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Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.

Encapsulation of small molecule drugs in long-circulating lipid nanoparticles (LNPs) can reduce toxic side effects and enhance accumulation at tumor sites. A fundamental problem, however, is the slow release of encapsulated drugs from these liposomal systems at the disease site resulting in limited therapeutic benefit. Methods to trigger release at specific sites are highly warranted. Here, it is demonstrated that incorporation of ultraviolet (UV-A) or red-light photoswitchable-phosphatidylcholine analogs (AzoPC and redAzoPC) in conventional LNPs generates photoactivatable LNPs (paLNPs) having comparable structural integrity, drug loading capacity, and size distribution to the parent DSPC-cholesterol liposomes. It is shown that 65-70% drug release (doxorubicin) can be induced from these systems by irradiation with pulsed light based on trans-to-cis azobenzene isomerization. In vitro it is confirmed that paLNPs are non-toxic in the dark but convey cytotoxicity upon irradiation in a human cancer cell line. In vivo studies in zebrafish embryos demonstrate prolonged blood circulation and extravasation of paLNPs comparable to clinically approved formulations, with enhanced drug release following irradiation with pulsed light. Conclusively, paLNPs closely mimic the properties of clinically approved LNPs with the added benefit of light-induced drug release making them promising candidates for clinical development., (© 2021 Wiley-VCH GmbH.)

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

Lipid nanoparticles (LNPs) containing short-interfering RNA (LNP-siRNA systems) are a promising approach for silencing disease-causing genes in hepatocytes following intravenous administration. LNP-siRNA systems are generated by rapid mixing of lipids in ethanol with siRNA in aqueous buffer (pH 4.0) where the ionizable lipid is positively charged, followed by dialysis to remove ethanol and to raise the pH to 7.4. Ionizable cationic lipids are the critical excipient in LNP systems as they drive entrapment and intracellular delivery. A recent study on the formation of LNP-siRNA systems suggested that ionizable cationic lipids segregate from other lipid components upon charge neutralization to form an amorphous oil droplet in the core of LNPs. This leads to a decrease in intervesicle electrostatic repulsion, thereby engendering fusion of small vesicles to form final LNPs of increased size. In this study, we prepared LNP-siRNA systems containing four lipid components (hydrogenated soy phosphatidylcholine, cholesterol, PEG-lipid, and 1,2-dioleoyl-3-dimethylammonium propane) by microfluidic mixing. The effects of preparation parameters [lipid concentration, flow rate ratio (FRR), and total flow rate], dialysis process, and complex formation between siRNA and ionizable cationic lipids on the physicochemical properties [siRNA entrapment on the particle size and polydispersity index (PDI)] were investigated using a design of experiments approach. The results for the preparation parameters showed no impact on siRNA encapsulation, but lipid concentration and FRR significantly affected the particle size and PDI. In addition, the effect of FRR on the particle size was suppressed in the presence of anionic polymers such as siRNA as compared to the case of LNPs alone. More intriguingly, unlike empty LNPs, a decrease in the PDI and an increase in the particle size occurred after dialysis in the LNP-siRNA systems. Such changes by dialysis were suppressed at FRR = 1. These findings provide useful information to guide the development and manufacturing conditions for LNP-siRNA systems.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity., (© 2020 by The American Society of Hematology.)

Gene therapy holds great potential for treating almost any disease by gene silencing, protein expression, or gene correction. To efficiently deliver the nucleic acid payload to its target tissue, the genetic material needs to be combined with a delivery platform. Lipid nanoparticles (LNPs) have proven to be excellent delivery vectors for gene therapy and are increasingly entering into routine clinical practice. Over the past two decades, the optimization of LNP formulations for nucleic acid delivery has led to a well-established body of knowledge culminating in the first-ever RNA interference therapeutic using LNP technology, i.e., Onpattro, and many more in clinical development to deliver various nucleic acid payloads. Screening a lipid library in vivo for optimal gene silencing potency in hepatocytes resulted in the identification of the Onpattro formulation. Subsequent studies discovered that the key to Onpattro's liver tropism is its ability to form a specific "biomolecular corona". In fact, apolipoprotein E (ApoE), among other proteins, adsorbed to the LNP surface enables specific hepatocyte targeting. This proof-of-principle example demonstrates the use of the biomolecular corona for targeting specific receptors and cells, thereby opening up the road to rationally designing LNPs. To date, however, only a few studies have explored in detail the corona of LNPs, and how to efficiently modulate the corona remains poorly understood. In this review, we summarize recent discoveries about the biomolecular corona, expanding the knowledge gained with other nanoparticles to LNPs for nucleic acid delivery. In particular, we address how particle stability, biodistribution, and targeting of LNPs can be influenced by the biological environment. Onpattro is used as a case study to describe both the successful development of an LNP formulation for gene therapy and the key influence of the biological environment. Moreover, we outline the techniques available to isolate and analyze the corona of LNPs, and we highlight their advantages and drawbacks. Finally, we discuss possible implications of the biomolecular corona for LNP delivery and we examine the potential of exploiting the corona as a targeting strategy beyond the liver to develop next-generation gene therapies.

