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Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models., (© 2022 by The American Society of Hematology.)

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.

Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery, and the PEG-lipids have been engineered to regulate LNP size and transfection potency. The roles of the other "helper" lipids, DSPC and cholesterol, remain less clear. Here we show that in empty LNP systems that do not contain siRNA, DSPC-cholesterol resides in outer layers, whereas in loaded systems a portion of the DSPC-cholesterol is internalised together with siRNA. It is concluded that the presence of internalised helper lipid is vital to the stable encapsulation of siRNA in the LNP and thus to LNP-siRNA function.

Delivering nucleic acid-based therapeutics to cells is an attractive approach to target the genetic cause of various diseases. In contrast to conventional small molecule drugs that target gene products (i.e., proteins), genetic drugs induce therapeutic effects by modulating gene expression. Gene silencing, the process whereby protein production is prevented by neutralizing its mRNA template, is a potent strategy to induce therapeutic effects in a highly precise manner. Importantly, gene silencing has broad potential as theoretically any disease-causing gene can be targeted. It was demonstrated two decades ago that introducing synthetic small interfering RNAs (siRNAs) into the cytoplasm results in specific degradation of complementary mRNA via a process called RNA interference (RNAi). Since then, significant efforts and investments have been made to exploit RNAi therapeutically and advance siRNA drugs to the clinic. Utilizing (unmodified) siRNA as a therapeutic, however, is challenging due to its limited bioavailability following systemic administration. Nuclease activity and renal filtration result in siRNA's rapid clearance from the circulation and its administration induces (innate) immune responses. Furthermore, siRNA's unfavorable physicochemical characteristics largely prevent its diffusion across cellular membranes, impeding its ability to reach the cytoplasm where it can engage the RNAi machinery. The clinical translation of siRNA therapeutics has therefore been dependent on chemical modifications and developing sophisticated delivery platforms to improve their stability, limit immune activation, facilitate internalization, and increase target affinity. These developments have resulted in last year's approval of the first siRNA therapeutic, called Onpattro (patisiran), for treatment of hereditary amyloidogenic transthyretin (TTR) amyloidosis. This disease is characterized by a mutation in the gene encoding TTR, a serum protein that transports retinol in circulation following secretion by the liver. The mutation leads to production of misfolded proteins that deposit as amyloid fibrils in multiple organs, resulting in progressive neurodegeneration. Patisiran's therapeutic effect relies on siRNA-mediated TTR gene silencing, preventing mutant protein production and halting or even reversing disease progression. For efficient therapeutic siRNA delivery to hepatocytes, patisiran is critically dependent on lipid nanoparticle (LNP) technology. In this Account, we provide an overview of key advances that have been crucial for developing LNP delivery technology, and we explain how these developments have contributed to the clinical translation of siRNA therapeutics for parenteral administration. We discuss optimization of the LNP formulation, particularly focusing on the rational design of ionizable cationic lipids and poly(ethylene glycol) lipids. These components have proven to be instrumental for highly efficient siRNA encapsulation, favorable LNP pharmacokinetic parameters, and hepatocyte internalization. Additionally, we pay attention to the development of rapid mixing-based methods that provide robust and scalable LNP production procedures. Finally, we highlight patisiran's clinical translation and LNP delivery technology's potential to enable the development of genetic drugs beyond the current state-of-the-art, such as mRNA and gene editing therapeutics.

Genetic drugs based on RNA or DNA have remarkable therapeutic potential as virtually any disease can be treated by silencing a pathological gene, expressing a beneficial protein, or by editing defective genes. However, therapies based on nucleic acid polymers require sophisticated delivery systems to deliver these macromolecules to the interior of target cells. In this study, we review progress in developing nonviral lipid nanoparticle (LNP) delivery systems that have attractive properties, including ease of manufacture, reduced immune responses, multidosing capabilities, larger payloads, and flexibility of design. LNP systems represent the most advanced delivery systems for genetic drugs as it is expected that an LNP-short interfering RNA (siRNA) formulation will receive clinical approval from the Food and Drug Administration (FDA) in 2018 for treatment of the hereditary condition transthyretin-mediated amyloidosis, a fatal condition for which there is currently no treatment. This achievement is largely due to the development of optimized ionizable cationic lipids, arguably the most important factor in the clinical success of LNP-siRNA. In addition, we highlight potential LNP applications, including targeting tissues beyond the liver and therapeutic approaches based on messenger RNA or Clustered Regularly Interspaced Short Palindromic Repeats/Cas.

Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed with siRNA in aqueous buffer at a pH (pH 4) where the ionizable lipid is positively charged. The resulting dispersion is then dialyzed against a normal saline buffer to remove residual ethanol and raise the pH to 7.4 (above the p K a of the cationic lipid) to produce the finished LNP-siRNA systems. Here we provide cryogenic transmission electron microscopy (cryo-TEM) and X-ray evidence that the complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures with siRNA sandwiched between closely apposed monolayers. Further, it is shown that ionizable lipid not complexed to siRNA promotes formation of very small vesicular structures at pH 4 that coalesce to form larger LNP structures with amorphous electron dense cores at pH 7.4. A mechanism of formation of LNP-siRNA systems is proposed whereby siRNA is first sandwiched between closely apposed lipid monolayers at pH 4 and subsequently trapped in these structures as the pH is raised to 7.4, whereas ionizable lipid not interacting with siRNA moves from bilayer structure to adopt an amorphous oil phase located in the center of the LNP as the pH is raised. This model is discussed in terms of previous hypotheses and potential relevance to the design of LNP-siRNA systems.

Objective: Hyperglucagonemia is present in many forms of diabetes and contributes to hyperglycemia, and glucagon suppression can ameliorate diabetes in mice. Leptin, a glucagon suppressor, can also reverse diabetes in rodents. Lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) effectively targets the liver and is in clinical trials for the treatment of various diseases. We compared the effectiveness of glucagon receptor (Gcgr)-siRNA delivered via LNPs to leptin in two mouse models of diabetes., Methods: Gcgr siRNA encapsulated into LNPs or leptin was administered to mice with diabetes due to injection of the β-cell toxin streptozotocin (STZ) alone or combined with high fat diet (HFD/STZ)., Results: In STZ-diabetic mice, a single injection of Gcgr siRNA lowered blood glucose levels for 3 weeks, improved glucose tolerance, and normalized plasma ketones levels, while leptin therapy normalized blood glucose levels, oral glucose tolerance, and plasma ketones, and suppressed lipid metabolism. In contrast, in HFD/STZ-diabetic mice, Gcgr siRNA lowered blood glucose levels for 2 months, improved oral glucose tolerance, and reduced HbA1c, while leptin had no beneficial effects., Conclusions: While leptin may be more effective than Gcgr siRNA at normalizing both glucose and lipid metabolism in STZ diabetes, Gcgr siRNA is more effective at reducing blood glucose levels in HFD/STZ diabetes., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required. Lipid nanoparticle (LNP) systems are currently the lead non-viral delivery systems for enabling the clinical potential of genetic drugs. Application will be made to the Food and Drug Administration (FDA) in 2017 for approval of an LNP siRNA drug to treat transthyretin-induced amyloidosis, presently an untreatable disease. Here, we first review research leading to the development of LNP siRNA systems capable of silencing target genes in hepatocytes following systemic administration. Subsequently, progress made to extend LNP technology to mRNA and plasmids for protein replacement, vaccine, and gene-editing applications is summarized. Finally, we address current limitations of LNP technology as applied to genetic drugs and ways in which such limitations may be overcome. It is concluded that LNP technology, by virtue of robust and efficient formulation processes, as well as advantages in potency, payload, and design flexibility, will be a dominant non-viral technology to enable the enormous potential of gene therapy., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The therapeutic applications of lipid nanoparticle (LNP) formulations of small interfering RNA (siRNA), are hampered by inefficient delivery of encapsulated siRNA to the cytoplasm following endocytosis. Recent work has shown that up to 70% of endocytosed LNP-siRNA particles are recycled to the extracellular medium and thus cannot contribute to gene silencing. Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal membrane protein required for efficient extracellular recycling of endosomal contents. Here we assess the influence of NP3.47, a putative small molecule inhibitor of NPC1, on the gene silencing potency of LNP-siRNA systems in vitro. Intracellular uptake and colocalization studies revealed that the presence of NP3.47 caused threefold or higher increases in accumulation of LNP-siRNA in late endosomes/lysosomes as compared with controls in a variety of cell lines. The gene silencing potency of LNP siRNA was enhanced up to fourfold in the presence of NP3.47. Mechanisms of action studies are consistent with the proposal that NP3.47 acts to inhibit NPC1. Our findings suggest that the pharmacological inhibition of NPC1 is an attractive strategy to enhance the therapeutic efficacy of LNP-siRNA by trapping LNP-siRNA in late endosomes, thereby increasing opportunities for endosomal escape.

