mRNA therapeutics is a rapidly changing area of drug development. From vaccines for COVID-19 to oligonucleotides to cure Spinal Muscular Atrophy, their potential applications are wide and deep. However, mRNA drugs have yet to become as common as small molecules and antibody pharmaceuticals. Major hurdles to their success lie with their biological characteristics. First, mRNA is a labile molecule, easily degraded and unstable. This is both an advantage, as there is an inherent clock on the efficacy of mRNA therapeutics, and a disadvantage, as re-administration is likely necessary to continue therapy. Second, mRNA must be in the cytoplasm of a cell in order to be “read” or translated by cellular machinery. Both of these challenges are addressed in the topic of “therapeutic mRNA delivery”. An ideal mRNA therapeutic delivery system will address the challenges of mRNA with an easily, repeatedly and effectively administered formulation. The concept of therapeutic mRNA delivery remains a major area of active research and development.
The formulation of an mRNA therapeutic is dependent on many features of the treatment, including inherent qualities of the drug, the target tissue and patient compatibility. One of the most common ways to deliver mRNA therapeutics is through a lipid nanoparticle or LNP. LNPs are generally comprised of an outer lipid layer that encapsulates the mRNA drug, protecting it from nuclease degradation. The composition of the outer membrane layer of LNPs has seen countless iterations for ideal cell targeting, plasma membrane fusion and release of contents into the cytoplasm. Further advances in LNP design are a critical step to the forward movement of mRNA therapeutics.
A recent paper from a group at Chiba University in Japan discussed a new structure of LNP for improved mRNA delivery. In their study, the researchers altered the chemical structure of their LNP to create a self-degrading particle that disassociates upon entry specifically into the cytoplasm of cells. They term their lipid-like degrading particle and mechanism “Hydrolysis accelerated by intra-Particle Enrichment of Reactant” or HyPER. In this way, their LNP formulation seeks to address the challenge of successful therapeutic mRNA delivery to the cytoplasm.
The research began with a description of the design and a proposed mechanism of their LNP self-degradation. Critical chemical components of their LNP included a disulfide bond and a phenyl ester linked to hydrophobic oleic acid chain. A high GSH concentration inside the cytoplasm reduced the thiols of the disulfide bond to become potent nucleophiles. Once reduced, the thiols attacked the phenyl ester electrophile efficiently due to their proximity within the molecule. Without the phenyl ester addition, ~75% of the chemical remained in a dimeric structure, connected by the disulfide bond. The group nicely described the kinetics of the degradation reaction and identified the products of degradation.
They went on to examine the ability of the LNP to deliver mRNA content in vitro and in vivo. In vitro, they demonstrated that the phenyl ester ring contributed to improved endosomal escape and rapid expression throughout the cytoplasm. These scientists convincingly showed how the phenyl ester was essential to the self-degradation by comparing four LNP structures: ssPalmO-Phe (+phenyl ester, +disulfide bond), ssPalmO-Ben (+benzene ester, so is non-self-degradable, +disulfide bond), ssPalmO (no ester, +disulfide bond) and a ccPalmO-Phe (no disulfide bond, so is non-cleavable). They used a combination of assays and these chemical structures to assess the LNP’s ability to facilitate cellular uptake, release, and gene expression.
For their in vivo experiments, the scientists relied on TriLink Biotechnologies’ modified EPO and Cas9 mRNAs. After tail vein injections into mice, this LNP facilitated gene expression of EPO mRNA in the liver to levels ten times higher than another commercial in vivo delivery product. The LNP also permitted successful genome editing of the transthyretin gene using a modified Cas9 mRNA: 55% of cells in the liver were edited and serum transthyretin levels decreased by 95% two weeks after the final dosing. Doses for both in vivo mRNA treatments were far below levels that showed toxicity. Overall, this novel self-degrading LNP structure permitted mRNA release and gene expression both in vitro and in vivo.
Although this novel LNP structure was effective, feasibility of LNP delivery to the liver in vivo is well-established. The bigger challenge remains: Can these LNPs robustly deliver mRNA therapeutics to other tissues in vivo? It would be very exciting to see comparisons between this LNP design and other LNPs in clinical trials or on the market. What modifications will this HyPER design need to target other tissues and cell types? With this self-degrading ssPalmO-Phe HyPER structure, LNP design is improved and has the potential to advance therapeutic mRNA delivery.
Featured product: modified EPO and Cas9 mRNA
Article Reference: Adv. Funct. Mater. 2020, 30, 1910575. https://doi.org/10.1002/adfm.201910575