Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression and .

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.

Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse.

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.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells.

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy.

The induction of a strong cytotoxic T cell response is an important prerequisite for successful immunotherapy against many viral diseases and tumors. Nucleotide vaccines, including mRNA vaccines with their intracellular antigen synthesis, have been shown to be potent activators of a cytotoxic immune response. The intracellular delivery of mRNA vaccines to the cytosol of antigen presenting immune cells is still not sufficiently well understood.

Poly(glycoamidoamine) brush nanomaterials for systemic siRNA delivery in vivo.

Delivery is the key challenge for siRNA based therapeutics. Here, we report the development of new poly(glycoamidoamine) brush nanomaterials for efficient siRNA delivery. GluN4C10 polymer brush nanoparticles, a lead material, demonstrated significantly improved delivery efficiency for siRNA against factor VII (FVII) in mice compared to poly(glycoamidoamine) brush nanomaterials reported previously.

Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system.

DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA.

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo.

Poly(glycoamidoamine) Brushes Formulated Nanomaterials for Systemic siRNA and mRNA Delivery in Vivo.

Safe and effective delivery is required for siRNA and mRNA-based therapeutics to reach their potential. Here, we report on the development of poly(glycoamidoamine) brush nanoparticles as delivery vehicles for siRNA and mRNA. These polymers were capable of significant delivery of siRNA against FVII and mRNA-encoding erythropoietin (EPO) in mice.

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs.

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency.

Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity.

One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.

Lipid-like nanomaterials for simultaneous gene expression and silencing in vivo.

New lipid-like nanomaterials are developed to simultaneously regulate expression of multiple genes. Self-assembled nanoparticles are capable of efficiently encapsulating pDNA and siRNA. These nanoparticles are shown to induce simultaneous gene expression and silencing both in vitro and in vivo.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates.

siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.

Delivery materials for siRNA therapeutics.

RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges.

In vitro-in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery.

A significant challenge in the development of clinically viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time associated with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examination of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple experimental systems.

Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice.

Rational design of cationic lipids for siRNA delivery.

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.

Lipid-like materials for low-dose, in vivo gene silencing.

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.

Development of lipidoid-siRNA formulations for systemic delivery to the liver.

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates.

Assessing siRNA pharmacodynamics in a luciferase-expressing mouse.

A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics.