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Chronic hepatitis B virus (HBV) infection remains a global health problem due to the lack of treatments that prevent viral rebound from HBV covalently closed circular (ccc)DNA. In addition, HBV DNA integrates in the human genome, serving as a source of hepatitis B surface antigen (HBsAg) expression, which impairs anti-HBV immune responses. Cytosine base editors (CBEs) enable precise conversion of a cytosine into a thymine within DNA. In this study, CBEs were used to introduce stop codons in HBV genes, HBs and Precore. Transfection with mRNA encoding a CBE and a combination of two guide RNAs led to robust cccDNA editing and sustained reduction of the viral markers in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Furthermore, base editing efficiently reduced HBsAg expression from HBV sequences integrated within the genome of the PLC/PRF/5 and HepG2.2.15 cell lines. Finally, in the HBV minicircle mouse model, using lipid nanoparticulate delivery, we demonstrated antiviral efficacy of the base editing approach with a >3log10 reduction in serum HBV DNA and >2log10 reduction in HBsAg, and 4/5 mice showing HBsAg loss. Combined, these data indicate that base editing can introduce mutations in both cccDNA and integrated HBV DNA, abrogating HBV replication and silencing viral protein expression.

mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types.

Intracellulardelivery of messenger RNA (mRNA) has the potential to induce proteinproduction for many therapeutic applications. Although lipid nanoparticleshave shown considerable promise for the delivery of small interferingRNAs (siRNA), their utility as agents for mRNA delivery has only recentlybeen investigated. The most common siRNA formulations contain fourcomponents: an amine-containing lipid or lipid-like material, phospholipid,cholesterol, and lipid-anchored polyethylene glycol, the relativeratios of which can have profound effects on the formulation potency.Here, we develop a generalized strategy to optimize lipid nanoparticleformulations for mRNA delivery to the liver in vivo using Design ofExperiment (DOE) methodologies including Definitive Screening andFractional Factorial Designs. By simultaneously varying lipid ratiosand structures, we developed an optimized formulation which increasedthe potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles7-fold relative to formulations previously used for siRNA delivery.Key features of this optimized formulation were the incorporationof 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly,the optimized lipid nanoparticle formulation did not improve siRNAdelivery, indicating differences in optimized formulation parameterdesign spaces for siRNA and mRNA. We believe the general method describedhere can accelerate in vivo screening and optimization of nanoparticleformulations with large multidimensional design spaces. [ABSTRACT FROM AUTHOR]