Rationale: Monocytes are key effectors of the mononuclear phagocyte system, playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into 2 major subsets termed conventional monocytes and patrolling monocytes (pMo) but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology; however, LYN's role in regulating specific monocyte subset homeostasis has not been investigated., Objective: We sought to comprehensively profile monocytes to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis., Methods and Results: Mass cytometric analysis of monocyte subsets and signaling pathway activation patterns in conventional monocytes and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN's role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn -deficient pMos. Furthermore, single-cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn -/- monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr -/- mice from high-fat diet-induced atherosclerosis., Conclusions: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a naturally derived flavonoid that is commonly found in fruits and vegetables. There is mounting evidence to suggest that quercetin has potential anticancer effects and appears to interact synergistically when used in combination with approved chemotherapeutic agents such as irinotecan and cisplatin. Unfortunately, quercetin has shown limited clinical utility, partly due to low bioavailability related to its poor aqueous solutions (< 10 μg/mL). In this study, liposomal formulations of quercetin were developed by exploiting quercetin's ability to bind copper. Quercetin powder was added directly to pre-formed copper-containing liposomes (2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 M ratio)). As a function of time and temperature, the formation of copper-quercetin was measured. Using this methodology, a final quercetin-to-lipid (mol:mol) ratio of 0.2 was achievable and solutions containing quercetin at concentrations of > 5 mg/mL were attained, representing at least a > 100-fold increase in apparent solubility. The resulting formulation was suitable for intravenous dosing with no overt toxicities when administered at doses of 50 mg/kg in mice. Pharmacokinetic studies demonstrated that the copper-quercetin formulations had an AUC 0-24H of 8382.1 μg h/mL when administered to mice at 50 mg/kg. These studies suggested that quercetin (not copper-quercetin) dissociates from the liposomes after administration. The resulting formulation is suitable for further development and also serves as a proof-of-concept for formulating other flavonoids and flavonoid-like compounds. Given that quercetin exhibits an IC 50 of >10 μM when tested against cancer cell lines, we believe that the utility of this novel quercetin formulation for cancer indications will ultimately be as a component of a combination product.

Objective: Genetic alterations have been identified in the CACNA1H gene, encoding the Ca V 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of Ca V 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN., Methods: Pathophysiological contributions of Ca V 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings., Results: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a Ca V 3.2 mutation-sensitive splice variant. In vivo knock-down of Ca V 3.2 using direct thalamic injection of lipid nanoparticles containing Ca V 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures., Significance: This supports a role for TRN Ca V 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

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.)

Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sclerostin is a protein secreted by osteocytes that is encoded by the SOST gene; it decreases bone formation by reducing osteoblast differentiation through inhibition of the Wnt signaling pathway. Silencing the SOST gene using RNA interference (RNAi) could therefore be an effective way to treat osteoporosis. Here, we investigate the utility of lipid nanoparticle (LNP) formulations of siRNA to silence the SOST gene in vitro and in vivo. It is shown that primary mouse embryonic fibroblasts (MEF) provide a useful model system in which the SOST gene can be induced by incubation in osteogenic media, allowing development of optimized SOST siRNA for silencing the SOST gene. Incubation of MEF cells with LNP containing optimized SOST siRNA produced significant, prolonged knockdown of the induced SOST gene in vitro, which was associated with an increase in osteogenic markers. Intravenous (i.v.) administration of LNP containing SOST siRNA to mice showed significant accumulation of LNP in osteocytes in compact bone, depletion of SOST mRNA and subsequent reduction of circulating sclerostin protein, establishing the potential utility for LNP siRNA systems to promote bone formation.

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen-targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer., (Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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.

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.

