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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., National Science Foundation (U.S.). Graduate Research Fellowship Program, Shire Pharmaceuticals, Wellcome Trust-MIT Postdoctoral Fellowship

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. Importantly, these nanoparticles were well-tolerated at their effective dose based on analysis of tissue histology, systemic cytokine levels, and liver enzyme chemistry. The polymer brush nanoparticles reported here are promising for therapeutic applications.

Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.

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 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

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. Here, we report on the development of a lipid nanoparticle formulation for the delivery of mRNA vaccines to induce a cytotoxic CD 8 T cell response. We show transfection of dendritic cells, macrophages, and neutrophils. The efficacy of the vaccine was tested in an aggressive B16F10 melanoma model. We found a strong CD 8 T cell activation after a single immunization. Treatment of B16F10 melanoma tumors with lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2 resulted in tumor shrinkage and extended the overall survival of the treated mice. The immune response can be further increased by the incorporation of the adjuvant LPS. In conclusion, the lipid nanoparticle formulation presented here is a promising vector for mRNA vaccine delivery, one that is capable of inducing a strong cytotoxic T cell response. Further optimization, including the incorporation of different adjuvants, will likely enhance the potency of the vaccine.

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. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

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 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

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. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

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. To this end, we synthesized 1400 degradable lipidoids and evaluated their transfection ability and structure function activity. Here we show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations upon IV administration to mice (siRNA EC50 values as low as 0.01 mg/kg). Surprisingly, we identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

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. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro experiments was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when, Alnylam Pharmaceuticals (Firm), National Institutes of Health (U.S.) (Fellowship Award F32EB009623)

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 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

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. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

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. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2′-O-methyl (2′-O Me) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.