High-Throughput Approach to Identify New Lipid Nanoparticle Formulations for Vaccine and Drug Delivery

High-throughput in vivo screening method to identify lipid nanoparticle (LNP) formulations for local and systemic delivery for mRNA vaccines and cancer immunotherapies.
Problem:
Nucleic acid delivery is an attractive and rapidly growing area of research, particularly for the development of vaccines and cancer immunotherapy. Historically, viral vectors are used for nucleic acid delivery; however, these possess several practical shortcomings. Lipid nanoparticles (LNPs) have become a promising non-viral nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process.
Solution:
High-throughput screening is used to investigate the influence of LNP composition on the uptake by different immune cell populations. The LNP formulations identified by the inventors hold promise for immunoengineering applications based on modified mRNA and demonstrate the value of the design criteria developed through high throughput in vivo screening.
Technology:
The inventors employed high-throughput in vivo LNP screening based on molecular barcoding to identify key parameters that influence mRNA LNP immune transfection such as ionizable lipid structure and lipid excipient composition. They screened a library of 75 LNPs under both intramuscular (i.m.) and intravenous (i.v.) injection and observed different compositional factors influencing LNP uptake by immune populations across the two administration routes. Validation studies were performed to test and confirm the potential of lead LNP candidates for in vivo immunoengineering using mRNA, with comparisons against clinical standards.
Advantages:

This screening provides insight into LNP design criteria for immune transfection and the interplay among administration route, ionizable lipid structure, and LNP composition in determining mRNA LNP efficacy and fate.
The lead LNP formulation for i.m. administration demonstrated substantial mRNA translation and a more favorable biodistribution profile than LNPs formulated with a clinical standard lipid, DLin-MC3-DMA (MC3).
The lead LNP formulations administered i.v. displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes.
Applications of LNPs include prophylactic vaccines and cancer immunotherapies.

Stage of Development:

Target Identified
Preclinical Discovery

A) Overview of the high-throughput screening approach used to develop immunotropic mRNA LNPs. A library of 75 LNPs encapsulating reporter mRNA along with barcoded DNA (b-DNA) was formulated and administered to healthy mice via either i.m. or i.v. administration. Blood, lymph nodes, and spleens were isolated as immune tissues of interest, and immune cells were isolated from each using fluorescence-activated cell sorting (FACS). High-throughput screening methodologies were employed to identify key parameters influencing LNP performance and lead candidates for each administration route. These lead candidates were then validated in isolation to evaluate their potential for in vivo immunoengineering using mRNA. B) Schematic overview of validation study to confirm the systemic immunoengineering potential of LNPs identified by high-throughput in vivo screening. LNPs from screening under i.v. administration were reformulated encapsulating Cre recombinase mRNA and administered systemically to Ai9 (CAG-loxP-STOP-loxP-tdTomato) mice. 72 h following injection, spleens and peripheral blood were processed for flow cytometric analysis of Cre mRNA transfection. C) Schematic overview of validation study to assess the immunoengineering potential of LNPs identified by high-throughput in vivo screening. LNPs from screening under i.m. administration were reformulated encapsulating FLuc mRNA and labeled with the DiD lipophilic fluorescent dye. Twelve hours following injection, organs were subjected to bioluminescence and fluorescence imaging to assess gross LNP accumulation and transfection.
Intellectual Property: