(132b) Engineering Advanced Delivery Vehicles for mRNA Therapies By Integrating High-Throughput Screening and Rational Design Approaches

Messenger RNA (mRNA) represents a promising new class of drugs that can be utilized not only as vaccines for infectious diseases and oncology, but also as protein replacement and gene editing therapies for the treatment of genetic diseases. However, major challenges for the clinical translation of mRNA drugs are safe and effective delivery of the mRNA to target tissues and cells. For mRNA vaccines, another challenge is to carefully control immune stimulation to enhance productive immune responses to the encoded antigen while minimizing reactogenic side effects. Recently, lipid nanoparticles (LNPs) have emerged as the most clinically translatable delivery vehicle for mRNA therapies and vaccines. However, engineering LNPs which optimally address the above challenges is complicated due to the many interactions between the multiple components comprising an mRNA-LNP formulation, including the four key lipid types which make up the LNP along with the mRNA cargo as well as the formulation buffer, and the complex interactions between the resultant LNP formulation and biological systems. Here, I will present an overview of my main projects where we address the above challenges to mRNA-LNP delivery by systematically engineering each of the mRNA-LNP components, including the lipids, the mRNA, and the buffer, using a combination of high-throughput and rational design approaches to improve the safety and efficacy of mRNA vaccines and inhaled mRNA therapies.

For inhaled mRNA therapies, there are both physical and biological barriers to developing clinically translatable mRNA delivery strategies. Inhaled mRNA requires nebulization to aerosolize the LNP formulation but this process imparts high shear forces on the LNP which causes them to release their mRNA cargo and also to aggregate, necessitating the development of LNPs with greater stability. Additionally, the lung epithelial microenvironment poses major cellular and extracellular transport barriers to intracellular mRNA delivery. I will present our work on addressing these barriers in which we develop nebulizer-stabilized lipid nanoparticle (LNP) formulations which demonstrate state-of-the-art delivery of mRNA to the lung epithelium in both healthy and muco-obstructive disease lung models. This is achieved through engineering of all of the LNP components, including high-throughput screening of novel ionizable lipids in ex vivo lung models to improve LNP transfectivity in the lung epithelium as well as rational design of the nebulization buffer and PEG-alternative polymeric modifications to the LNP surface for stabilizing LNPs to nebulization.

For mRNA vaccines, complex interactions between the immune system and the mRNA-LNP formulation play a central role in determining vaccine safety and efficacy. Thus, there is an opportunity to engineer improved mRNA vaccines through careful design of self-adjuvanting systems which target specific immune cells and pathways to promote antigen-specific immune responses while avoiding unwanted side effects. I will present how engineering adjuvanting properties into both the lipids of the LNP formulation as well as the mRNA encoded antigen results in synergistically enhanced immune responses to mRNA vaccination with promising safety profiles when compared to unadjuvanted vaccines following either intramuscular or intranasal administration. The ionizable lipids were designed to balance high transfection efficiency and immunostimulation with top-performing lipids identified using high-throughput in vivo screening. The mRNA encoded antigen was adjuvanted through genetic fusion of the antigen to molecular adjuvants adapted from naturally-occurring signaling proteins which are important in bridging the innate and adaptive immune response.

Finally, I will discuss how my future lab will conduct research at the intersection of chemical engineering, biomaterials, and drug delivery to engineer and implement a diverse toolbox of delivery vehicles, both viral and non-viral, for addressing biological barriers to therapeutic protein and nucleic acid delivery.