Current landscape of mRNA technologies and delivery systems for new modality therapeutics

Abstract

Realizing the immense clinical potential of mRNA-based drugs will require continued development of methods to safely deliver the bioactive agents with high efficiency and without triggering side effects. In this regard, lipid nanoparticles have been successfully utilized to improve mRNA delivery and protect the cargo from extracellular degradation. Encapsulation in lipid nanoparticles was an essential factor in the successful clinical application of mRNA vaccines, which conclusively demonstrated the technology's potential to yield approved medicines. In this review, we begin by describing current advances in mRNA modifications, design of novel lipids and development of lipid nanoparticle components for mRNA-based drugs. Then, we summarize key points pertaining to preclinical and clinical development of mRNA therapeutics. Finally, we cover topics related to targeted delivery systems, including endosomal escape and targeting of immune cells, tumors and organs for use with mRNA vaccines and new treatment modalities for human diseases.

Keywords: Lipid nanoparticles; New modality therapeutics; Targeting mRNA delivery systems; mRNA Technology; mRNA therapeutics.

Fig. 1

A Overview of new US FDA-approved therapeutic modalities. Recent technological breakthroughs have led to the introduction of several new therapeutic modalities and are expected to drive further innovation in the biopharmaceutical industry over the coming decades. The schematic illustrates a spectrum of new pharmaceutical modalities encompassing eight distinct categories: Antibody–Drug Conjugates (ADCs), gene therapy, chimeric antigen receptor T cell (CAR T) therapy, CRISPR-based therapeutics, messenger RNA (mRNA) therapeutics, small interfering RNA (RNAi), antisense oligonucleotides (ASOs), and bispecific antibodies. Prominent examples of pioneering US FDA-approved drugs within each modality are listed as follows. ADCs: Adcetris (brentuximab vedotin, approved 2011), bispecific antibodies: Blincyto (blinatumomab, approved 2014), RNAi: ONPATTRO (patisiran), ASOs: Exondys 51 (eteplirsen, approved 2016), CAR T therapy: Kymriah (tisagenlecleucel, approved 2017), Gene therapy: Luxturna (voretigene neparvovec-rzyl, approved 2017), mRNA therapeutics: Pfizer-BioNTech's Comirnaty (COVID-19 Vaccine, BNT162b2, approved 2021). Furthermore, the recent approval of a CRISPR/Cas9 gene editing therapy, Casgevy (exagamglogene autotemcel, approved December 8, 2023), underscores the continual expansion of therapeutic modalities. Numerous new cutting-edge technologies including mRNA technologies are currently under evaluation at various stages of drug development. B The graphic outline of milestones and development timeline in mRNA technologies and LNP delivery systems

Fig. 2

Synthesis and purification of three distinct mRNA types for mRNA-LNP drugs. A The in vitro transcription (IVT) process is illustrated. First, a plasmid is generated containing the target gene with a T7 promoter. After restriction enzyme (RE) digestion of the plasmid and purification of linear DNA, IVT is performed with T7 RNA polymerase, cap analogue, modified bases, and RNase inhibitor to generate transcribed linear mRNAs. The linear mRNAs may be traditional linear mRNAs, self-amplified mRNAs (saRNAs), or trans-amplified mRNAs (taRNAs). For production of circular RNAs (circRNAs), cyclization is achieved via intron-splicing reaction or T4 RNA ligase. Impurities within the mRNA products may be eliminated by DNA digestion and mRNA purification, along with other methods specific to the type of RNA product. The highly purified mRNAs are suitable for incorporation into mRNA-LNP formulations. (SEC: size exclusion chromatography, HIC: hydrophobic interaction chromatography, RP-HPLC: reverse phase HPLC). B Advantages and disadvantages of the three different mRNA types used for mRNA-LNP drugs are shown

Fig. 3

Strategies for enhancing the efficacy and stability of mRNAs and mRNA-LNPs. Diverse strategies may be used to preserve the integrity of mRNAs and mRNA-LNPs. Different characteristics of the mRNA can augment stability and prolong intracellular expression of the encoded product. Such characteristics include self-amplifying or circular mRNA, nucleotide sequences, and untranslated regions (UTRs). Furthermore, optimizing lipid formulations through adjustment of lipid ratios or inclusion of novel components can improve the stability of mRNA-LNPs. Recent studies indicate that lyophilization in the presence of appropriate cryoprotectants can facilitate the long-term storage of mRNA-LNPs at 4 ℃, which would be a major advantage for future clinical applications

Fig. 4

Strategies to facilitate endosomal escape of LNPs for enhanced mRNA release and protein translation. LNPs are taken up into cells via endocytosis. Targeted or modified LNPs can enhance endocytosis. Innovative ionizable lipid structures used in the formulation of LNPs can facilitate endosomal escape and release of the payload into the cytosol. The integration of cutting-edge ionizable lipid structures within LNPs can significantly enhance endosomal escape, facilitating the efficient release of the encapsulated payload into the cytosol. Novel ionizable lipids commonly feature distinctive characteristics, such as a polyamine head group with a pKa around 6.5, incorporation of aromatic ring moieties in linker chains, and diverse tail compositions (e.g., bioreducible disulfide, branched, or unsaturated tails). Different helper lipids, such as PE-containing phospholipids, pH-sensitive PEGylated lipids, and hydroxylated cholesterol derivatives can contribute to endosomal escape as well. Integration of other materials, such as the small molecule inhibitor NP3.47 or polyhistidine peptides can also promote this process. Novel LNPs with these features have been shown to enhance endosomal escape and reduce lysosomal degradation of mRNA cargoes, which eventually promotes mRNA escape from endosomes and facilitates mRNA release, ultimately increasing translation of the encoded protein. Created with BioRender.com

Fig. 5

Strategies for ligand-mediated surface modification to achieve mRNA-LNP targeting. The surface of an mRNA-LNP can be modified with various ligands to enhance targeting specificity. Addition of sugars, nucleotides, peptides, antibody fragments or full-length antibodies can facilitate the targeted delivery of mRNA-LNPs to specific cells, tissues, organs or tumors. mRNA-LNPs with enhanced targeting specificity hold promise for improving the efficacy of mRNA-based drugs and vaccines. Created with BioRender.com