modified nucleotides (6). This ‘gap’ region is necessary for the recruitment of RNase H (6).

Data have shown that these modifications increase the stability of the therapeutic, by a +2 degree increase in thermal stability per modification, compared to phosphorothioate chemistries (7). Isis’ lead therapeutic, mipomersen, is based on this chemistry and has demonstrated efficacy in a previous Phase 3 trial in patients with severe heterozygous familial hypercholesterolaemia (8). Importantly, this study also demonstrated the potential ability of a RNA-targeting therapeutic to rival a successful small molecule drug: statins. In a recent Phase 3 study, mipomersen reduced LDL-C in patients by 36 per cent compared with a 13 per cent increase for placebo (9). However, some critical off-target effects were reported in the clinic. Frequently observed adverse events included injection site reactions, flu-like symptoms and elevations in liver transaminases, which may ultimately limit the utility of this drug.

Locked Nucleic Acids (LNA)

Locked nucleic acids (LNA), an approach used by Santaris Pharma A/S, are another promising advanced chemistry with advantageous properties. LNAs are created when the furanose ring of the ribose sugar is chemically locked by the addition of an O2’, C4’ methylene linkage, creating a bicyclic sugar ring with an RNA-like conformation (10). Following binding, RNase H is recruited. These chemical modifications allow therapeutic oligonucleotides to be shorter, conferring added specificity and potentially minimising off-target effects, and they demonstrate increased affinity for the target mRNA, thereby improving drug efficacy. Increased affinity has been demonstrated by an increase in melting temperature from +2 to +8 degrees per LNA monomer, compared to unmodified duplexes (11). LNAs are also resistant to nucleases, improving the therapeutic’s stability (11).

Beyond their mRNA-targeting properties, LNAs also target microRNA. Results from a Phase 2a study evaluating Santaris’ miravirsen presented at the American Association for the Study of Liver Diseases demonstrated human proof-of-concept for the targeting of microRNA by LNAs (12). In this study, after four weeks of treatment with miravirsen, patients with hepatitis C showed reductions in virus from 2 to 3 logs from baseline, which was maintained for more than four weeks following the end of treatment. Four out of nine patients treated at the highest dose had undetectable levels of HCV during the study (12).

Due to their ability to act as master regulators of gene expression, microRNAs are a promising therapeutic target for future drug development and an attractive space for RNA-targeting therapeutics. However, as microRNAs can potentially control the expression of many genes, this approach may offer less exquisite control of a single target compared to other RNA-targeting approaches (13).

Phosphorodiamidate Morpholino Oligomers (PMOs)

Another promising next-generation RNAbased chemistry is phosphorodiamidate morpholino oligomers (PMOs). PMOs are created by modifying the five-membered ribose ring in the backbone of RNA to a six-membered morpholine ring (14). Additionally, the backbone utilises phosphorodiamidate linkages, instead of unstable phosophodiester or off-targetprone phosphorothioate bonds (14).

These structural modifications create a charge-neutral molecule, which confers multiple advantages over past negativelycharged chemistries. The lack of charge of the molecule decreases off-target effects, increasing the safety of therapeutics based on this technology. Previous studies have shown that PMOs do not activate the innate immune system, complement cascade or interact significantly with other proteins in the serum, such as coagulation factors (14). Therapeutics based on PMO chemistry are also more stable and have a higher affinity for the target mRNA sequence than therapeutics based on first-generation chemistries. These properties, combined with an improved safety profile that allows higher dosing, lead to an improved efficacy profile. A recent Phase 1/2 study demonstrated the safety of a Duchenne muscular dystrophy (DMD) therapeutic based on this chemistry, eteplirsen, which found that patients were able to be dosed safely up to 20mg/kg (15). An ongoing Phase 2 study of eteplirsen recently received approval from the data safety monitoring board (DSMB) to proceed to higher doses of up to 50mg/kg of eteplirsen (16).

Additionally, instead of engaging endogenous RNA degradation machinery, such as RNase H or the RISC complex, PMOs act through steric blockade (14). This approach allows the down-regulation of gene expression by preventing the ribosome from assembling on the mRNA and translating the protein (14), as well as the up-regulation of gene expression, through a process called exon skipping (15).

Pre-mRNA consists of exons interrupted with introns, the exons from which are spliced together to generate mature mRNA. A PMO complimentary to a region of the pre-mRNA required by the splicing machinery to define a particular exon will essentially ‘blind’ the machinery, causing the exon to be skipped. In the case of DMD, skipping certain exons restores the open reading frame of the affected dystrophin RNA, resulting in the expression of a truncated, yet functional dystrophin protein (15).

In a recent Phase 1/2 study in DMD patients, eteplirsen induced exon skipping and new dystrophin protein expression in all cohorts in a dose-dependent manner, in which background biopsies taken prior to dosing of the drug served as a baseline (15). In total, seven out of 17 biopsied subjects had a post-treatment increase in dystrophin protein expression compared to their pre-treatment biopsies. For example, two subjects in cohorts five (10mg/kg), and six (20mg/kg) exhibited increases in dystrophin-positive fibres to 15 per cent and 55 per cent of normal, respectively. Importantly, a statistically significant reduction in CD3 cell count in cohorts five and six (paired two-tailed t-test, p=0.0115) was observed. The decrease in inflammatory response in muscle tissue suggests not only that the newly produced dystrophin protein leads to a significant reduction in inflammation in the affected muscles, but that the eteplirsen PMO did not induce a nonspecific immunostimulatory response.

2’-O-methyl Phosphorothioate (2OMePS) Oligonucleotides

Prosensa, partnered with GlaxoSmithKline, is currently developing RNAtargeting therapeutics using 2OMePS oligonucleotides. These therapeutics utilise a ribose modified with a 2’O-methylsubstitution and a phosphorothioate backbone (17). This modified chemistry creates a compound that has increased stability against endogenous DNA and RNA nucleases, enhancing efficacy. Therapeutics based on this chemistry also have the ability to interfere with splicing and to block the expression of mRNA (17).

Proof-of-concept in humans for 2OMePSbased therapeutics was achieved with studies that evaluated therapeutics using exon skipping to treat DMD (18). A previous Phase 1/2 trial in DMD patients evaluating a 2OMePS oligonucleotide therapeutic, PRO051/GSK2402968, demonstrated that the drug induced exon skipping at a dose of 2.0mg/kg, and new dystrophin expression was observed in approximately 60 per cent and 100 per cent of muscle fibres in 10 out of the 12 patients (18). However, these data are difficult to interpret due to the lack of pre-treatment comparator biopsy samples in this study. In a 12-week extension study, clinical outcomes were observed, with a mean improvement of 35.2 metres (plus or minus 28.7 metres) on the six-minute walk test; however these data were not statistically significant (18).

Common adverse events reported during the extension study included proteinurea, found in all patient cohorts, including the lowest dose of 0.5mg/kg, as well as mild reactions at the injection site and increased urinary α1-microglobulin levels (18).

A Promising Future for RNATargeting Therapeutics

As with any transformative technology, difficult lessons have been learned in the process of developing RNA-targeting therapeutics. However, new chemistries in development have the advantage of applying knowledge gained from past development attempts and are able to potentially address past issues related to safety and efficacy. RNAtargeting therapeutics have reached a point in their development at which therapeutics based on next-generation chemistries are nearing approval and demonstrating positive outcomes in late-stage clinical trials. With these developments, the future is promising for RNA-targeting therapeutics.


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Peter Sazani is the Executive Director of Preclinical Development at AVI BioPharma, and focuses on the discovery and development of novel RNA-based therapeutics for rare and infectious diseases. Prior to AVI BioPharma, he was the director of discovery research at Ercole Biotech, a company developing oligonucleotide therapeutics that was acquired by AVI BioPharma in 2008. Peter received his PhD in Pharmacology from the University of North Carolina at Chapel Hill.
Peter Sazani
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