In a recent article published in Nature Biotechnology, researchers reviewed technology advancements that will unlock the promise of biologically targeted messenger ribonucleic acid (mRNA) therapeutics beyond vaccines.
The first part of the review focused on the design and purification of the mRNA cargos, including novel forms, such as circular (circRNA) and self-amplifying mRNA (saRNAs). The second part discussed improved mRNA packaging systems, including ionizable lipid nanoparticles (LNPs), to enhance cargo delivery.
In the third part, the researchers reviewed the engineering of packaging systems that will facilitate targeting mRNA therapeutics to specific tissues. The fourth and fifth parts discussed strategies enabling chronic disease treatment via mRNA therapeutics and a synopsis of present clinical trends in mRNA therapeutics. Lastly, the researchers emphasized the scope of the novel mRNA therapeutics in the near- and long term.
The unprecedented success of coronavirus disease 2019 (COVID-19) vaccines based on the mRNA technology platform has renewed interest in this therapeutic area. However, several challenges still prevent establishing mRNA technology as a general therapeutic modality with broad applicability against diverse clinical conditions.
Advancements in the area of protein expression, packaging systems, tissue targeting, and chronic dosing
Immunization requires minimal protein expression levels, while mRNA therapeutics requires a 1,000-fold-higher protein level to reach a therapeutic threshold. Efficient delivery to solid organs remains challenging. Even the tissue bioavailability, circulatory half-life, and efficiency of the LNP-based carrier could be rate-limiting when it is delivered to the target tissue. Even with optimized mRNA chemical modifications and advanced LNPs, chronic dosing eventually activates innate immunity, parallelly attenuating 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 advancements in the design of each of these components. Most notable of these are:
i) improved 5′ cap analogs that enhance translational capacity, but more importantly, the capping efficiency from 70% to 95%.
ii) the poly(A) tail length optimization has proven critical for balancing the synthetic capability of an mRNA cargo.
iii) UTR sequence optimization could improve protein expression of an mRNA cargo by a fewfold, allowing its customization to the targeted biological area and disease-elicited microenvironment.
iv) Studies have documented 130+ naturally occurring chemical modifications for mRNA so far. The chemically modified nucleosides, particularly uridine moieties, such as methylpseudouridine, can reduce recognition by the toll-like receptors of the innate immunity by up to 100-fold, which, in turn, markedly increases protein expression after in vivo transfection of mRNA cargos. In the future, clinically effective, unmodified therapeutic mRNAs might become available that will conceal themselves from the immune system and have enhanced translational efficiency in vivo, similar to chemically modified mRNA vaccines.
Likewise, saRNAs could prove favorable for enzyme replacement therapies. They require ~10-fold less RNA for a similar magnitude of protein expression compared to linear-modified mRNA and are under in vivo testing, a scalable process for vaccine production. Another alternative to linear mRNA is circRNA, that have been shown to extend mRNA life two-fold in vitro. The circRNA evades the need for expensive 5′ capping, tedious 3′ poly(A) tail and increases the total protein yield without increasing the protein expression levels compared to linear-modified mRNA.
Overcoming challenges concerning the amplitude of protein expression, parallel to the mRNA structural optimizations, could alleviate the need for repeated dosing, a primary requirement hindering the treatment of chronic diseases using mRNA therapeutics. The conventional treatment involves the systemic injection of recombinant clotting proteins (factor VIII/IX) three to seven times per week because of their relatively short half-life of ~12 hours. On the contrary, preclinical studies in mice have shown that a single weekly systemic injection of 0.2 to 0.5 mg kg−1 of linear-modified mRNA could treat hemophilia A and B while maintaining protein levels above a clinically relevant threshold.
There are four types of mRNA packaging systems - biomimetic, lipid- and cell-based packaging, and extracellular vesicle-based packaging. LNPs were first reported six decades ago and have since undergone several advancements, leading to their first clinical use as a small interfering RNA (siRNA) delivery vehicle. The other three packaging systems are still in the preclinical evaluation stage.
Cationic lipids induce cytotoxicity and exhibit low transfection efficiencies due to speedy splenic and hepatic clearance. On the contrary, ionizable cationic lipids are neutral, which sheaths them from cellular or molecular recognition. Thus, following cellular uptake, they fuse with the endosomes, releasing the mRNA cargo into the cell cytoplasm for translation. The MC3-composed LNPs, first receiving regulatory approval in 2018, shows a median effective dose (ED50) ~20-fold lower in animal models and are also currently used in COVID-19 mRNA vaccines.
The scope of mRNA therapeutics
Compared to mRNA vaccines which have completed successful phase III clinical trials, most mRNA therapeutics are in early clinical phase I trials, focused mainly on safety. The mRNA therapeutics could deliver any protein locally or systemically, including enzymatic, secreted, mitochondrial membrane, intracellular proteins, receptors, and gene-editing proteins. However, only two clinical studies have produced encouraging results about their efficacy and safety.
Secreted proteins offer ‘closest-neighbor’ effects beyond the few cells that are transfected. Similar to paracrine vascular endothelial growth factor (VEGF), they might have clinical applications via tissue-specific delivery. Recent studies on VEGF, with in vivo delivery systems, are extending the potential role of mRNA therapeutics in wound healing, peripheral vascular physiology, and bone repair.
The future of mRNA drugs may depend on rapid developments in the mRNA cargo, intracellular carriers, and in vivo delivery systems coupled with deep biological and clinical insights. Nevertheless, the versatility of mRNA could trigger therapeutic opportunities, and its other innovative applications, thus, are expectedly in the near future. For instance, recent research works have shown the utility of mRNA technology for in vivo expression of intracellular antibodies for heart failure treatment and as an in vitro disease-modeling tool.