Poster J10 - Optimization of poly(beta-amino ester) chemistry for T cell-targeted polymeric nanoparticle-based mRNA delivery

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Xinjie Yu

Research Assistant
Johns Hopkins University
Baltimore, Maryland, United States

Co-Author(s)

MJ

Manav Jain

Graduate Student
Johns Hopkins University, United States
SD

Sydney Dunham

Summer intern
Johns Hopkins University, United States

Primary Investigator(s)

Jordan J. Green, PhD (he/him/his)

Professor
Johns Hopkins University, Maryland, United States

Introduction: Chimeric antigen receptor (CAR) T cells have exhibited great therapeutic potential. However, wider clinical translation is undermined by the requirement of expensive viral vectors in the manufacturing process. In comparison, non-viral gene delivery vehicles based on poly(beta-amino ester)s (PBAEs) have greatly reduced cost. PBAEs are a family of biodegradable cationic polymers capable of complexing with nucleic acids to form nanoparticles. PBAEs are composed of acrylate backbone (B) monomers and amine sidechain (S) monomers, while terminated at both ends with amine-endcap (E) monomer. The wide range of available, B, S, and E monomers enables structural variability exhibiting distinct chemical properties. However, existing PBAE nanoparticle formulations were observed to transfect primary T cells with limited efficacy. Various observations have highlighted the importance of nanoparticle chemical properties in determining gene delivery performance. The uniquely more acidic endosomal pH of T cells linked the pKa of nanoparticles with endosomal escape outcome, an important step during transfection. Particle hydrophobicity was also implicated in the transfection of immune cells and dendritic cells. With such considerations, we describe a workflow to systematically explore the space of chemical properties accessible to the PBAE structure with the goal of selecting the optimal sidechain and endcap monomers for transfecting T cells.

Materials and

Methods: The PBAEs were synthesized through a Michael addition reaction. First, the B and S monomers were mixed in dimethylformamide and were allowed to react at 85℃ for 48h. Then, the chain growth reaction was terminated by a 2-hour end capping reaction, whereby a molar excess of E monomers was added to the chain growth reaction mixture. Afterwards, the mixture was added to excess diethyl ether to precipitate the PBAEs. The precipitate was washed with ether several times before drying in vacuum for 48 hours to remove the ether. Finally, the dried polymers were dissolved in THF. Two batches of PBAEs were synthesized as such, using different E and S monomers respectively, while keeping other components unchanged. The B monomer investigated in this study, B7, was kept unchanged in all PBAE formulations. S monomers evaluated were S3, S5, S90, Sc12, and Sc18. E monomers investigated were E1, E6, E7, E63, and E65.

Nanoparticles were formed via self-assembly through mixing an aqueous phase containing mRNA encoding green fluorescent protein (GFP) and an organic phase containing the synthesized PBAEs and poly(ethyl glycol). The PBAE nanoparticles formed were then dissolved in a 1X phosphate buffered saline (PBS) solution. The sizes of newly synthesized nanoparticles were determined via dynamic light scattering (DLS).

Primary murine T cells were isolated and co-incubated with the PBAE nanoparticles. After 24 hours, GFP fluorescence (as an indicator of the transfection efficiency) and cell viability were measured using flow cytometry.

Results, Conclusions, and Discussions: As shown in Figure 1 A-D, both endcap and sidechain monomer chemistry exhibited influence over the transfection efficiency of the resulting nanoparticles in primary T cells. Considering different endcap monomers, the PBAE with the endcap monomer designated E1 exhibited highest T cell transfection efficiency. The screen for sidechain monomers demonstrated that the PBAE lipophilic sidechain designated Sc18 yielded the formulation achieving the most robust transfection, which was considerably higher than the design previously used by our group. Also, the cell viability remained consistent across the treated and untreated groups, proving that the PBAE nanoparticles achieved transfection with low cytotoxicity.

These in vitro outcomes underscored the importance of optimizing polymer chemistry in creating the ideal T cell-targeting PBAE nanoparticle. Experiments investigating the combined effects of E and S components, particularly the PBAE consisting of E1 and Sc18 monomers, are underway. Due to accumulating evidence, we hypothesize that the variation in transfection efficiency conferred by PBAE endcap and sidechain chemistry is correlated with the endosomal escape capability of the resulting nanoparticles in an apparent pKa-dependent manner. Experiments exploring such a correlation are currently being conducted.

In conclusion, we have discovered a new PBAE polymer design that resulted in optimized gene delivery to primary T cells in vitro. Through a screening that systematically traversed the chemical space available to the modular structure of PBAEs, we were able to identify the best-performing E and S monomers. Our results also laid the foundation for future investigations concerning the exact chemical properties that determine the effectiveness of T cell-targeted gene delivery.

Acknowledgements (Optional):