Poster W20 - Expanding RNAi to lungs, kidneys, and spleen via Selective ORgan Targeting (SORT) siRNA lipid nanoparticles

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Amogh Vaidya

Graduate Student
University of Texas Southwestern Medical Center
Dallas, Texas, United States

Primary Investigator(s)

DS

Daniel Siegwart

Professor
UT Southwestern Medical Center, United States

Last Author(s)

DS

Daniel Siegwart

Professor
UT Southwestern Medical Center, United States

Introduction: Small interfering RNA (siRNA) is a therapeutic modality for achieving persistent genetic knockdown of conventionally undruggable disease-driving proteins that first entered the market in 2018. Crucial to Onpattro®’s clinical translation and for the remarkable success of COVID-19 vaccines were lipid nanoparticles (LNPs); a clinically viable, modular, and scalable platform for deploying genetic drugs. However, inadvertent accumulation of intravenously (IV) administered therapies in the liver posits a significant challenge for treating pathologies arising in other organs. Our lab addressed this roadblock through Selective Organ Targeting (SORT) technology where modulation of lipid chemistry resulted in delivery of mRNA and CRISPR-mediated gene editing machinery to mouse lungs and spleen. Although these LNPs were effective for non-liver delivery of mRNA, a long and flexible molecule, there are no IV-administered extrahepatic drug delivery strategies to carry smaller cargoes like siRNA, which is short, rigid, and rod-like. Building up on the clinical success of five FDA-approved RNA interference (RNAi) based medicines, we are broadening the siRNA therapeutic landscape by targeting presently unreachable tissues such as lungs, spleen, and for the first time, kidneys.

Materials and

Methods: Physicochemical attributes such as LNP size, surface charge, encapsulation efficiency, global apparent pKa, and protein corona, critical to the functionality of siRNA SORT LNPs, were validated and in vitro silencing efficiency of GAPDH and Luciferase genes was quantified. C57BL/6J mice were injected with LNPs containing fluorescently labeled siRNA to examine in vivo tissue biodistribution and organ targeting potential. LNP trafficking to extrahepatic tissues was corroborated by silencing organ-enriched endogenous genes - Tie2 and CD31. To investigate any unwarranted LNP side-effects, we evaluated their unbiased organ targeting capability by silencing TdTomato in an endogenous and constitutively expressing B6.129TdTom/eGFP mouse model. Through flow cytometry, key cell types amenable for transfection were elucidated across the targetable extrahepatic tissues. Finally, to test the physiological relevance of gene silencing facilitated by siRNA SORT LNPs, we tested the biological outcome of suppressing endogenous Tie2 in C57BL/6J mice.

Results, Conclusions, and Discussions: From a formulation engineering standpoint, lipid components and their relative proportions were optimized to obtain efficient loading of siRNA to achieve durable and potent gene knockdown in a tissue-targeted manner. All tested formulations outperformed off-the-shelf Lipofectamine 2000 and demonstrated 75% and 98% knockdown of GAPDH and Luciferase activity respectively in vitro. The in vivo biodistribution profile revealed that choice of SORT lipid altered the fate of LNPs and consequently established their feasibility to accomplish extrahepatic siRNA delivery. siRNA SORT LNPs were well tolerated in mice and enabled robust silencing of tissue-enriched endogenous gene targets. LNPs targeting the lungs and kidneys showed 88% and 61% reduction in Tie2 protein levels respectively, while 70% of CD31 was downregulated in the spleen. Ability to induce high knockdown levels can be leveraged to silence aberrant disease gene targets in extrahepatic tissues. Next, B6.129TdTom/eGFP mouse model served as an unbiased, endogenous, and ubiquitous target for testing TdTomato knockdown as these mice constitutively express membrane-tagged TdTomato in all cell/tissue types. A precipitous drop in TdTomato intensity (58% for lungs, 15% for kidneys, and 45% for spleen) showcased a potent and robust extrahepatic silencing profile for our platform. Therapeutically relevant cell types across the endothelial, epithelial, and immune populations of target organs were identified by flow cytometry. Obtaining a cell-level resolution of knockdown was paramount as silencing an impaired gene expressed in a cell population inaccessible by LNPs would be clinically futile and inconsequential. Ultimately, we demonstrated biological utility of siRNA SORT LNPs by silencing Tie2 to induce a phenotypic change specifically in mouse lungs. Acute Tie2 suppression facilitated by lung targeting LNPs resulted in a weakened and leaky pulmonary vascular barrier. We foresee retooling of such an endogenous gene silencing strategy for therapeutic benefit.

Collectively, we present a rational, predictable, and durable LNP strategy for delivering functionally active siRNA to hitherto inaccessible organs such as lungs, spleen, and kidneys via intravenous administration. This advancement holds immense potential in extrapolating RNAi platform beyond hepatic tissues to treat genetic disorders, fibrotic diseases, cancer, and potentially additional maladies.

Acknowledgements (Optional):