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European Biopharmaceutical Review

Two Pillars of Fledgling RNA Medicine

Recent advances in genetics and genomics have helped to establish the causes of genetic disorders. Emerging research revealed that many diseases could be treated if the expression of specific genes could be controlled – either by preventing them expressing proteins, or by altering the protein they produce. A promising solution was found in short pieces of RNA comprising 20-25 nucleotides (1), which can identify, inhibit and degrade mRNA. These are non-encoding sequences that are complementary to targeted mRNA regions, which renders them upstream RNA interference (RNAi) agents or small interfering RNA (siRNA).

In principle, such antisense therapeutics can suppress any disease-associated gene and eradicate the very root of a given disease: RNAi can block proteins responsible for an illness – thereby eliminating the need for small-molecule drugs that can only engage with a downstream target – whereas siRNA drugs can silence virtually any gene with specificity targeting even dominant oncogenic mutations. Other antisense approaches exist too – for example, based on antisense DNA or ribozymes. However, RNA drugs are far more potent, smaller, more specific and, as a consequence, would require lower therapeutic concentrations than non-RNA counterparts.

The mode of action of RNA agents is indeed universal, whereas their mechanistic differences are subtle and relate to their origin. For example, both micro RNA (miRNA) and siRNA can mediate RNAi. However, miRNA are naturally occurring (endogenous) agents that derive from self-complementary RNA transcripts forming short hairpins, while siRNA are longer, double-stranded and man-made (exogenous) molecules. Given the differences, it is not surprising that artificial RNA molecules are being favoured in research and industry. Furthermore, siRNA and miRNA are easily degradable and require constant protection, which justifies the development of more stable synthetic RNA analogues with non-natural molecular backbones.

Barriers and Solutions

Despite all the progress and attainable promise, using RNA as a drug remains very difficult because so little is taken into live cells and important tissues like nerve, heart and skeletal muscle. Current drugs that modulate genetic reactions can overcome the problems of stability, excretion and uptake by phagocytes. However, inefficient transfer and uptake by the target cells, release from endosomes, and entry into the nucleus remain major barriers. This problem sits atop two pillars.

Firstly, siRNA, miRNA or synthetic oligoribonucleotides (ORNs) cannot penetrate cellular membranes and require an auxiliary means for intracellular delivery. Tight connections among the endothelial cells make transfer into the central nervous system notoriously difficult. It is possible to use protein transduction domains, or cell-penetrating peptides, as conjugates to ORNs (2). However, it seems likely at present that they are more effective with neutral derivatives, such as phosphorodiamidate morpholino oligomers, which are inactive as bifunctional reagents because activator proteins do not bind.

Another strategy is the use of nanoparticles based on gold, polymers or lipids. The surface may adsorb plasma proteins – leading to phagocytosis – but it also presents an opportunity for introducing modifications such as cationic or basic groups for binding ORNs, penetrating cells and disrupting endosomes. Nonetheless, their size may restrict movement through tight vascular endothelia, and many formulations lead to levels of heterogeneity in size or composition that would compromise their acceptability as human drug carriers. Taken together, these issues prompt the development of RNA-encapsulating transporters.

Viruses – otherwise the most efficient gene transporters – pose safety concerns over immunogenicity and reversion to a pathogenic state, whereas synthetic gene packages greatly lack the structural integrity of viruses. An ideal vector is seen as a compromise between the two: a nanoscale device, which would mimic a virus and act as a virus, but would do this at will – that is, as designed (3). Viral architectures themselves offer an efficient strategy, with their functional reproducibility guaranteed by their exquisite assembly mechanisms.

Thus, to create an RNA-encapsulating virus is to re-create the way an RNA virus is built, or assembled. Hence, much research focuses on the development of artificial viruses that can enable the RNA to be administered as a drug. Although the area is relatively new, the current progress indicates substantial benefits – including the ease of production and control, diversity of self-assembling sequences, biocompatibility and predictable cell-targeting mechanisms. Engineering virus-like nanoscale capsules is arguably the most promising avenue. Like viruses, such nanocapsules are homogenous, monodispersing hollow shells that encapsulate RNA and deliver the cargo into live cells. They can be made of any size and, notably, an order of magnitude smaller than existing commercial transfection reagents. Unlike virus-derived transporters, these nanocapsules lack the inherent drawbacks of viruses. With all these properties, nanocapsules present promising candidates for developing reference materials to benchmark existing and emerging delivery technologies. But how quantitative can RNA carriers be?

