Notice and Invitation
Oral Defense of Doctoral Dissertation

Bioengineering Department
College of Engineering and Computing, George Mason University

Manuel Junior Carrasco Chamorro

Bachelor of Science, Genetics and Biotechnology, Universidad Nacional Mayor de San Marcos, Lima, Peru, 2015

Understanding the Effect of Ionization, Structural, and Manufacturing Parameters on Colloidal and Biological Properties of mRNA Lipid Nanoparticles

Monday, November 27, 2023, 12:00 pm – 2:00 pm
IABR 1004, SciTech campus

In-person preferred. Zoom if needed.

Meeting ID: 928 8626 1069

Passcode: 011386

All are invited to attend.

Committee
Dr. Mikell Paige, Director

Dr. Rémi Veneziano, Chair

Dr. Mohamad-Gabriel Alameh

Dr. Patrick Gillevet

Dr. Aarthi Narayanan

Abstract

Non-viral delivery systems have been developed for various clinical applications to ensure a safe and effective biological response to the payload. Efforts on Lipid nanoparticles (LNPs) were allocated to deliver siRNA to become the first FDA-approved drug. In addition to this splendid work, LNPs could be used as mRNA vaccines containing immunogens to provoke an immune response. They were considered the heroes that saved humankind from the current COVID-19 pandemic. LNPs are a multi-dynamic system comprising lipids that self-assemble based on their hydrophobicity and the increased solvent polarity, encapsulating the messenger RNA (mRNA) to efficiently deliver the mRNA for in vitro or in vivo applications. The development of novel mRNA LNPs is mainly focused on increasing the diversity of lipids to be used during formulation, even though the delivery efficiency is in part governed by the lipid structure, not primarily but mostly due to the overall LNP properties that dictate the potency, organ biodistribution, and immunogenicity. To date, previous research does not address more rigorous diligence in designing principles of mRNA LNPs for IM administration for a better understanding of significant role factors (ionization, structural, and manufacturing) that influence the optimization of more potent mRNA vaccines post-IM and avoid off-target effects.

This manuscript-based dissertation comprised three chapters with the following objectives: 1) Understand the drop in apparent LNP pKa by thermodynamic modeling of the ionization equilibrium in the aqueous phase and LNP phase to rationally design LNPs based on the ionizable lipid structure and ionization features, 2) Evaluate physicochemical, ionization, and structural properties of mRNA LNPs to correlate with transfection efficiency in vitro and in vivo post-IV and IM administration, 3) Investigate the effect of positively or negatively-charged LNPs on the muscle expression, and liver off-target expression post-IM administration. 4) Identify formulations with optimal features (pKa, size, charge, ultrastructure) that boost immunogenicity in mRNA vaccines post-IM, and 5) Assess novel manufacturing procedures with improved LNP parameters that enhanced potency, and adaptive immune response upon IM vaccination at lower doses. The first manuscript described the reduction of mRNA LNP apparent pKa to be 2-3 points lower than predicted ionizable lipid pKa. The study indicated the correlation between in vitro and in vivo expression for IM administration for LNPs in a pKa range of 6.5-7, and isoelectric point (pI) by Zeta potential (ZP) ~5.4-6.6. A more positive charge LNPs are more efficient for IM injections by increasing muscle expression and limiting liver off-targeting expression. To explore the role of LNP charge in potency and targeting, reducing the N:P ratio produced less polar, larger, more negatively charged LNPs with higher transfection efficiency in vitro due to increased protonation in the endosomal pH range. More negatively charged LNPs showed higher muscle expression with more liver off-targeting expression post-IM. These findings permitted better ionizable lipid candidates to be used for mRNA vaccines and the understanding of physicochemical, ionization, and structural properties toward potency. The second manuscript introduced a more stable novel ionizable Lipid (C24) LNP as a potential mRNA vaccine, which is a more positively charged LNP compared to gold standard MC3. C24 LNP resulted in larger sizes with higher transfection efficiency in vitro due to higher protonation levels in the endosomal range, in addition to improve in vivo muscle expression by ~4X with limited liver off-targeting post-IM. C24 LNP elicited higher immunogenicity ~10X against the S2P immunogen and was shown to be protective against lethal SARS-CoV-2 challenge. Furthermore, the features that correlated potency and immunogenicity were consistently tested using a novel LNP manufacturing procedure. The third manuscript described the mixing method by increasing mRNA concentration during LNP formulation and keeping the N:P ratio constant to formulate larger LNPs slightly negative at physiological pH without change in pKa and pI but with higher protonation level in the endosome range. Ultrastructural analysis reveals an LNP population with larger LNPs that contain higher bleb frequency, larger blebs, relative blebs per LNP, and multiple blebs in one LNP. The LNPs were ~10X more potent in vitro, with higher in vivo muscle expression (up to ~150%), limited liver off-targeting expression, and increased humoral and cellular immune response in an ionizable lipid identity-dependency manner post-IM.

Carol McHugh

Academic Program Assistant

Department of Bioengineering

3100 Peterson Hall

703-993-5846