The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in...

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Self-amplifying mRNA (saRNA) offers scalable protein expression for biotherapeutics.
• Current mRNA therapeutic efficacy in humans does not linearly scale from small animal models.
• Optimization of saRNA formulations is critical for clinical translation.

Purpose of the Study:

• To optimize self-amplifying mRNA (saRNA) lipid nanoparticle formulations for enhanced protein expression in human skin explants.
• To identify key formulation parameters influencing saRNA delivery and expression using a design of experiments (DoE) approach.
• To evaluate the cellular uptake and expression profile of optimized saRNA formulations in human skin.

Main Methods:

• A design of experiments (DoE) approach was employed to systematically vary formulation parameters.
• Lipid nanoparticle formulations containing saRNA were tested in human skin explants.
• Luciferase expression was quantified to assess saRNA activity.
• Flow cytometry was used to analyze cellular delivery and RNA expression in different cell types.

Main Results:

• Luciferase expression from saRNA peaked at 11 days post-administration in human skin.
• Lipid identity and lipid concentration were identified as significant parameters in the DoE model.
• The optimized formulation yielded a ~7-fold increase in luciferase expression compared to the initial DOTAP formulation.
• Optimized saRNA formulations delivered to ~2% of resident skin cells, with immune cells showing disproportionately high RNA expression (~50% of total RNA).

Conclusions:

• A DoE approach combined with human skin explants is a powerful system for optimizing nucleic acid formulations.
• The optimized saRNA formulations demonstrate significantly enhanced protein expression in a clinically relevant model.
• This methodology can accelerate the development of translational nucleic acid vaccines and therapeutics.