Sam Chen

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.

Advances in Lipid Nanoparticles for siRNA Delivery

Technological advances in both siRNA (small interfering RNA) and whole genome sequencing have demonstrated great potential in translating genetic information into siRNA-based drugs to halt the synthesis of most disease-causing proteins. Despite its powerful promises as a drug, siRNA requires a sophisticated delivery vehicle because of its rapid degradation in the circulation, inefficient accumulation in target tissues and inability to cross cell membranes to access the cytoplasm where it functions. Lipid nanoparticle (LNP) containing ionizable amino lipids is the leading delivery technology for siRNA, with five products in clinical trials and more in the pipeline. Here, we focus on the technological advances behind these potent systems for siRNA-mediated gene silencing.

Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles.

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.

Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA.

UNLABELLED 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.

Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration

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.

Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA

UNLABELLED 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.

Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA

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.

IGFBP2 Is Neither Sufficient nor Necessary for the Physiological Actions of Leptin on Glucose Homeostasis in Male ob/ob Mice

The ability of leptin to improve metabolic abnormalities in models of leptin deficiency, lipodystrophy, and even type 1 diabetes is of significant interest. However, the mechanism by which leptin mediates these effects remains ill-defined. Leptin was recently reported to regulate insulin-like growth factor-binding protein-2 (IGFBP2), and adenoviral overexpression of pharmacological levels of IGFBP2 ameliorates diabetic symptoms in many models of diabetes. We sought to determine the role of physiological levels of IGFBP2 in the glucoregulatory action of leptin. To investigate whether physiological levels of IGFBP2 are sufficient to mimic the action of leptin, we treated male ob/ob mice with low-dose IGFBP2 adenovirus (Ad-IGFBP2) or low-dose leptin. Despite similar levels of circulating IGFBP2, leptin but not Ad-IGFBP2 lowered body weight and plasma insulin and improved glucose and insulin tolerance. To elucidate the role of IGFBP2 in normal glucose homeostasis, we knocked down IGFBP2 in male C57BL/6 mice using small interfering RNA to determine whether this would recapitulate any aspect of the ob/ob phenotype. Despite successful IGFBP2 knockdown, body weight, blood glucose, and plasma insulin were unchanged. Finally, to determine whether IGFBP2 is required for the glucoregulatory actions of leptin, we prevented leptin-mediated increases in IGFBP2 in male ob/ob mice using RNA interference. Even though increases in IGFBP2 were blocked, the ability of leptin to decrease body weight, blood glucose, and plasma insulin levels were unaltered. In conclusion, physiological levels of IGFBP2 are neither sufficient to mimic nor required for the physiological action of leptin.

Identification and biochemical characterization of the SLC9A7 interactome

Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.

Microfluidic-Based Manufacture of siRNA-Lipid Nanoparticles for Therapeutic Applications

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.