Poster V5 - Transcriptomics-Driven De Novo Design of Lipid Nanoparticle for RNA Delivery to Disease-Associated Cellular Subtypes

Introduction: Identifying precise transcripts governing pathological cells and fundamental disease induction process is of paramount significance in developing targeted therapeutics. Here, we present a transcriptomics-driven de novo design of lipid nanoparticle (LNP)-mediated therapeutic (TRANSMIT) targeting strategy, utilizing next-generation transcriptomics to discern representative pathological feature and identify pathogenic-associated genes and cell subtypes to motivate precise LNP-mediated RNA delivery for disease treatment. As a proof-of-concept, we use a preclinical hepatic fibrosis model, verifying elevated levels of ROS and identifying a novel fibrogenic macrophage subtype (CD63+ MΦ), to de novo design antibody-conjugated ROS-scavenging (RS) LNP for precise delivery of small interfering RNA (siRNA) to alleviate liver fibrosis.

Materials and

Methods: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) were used to identify key pathological transcripts and characterize the phenotype of the fibrogenic MΦ in a murine liver fibrosis model. A library of 90 novel RS ionizable lipids were designed, formulated into RS LNP, and screened in vitro. Anti-F4/80 conjugated top-performed LNP delivering therapeutic siRNA to murine liver fibrosis model were used to mitigate liver fibrosis.

Results, Conclusions, and Discussions: The identified fibrogenic MΦ, enriched in the livers of fibrotic mice, represent a subtype of CD63+ MΦ characterized by high expression of Fabp5, Gpnmb, and Spp1. Through high-throughput screening in vitro, we identified 6-S-9b2 LNP as the top-performer, which reduced ROS levels both in vitro and in vivo. In a preclinical mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, F4/80-conjugated 6-S-9b2 LNP demonstrated significantly higher uptake by the identified fibrogenic MΦ. By delivering therapeutic siFabp5, targeted 6-S-9b2 LNPs achieved potent knockdown of fibrosis-associated features to mitigate liver fibrosis, outperforming the benchmark MC3 LNP.
TRANSMIT targeting strategy presents a highly promising and comprehensive approach for de novo design LNP system for the treatment of liver fibrosis. These findings underscore the potential of TRANSMIT targeting strategy for de novo designing of LNP aiming at specific diseases and advancing the field of targeted gene therapeutics in liver fibrosis and potentially other diseases across various organs and species.

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