Quantitative Intracellular Delivery

Indeed, RNA-based therapies have reached a point where quantitative control over intracellular transfer is necessary for any further progress. RNA therapeutics are available, having grown in numbers and diversity, with many companies having a portfolio of RNA-based drugs. However, it is the systemic use of RNA drugs in the clinic that is hampered by uncertainties regarding delivery into live cells and tissues, and it is the structural inconsistency of delivery vectors that compromise drug manufacturability (4). Collectively, these issues constitute the second pillar hampering RNA medicine.

In overcoming it, the industry is concerned with more efficient and quantitative intracellular delivery, the need for which is echoed by regulatory agencies calling for a harmonised legislation and suitable standards for gene therapy products (5).

To enable the systemic assessment of the safety and efficacy of delivery technologies, correlative measurements of intracellular delivery are necessary. Such metrics can quantitatively probe delivery vectors, membrane permeability, efficiency and specificity of cell targeting. Combined with temporal monitoring in live cells, the measurements are anticipated to provide an exploitable range of intracellular RNA delivery. In this vein, biotechnologists in the National Physical Laboratory are developing an advanced measurement capability to provide quantitative correlations between physico-chemical and structural properties of gene delivery vectors – existing and emerging – and the efficiency of their uptake to the targeted cells and tissues. The capability is being developed as a reference methodology alongside the formulation of reproducible, non-viral delivery vectors as standard reference materials for gene transfer.

Impact and Benefits

The outlined issues are a major source of inconsistent results in clinical trials. Encouragingly, however, the same studies suggest that more effective delivery carriers hold the future of RNA-based therapies. To an equal extent, the lack of progress is attributed to the lack of innovative measurements that could advance the understanding of factors influencing uncertainty and reproducibility of intracellular delivery. Concerns over this will remain – there are no ISO standards for vector materials – unless such metrics are developed. The gene and drug delivery industry would benefit from quantitative metrics of intracellular delivery, which should be adaptable for a specific disease target. This will facilitate the timely market entry of new technologies and will further pave the way for future molecular therapy standards.

The impact on industry is integral to that on healthcare and clinic. Genetic disorders, let alone cancer, comprise more than 15,000 different diseases. The most vulnerable of age groups is children, with over 30% of all infant deaths due to single-gene disorders – each of which might be cured by a single RNA drug if delivered into the cell. Up to 20% of all paediatric hospital admissions (the average for the developed world) are for children with genetic disorders, whereas the mortality rate for boys with Duchenne muscular dystrophy – which can be treated only by RNA – remains at 100%. This is where further advancements are needed and where fledgling RNA medicine will impact most.


1. Kanasty R, Dorkin JR, Vegas A and Anderson D, Delivery materials for siRNA therapeutics, Nat Mater 12: pp967-977, 2013
2. Boisguérin P et al, Delivery of therapeutic oligonucleotides with cell-penetrating peptides, Adv Drug Deliv Rev 87: pp52-67, 2015
3. Lamarre B and Ryadnov MG, Self-assembling viral mimetics: One long journey with short steps, Macromol Biosci 11: pp503-513, 2011
4. Wittrup A and Lieberman J, Knocking down disease: A progress report on siRNA therapeutics, Nat Rev Genetics 16: pp543-552, 2015
5. EMEA Guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products, EMEA/273974/05

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Max Ryadnov leads the Biotechnology area of the National Physical Laboratory (NPL). After obtaining his PhD from Moscow State University and the Russian Academy of Sciences, he moved to the UK to take up a postdoctorate in Sussex, following which he pursued independent academic careers in Bristol and Leicester before joining NPL in 2010. His current research aims at revealing exploitable design principles of biomolecular structure and function for prescriptive molecular diagnostics and therapy.
Max Ryadnov
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