(108f) Are Structure-Function Relationships Governing Polymeric Gene Delivery Payload-Specific?

Polymeric carriers have proven to be safe, economical, and versatile platforms for the intracellular delivery of therapeutic nucleic acid payloads such as DNA, RNA, and genome editing payloads (e.g., ribonucleoproteins or RNP). Clustered regularly interspaced short palindromic repeats (CRISPR) technology requires polymeric carriers to package, protect, and deliver diverse therapeutic payloads, ranging from plasmids (pDNA), RNA, and ribonucleoproteins. Whether structure-function trends identified for a specific nucleic acid payload are broadly applicable to cargoes with contrasting molecular characteristics, sizes, or biological functionalities remains unresolved. Screening a combinatorially designed polymer library, we discovered that P38, which had earlier been identified as the lead polymer for delivering RNPs, proved to be the most promising carrier for pDNA as well. In addition to mediating high levels of transgene expression, P38 effected homology-directed repair (HDR) at higher rates than the commercial polymeric reagent JetPEI while co-delivering RNPs and pDNA donors to HEK293 cells. Further, the superlative pDNA delivery performance of P38 was generalizable to two therapeutically relevant cell types, human mesenchymal stem cells and retinal pigment epithelial cells, thereby validating the broad applicability of P38 across diverse clinical contexts. The versatility of P38 in delivering multimodal cargoes initially suggested that design specifications for RNP and pDNA carriers overlap considerably. However, in contrast to intracellular RNP delivery, polymeric delivery of pDNA is far less reliant on hydrophobic-hydrophilic phase balance and instead exploits electrostatic interactions to realize functional pDNA delivery. By elucidating the physicochemical basis for polyplex internalization, toxicity, and transfection efficiency through statistical learning, we derived payload-specific guidelines that inform the design of bespoke polymers for specific therapeutic contexts.