Lipid nanoparticles (LNP) can provide a clinically effective method for delivering small interfering RNA (siRNA) to silence pathological genes in hepatocytes. The gene silencing potency of these LNP-siRNA systems has been shown to depend on a variety of factors including association with serum factors such as ApoE and the pKa of component ionizable lipids. Here we investigate the influence of LNP size, an important parameter affecting tissue penetration of LNP systems, on the pharmacokinetics, biodistribution, and hepatic gene silencing potency of LNP-siRNA systems following intravenous administration. For LNP systems stabilized by a polyethylene glycol (PEG)-lipid that can dissociate from the LNP following injection, it is shown that small (diameter≤30nm) systems are considerably less potent than their larger counterparts. This is attributed in part to the ability of other lipid components, particularly the ionizable amino-lipid, to dissociate from the LNP following dissociation of the PEG-lipid. Small LNP stabilized by PEG-lipids with slow dissociation rates exhibited much reduced amino-lipid dissociation rates, however such systems are relatively impotent due to the continued presence of the PEG coating. These results demonstrate the delicate balance between the in vivo potency of LNP-siRNA systems and the residence times of component lipids in the LNP particle itself and suggest new directions to optimize the in vivo gene silencing potency of small LNP-siRNA systems., (Copyright © 2016. Published by Elsevier B.V.)

Purpose: Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model., Experimental Design: Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models., Results: LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO., Conclusions: LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics., (©2015 American Association for Cancer Research.)

Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4(+) T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4(+) T lymphocytes. Ex vivo, tLNPs demonstrated specificity by targeting only primary CD4(+) T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4(+) T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4(+) T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs' efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases.

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP-siRNA formulations in clinical trials. The aim of this work was to develop LNP-siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.). Three parameters were investigated, namely LNP size, residence time of the polyethylene glycol (PEG)-lipid coating and the influence of hepatocyte-specific targeting ligands. LNP sizes were varied over the range of 30 to 115 nm in diameter and PEG-lipid that dissociates rapidly (PEG-DMG) and slowly (PEG-DSG) were employed. In mice, results show that large (~80 nm) LNP exhibited limited accumulation in the liver and poor Factor VII (FVII) gene silencing at 1mg siRNA/kg body weight. Conversely, small (~30 nm) LNP systems showed maximal liver accumulation yet still had minimal activity. Interestingly, intermediate size (~45 nm) LNP containing PEG-DSG exhibited nearly equivalent liver accumulation as the smaller systems following s.c. administration but reduced FVII levels by 80% at 1mg siRNA/kg body weight. Smaller systems (~35 nm diameter) containing either PEG-DMG or PEG-DSG were less active; however addition of 0.5 mol.% of a GalNAc-PEG lipid to these smaller systems improved activity to levels similar to that observed for the ~45 nm diameter systems. In summary, this work shows that appropriately designed LNP-siRNA systems can result in effective hepatocyte gene silencing following s.c administration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).

Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na(+)/K(+) ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency., From the Clinical Editor: In this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species. In particular, the low potency of LNP containing DLinDAP can be attributed to hydrolysis by endogenous lipases following uptake. LNP containing DLinKC2-DMA, DLinKDMA, or DLinDMA, which lack ester linkages, are not vulnerable to lipase digestion and facilitate much more potent gene silencing. The superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to higher uptake and improved ability to stimulate siRNA release from endosomes subsequent to uptake., From the Clinical Editor: This study reports on the in vivo gene silencing potency of lipid nanoparticle-siRNA systems containing ionizable cationic lipids. It is concluded that the superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to their higher uptake thus improved ability to stimulate siRNA release from endosome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo., (Copyright © 2011 UICC.)

Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005 mg kg(-1) in mice and less than 0.03 mg kg(-1) in non-human primates., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

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.