In a recent article published in Natural Biotechnologiesresearchers reviewed technological advances that will unlock the promise of biologically targeted ribonucleic acid (mRNA) therapy beyond vaccines.
The first part of the review focuses on the design and purification of mRNA cargoes, including novel forms such as circular (circRNA) and self-amplifying mRNA (saRNA). The second part discussed improved mRNA packaging systems, including ionizable lipid nanoparticles (LNPs), to improve cargo delivery.
In the third part, the researchers reviewed the engineering of packaging systems that will facilitate the targeting of mRNA therapeutics to specific tissues. The fourth and fifth parts discussed strategies enabling the treatment of chronic diseases by mRNA therapy and a summary of current clinical trends in mRNA therapy. Finally, the researchers highlighted the near- and long-term scope of new mRNA therapies.
The unprecedented success of vaccines against coronavirus disease 2019 (COVID-19) based on the mRNA technology platform has renewed interest in this therapeutic area. However, several challenges still hinder the establishment of mRNA technology as a general therapeutic modality with broad applicability against various clinical conditions.
Advances in protein expression, packaging systems, tissue targeting and chronic dosing
Immunization requires minimal levels of protein expression, while mRNA therapy requires a 1000-fold higher level of protein to reach a therapeutic threshold. Effective delivery to solid organs remains a challenge. Even the tissue bioavailability, circulation half-life, and efficiency of the LNP-based carrier can be rate-limiting when delivered to the target tissue. Even with optimized chemical modifications of mRNA and advanced LNPs, chronic dosing ultimately activates innate immunity while simultaneously reducing therapeutic protein expression.
An individual mRNA has a cap, 5′ and 3′ untranslated regions (UTRs), an open reading frame (ORF), and a polyadenylated (poly(A)) tail. There have been advances in the design of each of these components. The most notable of them are:
i) improved 5′ cap analogues that improve translational capacity but more importantly restriction efficiency from 70% to 95%.
ii) optimization of the length of the poly(A) tail proved critical for balancing the synthetic ability of the mRNA cargo.
iii) UTR sequence optimization can enhance the protein expression of an mRNA cargo several-fold, allowing its customization to the target biological domain and disease-induced microenvironment.
iv) Studies have so far documented 130+ naturally occurring chemical modifications of mRNA. Chemically modified nucleosides, particularly uridine moieties such as methylpseudouridine, can reduce recognition by toll-like receptors of innate immunity by up to 100-fold, which in turn greatly increases protein expression after in vivo transfection of mRNA cargoes. In the future, clinically effective, unmodified therapeutic mRNAs may emerge that will hide from the immune system and have increased translational efficiency in vivosimilar to chemically modified mRNA vaccines.
Likewise, saRNAs may prove beneficial for enzyme replacement therapies. They require ~10-fold less RNA for a similar magnitude of protein expression compared to linearly modified mRNA and are under in vivo testing, scalable vaccine manufacturing process. Another alternative to linear mRNA is circRNA, which has been shown to extend the lifetime of mRNA by twofold IVF. CircRNA avoids the need for an expensive 5′ capping, tedious 3′ poly(A) tail and increases overall protein yield without increasing protein expression levels compared to linearly modified mRNA.
Overcoming the challenges of protein expression amplitude in parallel with mRNA structural optimizations may alleviate the need for multiple dosing, a major requirement impeding the treatment of chronic diseases using mRNA therapeutics. Conventional treatment involves systemic injection of recombinant clotting proteins (factor VIII/IX) three to seven times per week due to their relatively short half-life of ~12 hours. On the contrary, preclinical studies in mice showed that a weekly systemic injection of 0.2 to 0.5 mg kg−1 of linearly modified mRNA can treat hemophilia A and B while maintaining protein levels above a clinically relevant threshold.
There are four types of mRNA packaging systems—biomimetic, lipid-cell-based packaging, and extracellular vesicle-based packaging. LNPs were first reported six decades ago and have since undergone several improvements leading to their first clinical use as a means of delivering small interfering RNA (siRNA). The remaining three packaging systems are still in preclinical evaluation.
Cationic lipids induce cytotoxicity and show low transfection efficiency due to rapid clearance in the spleen and liver. In contrast, ionizable cationic lipids are neutral, which protects them from cellular or molecular recognition. Thus, after cellular uptake, they fuse with endosomes, releasing the mRNA cargo into the cell cytoplasm for translation. MC3-derived LNPs, which first received regulatory approval in 2018, showed a mean effective dose (ED50) ~20-fold lower in animal models and are currently also being used in mRNA vaccines against COVID-19.
The scope of mRNA therapy
Compared to mRNA vaccines that have completed successful phase III clinical trials, most mRNA therapeutics are in early phase I clinical trials focused primarily on safety. mRNA therapeutics can deliver any protein locally or systemically, including enzymatic, secreted, mitochondrial membrane, intracellular proteins, receptors, and gene editing proteins. However, only two clinical trials have provided encouraging results regarding their efficacy and safety.
Secreted proteins offer “nearest neighbor” effects beyond the few cells that are transfected. Like paracrine vascular endothelial growth factor (VEGF), they may have clinical applications through tissue-specific delivery. Recent studies on VEGF, p in vivo delivery systems, expand the potential role of mRNA therapeutics in wound healing, peripheral vascular physiology, and bone repair.
The future of mRNA drugs may depend on the rapid development of mRNA cargo, intracellular carriers, and in vivo delivery systems coupled with deep biological and clinical insights. Nevertheless, the multifunctionality of mRNA may give rise to therapeutic opportunities, and thus other innovative applications of it are expected in the near future. For example, recent research has shown the utility of mRNA technology for in vivo expression of intracellular antibodies for the treatment of heart failure and as IVF disease modeling tool.