Targeting dendritic cells with RNA-loaded nanoparticles grafted with short peptides

Nanoparticles encapsulating therapeutic RNA have emerged as a transformative strategy in precision medicine, capable of mobilizing the immune system to induce specific responses, ranging from immune tolerance to fighting tumors. However, most current preclinical and clinical efforts rely on non-targeted delivery systems, limiting their safety, therapeutic efficacy, and selectivity. To enhance the therapeutic index of RNA-based therapeutic systems for immunomodulatory purposes, we report on the design of a novel Clec9A-targeted polymeric nanoparticle, aimed at selectively engaging dendritic cells responsible for antigen presentation. We began by evaluating in silico the binding potential of the previously reported 12-amino-acid WH peptide, known for its high affinity to mouse Clec9A, the human ortholog. Using computational tools, we designed and screened truncated variants of the peptide and identified promising candidates with retained or enhanced binding capacity to human Clec9A. These optimized short peptides were synthesized and covalently conjugated to our proprietary poly(beta amino ester) (pBAE) polymers. We evaluated the impact of conjugation site, comparing terminal versus lateral chain attachment on receptor targeting and confirmed in vitro that peptide orientation significantly influences binding efficiency. Additionally, we computationally generated and validated shorter mutant peptide variants with improved Clec9A affinity over the original sequences. Our findings demonstrate that rationally engineered short peptides, when site-specifically conjugated to pBAE polymers, can provide high-affinity, selective targeting of dendritic cells via Clec9A. This strategy lays the groundwork for the next generation of targeted RNA-based immunotherapeutics, offering improved selectivity, immune activation, and therapeutic potential.