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.Molecular Therapy-Nucleic Acids (2013) 2, e136; doi:10.1038/mtna.2013.65; published online 3 December 2013.

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.

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.)

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.

The use of targeting ligands to enhance the delivery of liposomal nanoparticles (LNs) has moved slowly toward clinical application. This relative lack of clinical progression is further complicated by the existence of conflicting in vivo results in the literature. In this work, we describe new formulations of LNs that are targeted with an arginine-glycine-aspartic acid-containing peptide, cRGDfK, conjugated to the lipid distearoyl phosphatidylethanolamine (DSPE). These formulations may be able to circumvent some of the challenges encountered during the development of targeted-LNs. Of the constructs studied, a fluorescently labeled peptide-lipid conjugate was incorporated into LNs with high yield and accuracy. It is shown that the resulting targeted-LNs bind to human umbilical vein endothelial cells (HUVECs) with increasing avidity as the amount of peptide displayed on the LN surface increases. We specifically demonstrate the ability of targeted-LNs loaded with doxorubicin and incubated with HUVECs to deliver the drug to the cytosol. The cell does not internalize nontargeted LNs, supporting the notion that the RGD motif is associated with internalization of the targeted LN.

The specific binding characteristics of alphavbeta3 integrins with an arginine-glycine-aspartic-acid (RGD) containing fluorescently labeled cyclic peptide is investigated with capillary electrophoresis-frontal analysis method. The new algorithm used to calculate the binding constants and binding stoichiometry was derived without the assumptions made in the commonly used Scatchard Plot method, thus enabling the determination of specific binding parameters in the presence of nonspecific binding. The alphavbeta3 integrin, a membrane protein, was studied in solution, without the need of immobilization or any other kind of modification. An RGD containing fluorescently labeled cyclic pentapeptide is used as the ligand with both specific and nonspecific binding characteristics, and an arginine-alanine-aspartic-acid (RAD) containing peptide is used as the control for nonspecific binding. While a typical specific binding isotherm has a shape of a rectangular hyperbola, a nonspecific binding isotherm is linear in the same ligand concentration region. A 1:2 specific binding stoichiometry was revealed with the second binding having a similar affinity compared to the first binding event.

The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of tumor transfection properties following systemic administration. For example, the PEG coating of SPLP is necessary to engender the long circulation lifetimes required to achieve tumor delivery. However, PEG coatings have also been shown to inhibit cell association and uptake required for transfection. The dissociation rate of the PEG coating from SPLP can be modulated by varying the acyl chain length of the ceramide anchor, suggesting the possibility of developing PEG-Cer molecules that remain associated with SPLP long enough to promote tumor delivery, but which dissociate quickly enough to allow transfection. Alternatively, improvements may be expected from inclusion of cell-specific targeting ligands in SPLP to promote cell association and uptake. Finally, the nontoxic properties of SPLP allow the possibility of higher doses. A dose of 100 micrograms plasmid DNA per mouse corresponds to a dose of approximately 5 mg plasmid DNA per kg body weight. This compares well to small molecules used for cancer therapy, which typically are used at dose levels of 10 to 50 mg per kg body weight. In summary, SPLP consist of plasmid encapsulated in a lipid vesicle that, in contrast to naked plasmid or complexes, exhibit extended circulation lifetimes following intravenous injection, resulting in accumulation and transgene expression at a distal tumor site in a murine model. The pharmacokinetics, biodistribution, and tumor transfection properties of SPLP are highly sensitive to the nature of the ceramide anchor employed to attach the PEG to the SPLP surface. The SPLP-CerC20 system in which the PEG-Cer does not readily dissociate exhibits good serum stability, long circulation lifetimes, and high levels of tumor accumulation and mediates marker gene expression at the tumor site. The flexibility of the SPLP system offers the potential of further optimization to achieve therapeutically effective levels of gene transfer and clearly has considerable potential as a nontoxic systemic gene therapy vehicle with general applicability. These features of SPLP contrast favorably with previous plasmid encapsulation procedures. Plasmid DNA has been encapsulated by a variety of methods, including reverse phase evaporation, ether injection, detergent dialysis in the absence of PEG stabilization, lipid hydration and dehydration-rehydration techniques, and sonication, among others. The characteristics of these protocols are summarized in Table I. None of these procedures yields small, serum-stable particles at high plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmid-encapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique that can shear nucleic acids. Size ranges of 100 mm diameter or less can be achieved by reverse-phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure.