Engineering Ionizable Lipid Nanoparticles for Gene Editing in the Liver

Introduction::
Inborn errors of metabolism (IEM) are a group of heterogenous congenital disorders estimated to impact 1 in 2500 births worldwide (1). Loss-of-function mutations in enzymes essential for hepatocyte metabolism play a role in most inborn liver diseases. The management of IEMs has traditionally consisted of diet management, enzyme replacement, and supportive therapy, yet these do not permanently compensate for the primary genetic defects (1). In contrast, CRISPR-mediated editing of the genome enables correction of pathogenic mutations prior to disease onset and offers a “one-and-done” therapeutic avenue for IEMs (2). However, a key challenge for the translation of CRISPR-based gene therapies is safe and effective intracellular delivery. Although ionizable lipid nanoparticles (LNPs) are the most clinically advanced non-viral platforms for mRNA delivery, LNPs have been minimally studied for the co-delivery of the large Cas9 mRNA and disease-specific single guide RNA (sgRNA) necessary to perform gene editing. The objective of this study was to utilize a sequential orthogonal design of experiments (DOE) approach to identify novel LNP formulations that facilitate potent gene editing of hepatocytes.




Materials and Methods::
Computationally-directed orthogonal DOE was used to engineer two sequential libraries of LNPs with varying molar ratios of the four organic excipients: ionizable lipid, phospholipid, cholesterol, and PEG-lipid. LNPs were formulated to encapsulate Cas9 mRNA and an sgRNA to disrupt a reporter GFP gene, characterized for physiochemical properties, and screened in vitro in GFP-expressing HepG2 liver cells. Treated cells were harvested, and the genomic region of interest was sequenced. Impact trend curves for each organic component as a function of LNP-mediated gene editing efficacy were generated and used to inform the design of a second-generation combinatorial library, which was similarly screened in vitro. With an eye toward translation, the top-performing LNP formulation was then reformulated with a therapeutic sgRNA and assessed in vitro for toxicity and gene editing efficacy.




Results, Conclusions, and Discussions::
Our initial screen investigated a design space of 256 distinct LNPs with a representative library (Fig. 1A) and revealed three LNP formulations that mediated a 1.5-fold increase in gene knockout efficiency in hepatocytes compared to the initial formulation (Fig. 1B). To further optimize this formulation, a second library of LNPs was engineered using trends observed in the first library, notably high molar fractions of the ionizable lipid, low molar fractions of PEG-lipid, and moderate molar fractions of the phospholipid and cholesterol (Fig. 1A, 1C). The top-performing LNP in the second library facilitated nearly a 3-fold increase in gene knockout efficiency relative to the initial formulation (Fig. 1B). Reformulation of the lead LNP with a therapeutic sgRNA resulted in greater than 35% knockout at the target locus in hepatocytes with minimal toxicity at a low dose (Fig. 1D).




Acknowledgements (Optional): :

References (Optional): : 1) Jeanmonod, R., Asuka, E., Jeanmonod, D. Inborn Errors of Metabolism. (STATPearls, 2022)
2) Palanki, R., Peranteau, W. Mitchell, M. Delivery technologies for in utero gene therapy. Advanced Drug Delivery Technologies, 169: 51-62 (2021).