Abstract Details

E2 - Other Nonviral Delivery (including lipid nanoparticles and exosomes)

746: Optimization of Lipid Nanoparticles for Gene Editing of the Liver via Intraduodenal Delivery

Type: Poster Session

Poster Board Number: 746

Presentation Details

Session Title: Wednesday Posters: Other Nonviral Delivery






Introduction: Lipid nanoparticles (LNPs) have emerged as a promising non-viral gene delivery vehicle for diverse therapeutic strategies, including in vivo gene editing. Despite notable progress in gene delivery research, the predominant focus remains on optimizing traditional administration modes, such as intravenous and intramuscular injections. The potential of oral delivery to enhance the quality of life for patients is constrained by challenges associated with physiological barriers. In this study, we explore the feasibility of gene editing in the liver using pDNA LNPs through intraduodenal injection as an indicative route for oral administration. Our optimized LNP-based all-in-one CRISPR-Cas9 pDNA gene-editing tool demonstrated targeted liver delivery, efficient gene editing, therapeutic efficacy, and safety in a hypercholesterolemia-related mouse model following intraduodenal injection, underscoring the potential for an oral gene editing approach in the treatment of familial hypercholesterolemia and other genetic diseases.
Methods: By employing a multi-step screening approach that addresses various physiological barriers and a machine learning-guided framework, we systematically assessed a diverse library of ca. 1,000 pDNA LNP formulations and optimized the type of helper lipid and component ratios for liver-targeted gene delivery via intraduodenal injection. Using the optimized LNP encapsulating a pDNA construct encoding both Strep. pyogenes Cas9 and gRNAs targeting loxP sites, we demonstrated successful non-viral in vivo gene editing of the liver post intraduodenal injection in an Ai9 reporter mouse model. Utilizing an all-in-one SpCas9 pDNA construct containing gRNAs targeting PCSK9 and ANGPTL3, we evaluated the therapeutic efficacy of the optimized pDNA LNP formulation in a hypercholesterolemia-related mouse model.
Results: The top-performing LNPs from the library screening achieved highly preferential liver transfection one week post intraduodenal injection, with over 87% of the bioluminescent flux localized in the liver (Fig. 1a). At a dose of 1 mg/500 µL, CII-β3, a DOPE-based LNP with over 95% liver accumulation one week after intraduodenal administration, achieved the highest transfection efficiency in the liver among all formulations and doses, and demonstrated efficient gene editing capability in the liver in the Ai9 model (Fig. 1b-c). In the hypercholesterolemia-related mouse model, mice with elevated serum cholesterol levels (> 300 ng/µL) experienced a significant reduction in serum cholesterol following treatment with CII-β3 pDNA LNPs, with no significant change in serum ALT (a liver toxicity marker) compared to the untreated group (Fig. 1d). Notably, there was an average of 27% decrease in serum cholesterol (p < 0.01) one week post intraduodenal LNP administration in comparison to the untreated group (Fig. 1e).
Conclusion: Our optimized pDNA LNP formulation, CII-β3, demonstrated efficient gene editing capability in the liver and therapeutic efficacy in lowering serum cholesterol levels post intraduodenal administration. This all-in-one LNP-based pDNA-mediated gene editing strategy via intraduodenal administration not only showcases its potential as an oral therapeutic solution for familial hypercholesterolemia, but also unveils new possibilities for DNA-based medicine.

Plain Language Summary

The success of COVID-19 vaccines has highlighted the potential of lipid nanoparticles (LNPs) in delivering nucleic acid therapeutics to combat diseases. However, most research efforts have been focused on traditional delivery modes, such as intravenous and intramuscular injections, which involve the use of needles. Here we explored the potential of editing genes in the liver using LNPs through an administration route indicative of oral delivery. Our optimized LNP-based gene-editing tool, when administered directly into the duodenum, demonstrated therapeutic success in reducing cholesterol levels in a mouse model of hypercholesterolemia. This suggests that our LNP formulation holds the promise of treating genetic diseases like familial hypercholesterolemia through a simple oral LNP pill. This advancement could make the next generation of gene therapies needle-free, potentially enhancing the quality of life for patients.

Christine Wei¹, Yining Zhu¹, Shuting S. Cai², Jingyao Ma¹, Leonardo Cheng¹, Kam W. Leong², Hai-Quan Mao<sup>1

1</sup>Johns Hopkins University, Baltimore, MD,²Columbia University, New York, NY"