Versatile polymeric nanoparticle formulations for fibroblast-targeted mRNA delivery for gene editing

ABSTRACT

Background: Messenger RNA (mRNA) has been regarded as a highly promising tool for gene therapy owing to its transient nature and biodegradability, which offers the possibility of controlling protein production by modulating mRNA pharmacokinetics. This technology has recently emerged to the clinic, following the recent developments of vaccines against SARS-CoV-2 infection (Pfizer/BioNTech, Moderna). In order to further expand its therapeutic potential, it is paramount to engineer vectors capable of effectively delivering mRNA to specific targets. Polymeric nanoparticles are attractive candidates owing to their biocompatibility, biodegradability, and low production costs. Their chemical versatility has enabled the rapid generation of libraries by combining different amines and acrylates/acrylamides via Michael addition. These libraries could guide the design of systems with tropism towards specific cell types, without the need of complicated formulation steps. Herein, we designed a high-throughput screening strategy for rapid in vitro identification of polymeric nanoparticles for safe and effective delivery of mRNA. Materials and Methods: Polymers were prepared by addition of different monomers (bisacrylamides, amines) diluted in DMSO at 1.6 M for 5 days at 60°C, followed by an end-capping step with 20% molar excess of their respective amines for 2 hours. Polymeric nanoparticles were prepared by electrostatic complexation in water of the resulting polymers with mRNA encoding Cre recombinase. Transfection efficiency of the generated 152 formulations was screened using reporter fibroblasts expressing GFP upon Cre-mediated recombination. The top 7 hits were selected for subsequent investigations. After purification by dialysis (MWCO = 2 kDa), these hits were tested for mRNA transfection across several cell types populating the skin (dermal fibroblasts, keratinocytes, endothelial cells, macrophages). Results: Our high-throughput screening strategy identified a lead polymer candidate which transfected dermal fibroblasts much more effectively than other cell types. It also exhibited superior performance than lipid-based transfection agents in the delivery of different mRNAs (Cre recombinase, GFP), including the co-delivery of Cas9 mRNA and a guide RNA. Structure-activity analysis revealed that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. Conclusions: High-throughput screening strategies can rapidly identify chemical features towards the design of highly efficient mRNA delivery systems. Polymeric nanoparticles are versatile systems which can unveil physicochemical properties required for efficient mRNA delivery to target cells. Here, we identified a lead candidate targeting fibroblasts, which could be promising for the treatment of fibrosis. This strategy could be expanded by developing additional reporter cells to unveil novel nanoformulations targeting other cell types.