Garcia-Fernandez, C. | Search bioRxiv

bioRxiv2025

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. Graphical abstractSchematic representation of the workflow used in this work. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/686747v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@17f2d4eorg.highwire.dtl.DTLVardef@779825org.highwire.dtl.DTLVardef@1a5ded2org.highwire.dtl.DTLVardef@c7cb96_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health

BackgroundTargeted amplicon sequencing is a powerful and efficient tool for interrogating the Plasmodium falciparum genome, generating actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive, and reproducible. MethodsWe developed, characterized, and implemented MAD4HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. Additionally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. Laboratory control and field samples were used to demonstrate the panels high sensitivity and robustness. ResultsMAD4HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes, key markers for drug and diagnostic resistance (including duplications and deletions), and csp and potential vaccine targets. The panel can also detect non-falciparum Plasmodium species. MAD4HatTeR successfully generated data from low-parasite-density dried blood spot and mosquito midgut samples, and detected minor alleles at within-sample allele frequencies as low as 1% with high specificity in high-parasite-density dried blood spot samples. Gene deletions and duplications were reliably detected in mono- and polyclonal controls. Data generated by MAD4HatTeR were highly reproducible across multiple laboratories. ConclusionsThe successful implementation of MAD4HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD4HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.

genomics↗