Development of siRNA-peptide libraries for delivery screening - Project Summary/Abstract Viral infections pose a major public health concern, affecting millions and contributing significantly to global mortality, as evidenced by the COVID-19 pandemic, which cut short over 7 million lives worldwide. The success of mRNA vaccines during this pandemic showcased the power of nucleic acid therapeutics in combating emerging viral threats. However, vaccines rely on the adaptive immune response, requiring time to mount protection and unable to treat ongoing infections. Small interfering RNAs (siRNAs) represent a promising class of nucleic acid therapeutics with the potential to overcome these limitations. siRNAs induce gene silencing by directly degrading targeted RNAs, offering rapid action and adaptability to emerging viral strains. These properties make siRNAs attractive for treating viral infections, with positive indications in models for many viruses on NIAID’s priority list, including Dengue Fever, Ebola, Hepatitis, Herpes, Human Papillomavirus, Influenza, Respiratory Syncytial Virus (RSV), Smallpox, West Nile Virus, and Zika Virus. Despite demonstrated efficacy in tissue culture and model systems, clinical translation of siRNAs is hampered by challenges in efficient delivery to target tissues. While the development of GalNAc-siRNA conjugates has led to FDA-approved liver-targeted therapies, scalable methods for screening siRNA delivery strategies in tissues beyond the liver are lacking. This proposal aims to address these limitations by developing high-throughput technologies for generating and screening siRNA-peptide conjugates—a novel approach to improving delivery efficiency and overcoming key bottlenecks of cellular uptake, endosomal escape, and siRNA activation. In Aim 1, we will adapt mRNA display, an established method for creating diverse mRNA-protein libraries, to synthesize compact siRNA-peptide conjugates. By encoding each peptide's amino acid sequence within its conjugated siRNA nucleotide sequence, we will create libraries with millions of variants, enabling high-throughput functional screening. In Aim 2, we will evaluate the functionality of these siRNA-peptide conjugates using biochemistry and small RNA sequencing (sRNA-seq) to identify those that successfully load into Argonaute 2 (Ago2), the enzyme that mediates silencing directed by siRNAs. This method allows for a direct, highly sensitive assessment of delivery efficiency and quantitative identification of the most promising siRNA conjugates for further development. Completion of this project will establish new technologies for high-throughput siRNA conjugate screening, facilitating the discovery of efficient delivery strategies for siRNA-based antiviral therapeutics across diverse biological contexts. This will significantly impact the treatment of viral infections, providing a pathway toward developing siRNA therapies that can rapidly respond to emerging viral threats, treat chronic infections, and protect vulnerable populations such as immunocompromised individuals. This work aligns with NIAID’s mission to combat infectious diseases through innovative therapeutics and the development of broadly applicable antiviral treatments.
