Thiol-Reactive PEGylation Strategy for Antibody-Functionalized Polymeric Micelles in Nonviral Jurkat T Cell Engineering
mRNA therapeutics enable direct cytoplasmic protein expression without genomic integration, which makes them safer than DNA-based methods. But mRNA is unstable, therefore it needs delivery vehicles. Although viral vectors and lipid nanoparticles (LNPs) are highly efficient, concerns about immunogenicity and cost have driven interest in alternatives. Polymeric micelles, formed from amphiphilic block copolymers, offer tunable design and scalable production. It is still very hard to get nucleic acids to T cells, the approach used to engineer cells for CAR-T therapies, since they don't take in enough of them, their membranes aren't particularly permeable, and they are sensitive to cationic compounds. We tested a group of linear polymers and polymeric micelle formulations for delivering mRNA into Jurkat T cells. The A7, polyamine ampiphile copolymer micelle formulation, had the best balance of transfection efficiency, with only 5-7% of cells being GFP-positive and the greatest MFI across micelle systems. Nonetheless, no formulations surpassed JET-PEI, the commercial positive control, underscoring the necessity for T cell-targeted delivery techniques. To overcome this constraint, we are creating an anti-CD3 targeted micelle platform to improve T-cell uptake through CD3 engagement. This involves attaching partly reduced anti-CD3 to an OPSS-PEG-DDMAT micelle copolymer using thiol chemistry or layering it over premade A7 micelles using OPSS-PEG-DDMAT-CEA conjugate. As a feasibility study, thiol-reactive chemistry effectively attached 5 kDa OPSS-PEG-OH to bovine serum albumin (BSA). SDS-PAGE and Ellman's assay both verified conjugation. SDS-PAGE showed a change in molecular weight from 66.5 kDa to 70 kDa, and Ellman's assay showed that free thiols (BSA Cys-34) were reduced in the reaction sample by roughly 50% compared to fresh BSA control and by 30% compared to BSA left in reaction buffer overnight. After that, DCC chemistry was used to link OPSS-PEG to DDMAT, and RAFT was used to polymerize the two to make a PEG-functionalized copolymer. Current efforts are directed towards the conjugation of both BSA and anti-CD3 antibodies to OPSS-PEG-functionalized polymers, thereafter undergoing purification using size exclusion chromatography and validation via SDS-PAGE and Ellman’s test. We will add these constructs to micelle formulations and test them for size, stability, and mRNA encapsulation (using DLS and RiboGreen), as well as how well they work for transfection in Jurkat T cells. This approach allows for tailored, nonviral mRNA delivery for T-cell engineering and may make polymer-based delivery more effective in Jurkat T cells.