# Efficiency and safety of five different agents for in vivo delivery of novel bioengineered RNAi molecules

## TL;DR

This study compares five delivery agents for RNAi molecules in mice, finding differences in their effectiveness and safety.

## Contribution

The study evaluates a novel bioengineered RNA delivery system in a transgenic mouse model.

## Key findings

- All five agents delivered RNAi molecules to mouse livers, but LNP, Invivofectamine, and nanoparticle formulations were more effective.
- MC3-based LNP caused significant weight loss, organ enlargement, and elevated cytokines, indicating toxicity.
- In vitro delivery efficiency did not predict in vivo performance, emphasizing the need for in vivo testing.

## Abstract

RNA molecules have emerged as an addition to existing entities for therapy and vaccination, whose success may be hindered by inefficient in vivo delivery or induction of excessive toxicities, such as severe cytokine release syndrome. In this study, we used a novel, bioengineered RNA (BioRNA) bearing payload siRNA against green fluorescent protein (GFP) (BioRNA/GFP-siRNA) and a GFP-transgenic mouse model to compare the efficiency and safety of five commercial agents, namely lipid nanoparticles (LNP) and Invivofectamine, as well as Nanoparticle, LIPID-, and PEG-Liposome based In Vivo Transfection Reagents. The results showed that all products provided effective delivery of BioRNA/GFP-siRNA into mouse livers to elicit RNA interference (RNAi) effects. Among them, the LNP, Invivofectamine, and nanoparticle formulations showed relatively greater efficacy, as manifested by higher siRNA accumulation or lower GFP mRNA levels and fluorescence intensity. However, the MC3-based LNP-BioRNA treatment led to an 8% decrease in body weights and obvious hepatosplenomegaly, as well as statistically significant changes in liver and kidney function biomarkers and elevation of multiple pro-inflammatory cytokines, while all other formulations were generally well tolerated. In addition, delivery efficiency of these in vivo transfection agents determined in cells in vitro were not proportional to their performance in mice in vivo. These findings highlight the differences among these RNA delivery systems examined herein and underscore the importance of rigorous evaluation of both efficacy and safety when selecting appropriate platforms for RNA agents.

Scientific infographic showing mice injected with BioRNA/GFP-siRNA formulated with five agents, grouped efficacy and safety data, GFP fluorescence images, and bar and scatter plots for body weight, organ weights, liver and kidney function, and pro-inflammatory cytokines, highlighting greater efficacy and pronounced adverse effects with MC3-based LNP formulations.