RNA Therapeutics

While RNA molecules have demonstrated promising results as therapeutics for difficult-to-treat disease indications, their current limitations in terms of delivery mean their full potential is not being realised. How can this be overcome?

Nigel Theobald at N4 Pharma

Ribonucleic acid (RNA) molecules have gained attention as promising therapeutics due to their ability to precisely target proteins or molecules that have previously been seen as ‘undruggable’ with minimal side effects. While RNA therapeutics have the potential to transform the treatment of a range of diseases, their clinical translation faces considerable challenges. Primary hurdles include their rapid degradation and clearance from circulation, an inability to traverse cell membranes and the inefficiency of intracellular delivery of bioactive RNA molecules.11 Overcoming these delivery barriers is crucial to unlocking the full therapeutic benefits of RNA-based medicines and to address this, alternative formulation-based delivery strategies such as mesoporous silica nanoparticles (MSNs) are being actively explored.

Evolution of RNA therapeutics

The rapid advancement of RNA therapeutics started in the early 1980s with the success of antisense oligonucleotides (ASO), paving the way for small interfering RNA (siRNA) and messenger RNA (mRNA) technologies. This culminated in the approval of the first siRNA drug in 2018 and the deployment of the first mRNA-based vaccine during the COVID-19 pandemic.2,3 These therapeutic breakthroughs highlight the unique potential of RNA to modulate splicing, silence specific genes, and replace or block defective proteins without altering the genome. By leveraging these properties, RNA therapeutics are expanding the scope of treatable diseases and offering hope for conditions previously considered untreatable with traditional therapies.4 This promise is reflected by the rapidly growing RNA therapeutics market, evaluated at $6.83bn in 2023 and expected to grow to around $40.71bn by 2034.5

Limitations of siRNA therapeutics

Recent studies recognise siRNA as particularly valuable for therapies targeting cancers and genetic disorders due to its unparalleled specificity in targeting mRNA sequences.6,7 However, the inherent properties of siRNA molecules, including their unstable negative charge and hydrophilicity, pose several challenges. siRNA’s varying ability to cross cell membranes makes them susceptible to rapid degradation in the wrong location, necessitating the use of a delivery carrier to safely transport it to the intended site of action in vivo.8 Without a delivery system, the siRNA can bind to unintended mRNA sequences with partial complementarity and lead to off-target effects and disruption of normal cellular functions, triggering unwanted side effects. Precision in siRNA targeting is therefore critical to avoiding unintended gene silencing.

Another limitation is in the large-scale manufacturing of siRNA therapies, which presents logistical and technical hurdles. Maintaining high purity and quality in production, especially for chemically modified siRNA, can result in demanding production costs, further complicating widespread adoption.

Formulation-based delivery strategies

To overcome the challenges associated with the safe delivery of siRNA, various formulation strategies are currently being explored.9,10 While viral vectors are effective at delivering genetic material, they often present safety concerns, including immunogenicity and possible long-term effects. Non-viral delivery systems such as lipid-based nanoparticles (LNPs) and MSNs offer promising alternatives for siRNA delivery as these systems provide enhanced stability, protection and targeting capabilities while reducing toxicity risks.11

Formulating siRNA into nanoparticles requires careful consideration of multiple factors to ensure safe and effective delivery, including:12,13

• Particle size and formulation: the size influences cellular uptake, biodistribution and the ability to traverse biological barriers, such as the vascular endothelium, to enter the bloodstream

• Compatibility and safety: ensuring the nanoparticle is biocompatible and non-immunogenic to minimise adverse reactions once inside the body in vivo

• Protection and targeting: the nanoparticle must shield siRNA from enzymatic degradation while incorporating ligands or surface modifications to enable precise delivery to target tissues or cells

• Uptake and efficacy: once internalised, the siRNA must effectively escape the endosome to reach the cytoplasm, a significant challenge that can limit therapeutic activity

• Metabolism and clearance: nanoparticles must be metabolised in a controlled manner without accumulating in non-target tissues or causing systemic toxicity

• Cost and scale-up: reproducible manufacturing processes that ensure robust quality and simplify analysis are critical for clinical translation

• Bioavailability: determines the therapeutic potential of the siRNA formulation, necessitating designs that maximise the safe and efficient delivery of active siRNA to its site of action.

Addressing these factors is particularly problematic with viral delivery systems, underscoring the importance of advancing non-viral nanoparticle-based approaches for siRNA therapeutics.

A novel delivery system

Nanocarriers composed of LNPs, often the first choice for siRNA delivery, are generally recognised for their biodegradability, biocompatibility and minimal immunogenicity and toxicity. Despite these advantages, these nanocarriers are not always completely inert.

Additionally, LNPs face other limitations including limited stability, a relatively low siRNA-loading capacity, and the potential degradation or interaction of encapsulated nucleic acids.14 These drawbacks underscore the need for alternative nanoparticles, such as MSNs, which have emerged as a promising solution for siRNA delivery.

MSNs offer unique structural and functional properties that make them an ideal candidate for addressing key siRNA delivery challenges, including protection against degradation, efficient cellular uptake and targeted delivery. For RNA gene delivery, such nanoparticles can address many of the challenges associated with manufacturing and targeted delivery of RNA therapeutics. These include the ability to target specific cells and tissues using surface modifications with no undesirable immune response.

Multiple delivery

Future evolution of RNA-therapeutics may focus on delivering multiple types of RNA intracellularly, for instance two particles carrying different siRNAs, or a mixture of siRNA and mRNA targeting the same cell. This kind of approach could address multiple pathways simultaneously and potentially enhance therapeutic outcomes for complex diseases like cancer or genetic disorders.

Combination therapies have long proven effective in the first-line treatment of a range of conditions such as human immunodeficiency virus (HIV) and hepatitis, which are routinely treated with synergistic combinations of drugs to maximise therapeutic outcomes. This success suggests a promising opportunity to integrate combination strategies into RNA delivery systems, potentially by co-delivering immunotherapies. Emerging research strongly supports the enhanced efficacy of combination therapies. For example, Zhao Y et al showed that co-delivering siRNA and the chemotherapy drug doxorubicin (DOX) significantly improved cancer treatment outcomes compared to delivering a single agent, highlighting a powerful synergistic effect.15 Similarly, researchers at the University of Pittsburgh found that simultaneously delivering a chemotherapy drug and siRNA could effectively eliminate residual cancer cells that evade traditional therapies such as chemotherapy and radiation. They concluded that this dual approach holds potential for broader applications.16

Currently, the delivery of multiple RNAs requires an additional carrier, decreasing the probability of transfecting the same cell with more than one RNA, thus hitting multiple aspects of a cellular pathway is less likely. Delivery of two RNAs acting on two complementary pathways in the same cell has the potential to provide an improved clinical response with fewer adverse events. In vitro research has shown that it is possible to load more than one siRNA or a mixture of mRNA and siRNA onto the same nanoparticle for combination therapies.

Conclusion

As nucleic acid chemistry and delivery methods continue to evolve, RNA-based drugs targeting new therapeutic areas can be developed more rapidly. Recent advances in the production, purification and cellular delivery of RNA have paved the way for RNA-based therapies that could redefine disease treatment and enable more personalised medicine. Non-viral nanoparticle delivery systems offer a promising solution to address key challenges in RNA drug delivery and manufacturing. By overcoming these hurdles, RNA-based therapeutics are positioned to revolutionise medicine, providing precise, targeted and effective treatments for a wide range of diseases.

References:
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3. Visit: berthub.eu/articles/11889.doc

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5. Visit: towardshealthcare.com/insights/rna-basedtherapeutic-market-sizing

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11. Visit: insideprecisionmedicine.com/news-andfeatures/dual-action-nanoparticles-deliverchemoimmunotherapy/

12. Gao H et al (2021), ‘Nanoparticle-mediated siRNA delivery systems for cancer therapy’, VIEW, 2, 20200111

13. Yadav D N et al (2022), ‘Recent advancements in the design of nanodelivery systems of siRNA for cancer therapy’, Molecular Pharmaceutics, 19(12), 4506-4526

14. Morales-Becerril A et al (2022), ‘Nanocarriers for delivery of siRNA as gene silencing mediator’, EXCLI J, 1(21), 1028-1052

15. Zhao Y et al (2022), ‘Investigation ofadual siRNA/ chemotherapy delivery system for breast cancer therapy’, ACS Omega, 7(20), 17119-17127
16. Visit: insideprecisionmedicine.com/news-andfeatures/dual-action-nanoparticles-deliverchemoimmunotherapy/

Nigel Theobald, chief executive officer at N4 Pharma, has over 25 years’ experience in healthcare and in building businesses, strategy development and its implementation, and a strong network covering all aspects of pharmaceutical product development and commercialisation. He was the head of healthcare brands at Boots Group plc in 2002 before leaving to set up a series of successful businesses, including Oxford Pharmascience Group plc. Nigel formed N4 Pharma in 2014.