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Nucleic acid therapeutics represent a major advance toward treating diseases at their root cause. However, nucleic acids are prone to degradation by serum endonucleases, clearance through the immune system, and rapid degradation in complex medium. To overcome these barriers, nucleic acids frequently include chemical modifications to improve stability or decrease immune responses. Lipid nanoparticles (LNPs) have enabled a dramatic reduction in the dose required to achieve a therapeutic effect by protecting these nucleic acids and improving their intracellular delivery. It has been assumed thus far that nonspecific ionic interactions drive LNP formation and chemical modifications to the nucleic acid backbone to confer improved stability do not impact LNP delivery in any way. Here, we demonstrate that these chemical modifications do impact LNP morphology substantially, and phosphorothioate modifications produce stronger interactions with ionizable amino lipids, resulting in enhanced entrapment. This work represents a major first step toward greater understanding of the interaction between the lipid components and nucleic acids within an LNP.

Hepatitis B virus (HBV) can rapidly replicate in the hepatocytes after transmission, leading to chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma. Interferon-α (IFN-α) is included in the standard treatment for chronic hepatitis B (CHB). However, this therapy causes serious side effects. Delivering IFN-α selectively to the liver may enhance its efficacy and safety. Imiquimod (IMQ), a Toll-Like Receptor (TLR) 7 agonist, stimulates the release of IFN-α that exhibits potent antiviral activity. However, the poor solubility and tissue selectivity of IMQ limits its clinical use. Here, we demonstrated the use of lipid-based nanoparticles (LNPs) to deliver IMQ and increase the production of IFN-α in the liver. We encapsulated IMQ in two liver-targeted LNP formulations: phospholipid-free small unilamellar vesicles (PFSUVs) and DSPG-liposomes targeting the hepatocytes and the Kupffer cells, respectively. In vitro drug release/retention, in vivo pharmacokinetics, intrahepatic distribution, IFN-α production, and suppression of serum HBV surface antigen (HBsAg) were evaluated and compared for these two formulations. PFSUVs provided95% encapsulation efficiency for IMQ at a drug-to-lipid ratio (D/L) of 1/20 (w/w) and displayed stable drug retention in the presence of serum. DSPG-IMQ showed 79% encapsulation of IMQ at 1/20 (D/L) and exhibited ∼30% burst release when incubated with serum. Within the liver, PFSUVs showed high selectivity for the hepatocytes while DSPG-liposomes targeted the Kupffer cells. Finally, in an experimental HBV mouse model, PFSUVs significantly reduced serum levels of HBsAg by 12-, 6.3- and 2.2-fold compared to the control, IFN-α, and DSPG-IMQ groups, respectively. The results suggest that the hepatocyte-targeted PFSUVs loaded with IMQ exhibit significant potential for enhancing therapy of CHB.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest. Here we examine the ability of small interfering RNA (siRNA) to enhance osteoblast differentiation and bone formation by silencing the negative suppressor gene GNAS in bone MSCs. Using clinically validated lipid nanoparticle (LNP) siRNA delivery systems, we show that silencing the suppressor gene GNAS in vitro in MSCs leads to molecular and phenotypic changes similar to those seen in osteoblasts. Further, we demonstrate that these LNP-siRNAs can transfect a large proportion of mice MSCs in the compact bone following intravenous injection. Transfection of MSCs in various animal models led to silencing of GNAS and enhanced differentiation of MSCs into osteoblasts. These data demonstrate the potential for LNP delivery of siRNA to enhance the differentiation of MSCs into osteoblasts, and suggests that they are a promising approach for the treatment of osteoporosis and other bone diseases.

Supplementary Figure Legends S1-4. Supplementary Materials and Methods: Antibodies used for western blotting.

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. Clin Cancer Res; 21(21); 4845–55. ©2015 AACR.

Supplemental Table 1: Source and authentication of cell lines. Supplemental Table 2: Primers used for quantitative real-time PCR. S1: PC-3M-luc cell numbers correlated with the expression of LUC in vitro. S2: PC-3M-luc tumor volume correlated with the expression of LUC in vivo. S3: DMAP-BLP LNP LUC-siRNA decreases LUC expression in a dose dependent manner in PC-3M-luc xenograft model in vivo. S4: LNP CLU-siRNA enhances AR-ASO-induced inhibition of LNCaP cell growth.

Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-β1 (TGF-β1). To determine the role of FAM13A in TGF-β1 signaling, FAM13A−/− airway epithelial cells were generated using CRISPR-Cas9, whereas overexpression of FAM13A was achieved using lipid nanoparticles. Wild-type (WT) and FAM13A−/− cells were treated with TGF-β1, followed by gene and/or protein expression analyses. FAM13A−/− cells augmented TGF-β1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in β-catenin (CTNNB1) expression in FAM13A−/− cells compared with WT cells after TGF-β1 treatment. FAM13A overexpression was partially protective from TGF-β1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared with control nonsmokers, and negatively correlated with lung function. In contrast, β-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared with non-COPD subjects. Together, our data showed that FAM13A may be protective from TGF-β1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.

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.

Hybrid lipid nanoparticles containing gold nanoparticles (LNP-GNPs) and drugs have potential for imaging applications as well as triggered release of LNP contents in response to pulsed laser or X-ray radiation mediated by the GNPs. However, methods to synthesize LNP-GNP systems that efficiently entrap GNPs (the potential triggered release and imaging agent) and then load and retain the drug cargo in a manner that may have clinical applications have proven elusive. Here, we develop a straightforward "bottom-up" approach to manufacture drug-loaded LNP-GNP systems. We show that negatively charged GNPs of 5 nm diameter can be stably loaded into LNPs containing 10 mol % ionizable cationic lipid using an ethanol dilution, rapid mixing approach and that these systems also exhibit aqueous compartments. Further, we show that such systems can also entrap ammonium sulfate, enabling pH-dependent loading of the weak base anti-cancer drug doxorubicin into the aqueous compartments. Cryo-transmission electron microscopy (Cryo-TEM) imaging clearly demonstrates the presence of GNPs in the interior of the resulting hybrid nanostructures as well as the formation of electron-dense drug precipitates in the aqueous core of the LNP-GNPs. The approach described here is a robust and straightforward method to generate hybrid LNP-GNP-drug and other LNP-metal nanoparticle-drug systems with potential applications for a variety of triggered release protocols.

Many medicines are only available in solid dosage forms suitable for adults, and extemporaneous compounding is required to prepare formulations for children. However, this common practice often results in inaccurate dosing and unpleasant taste, reducing the medication adherence. Here, we report the development of a new method to prepare and compound child-friendly oral formulations based on a liposomal multilamellar vesicle (MLV) platform. MLVs composed of a phospholipid (DSPC) and cholesterol (55/45, molar ratio) were prepared using the standard thin film hydration method with 300 mM citric acid (pH 2), followed by an addition of aqueous sodium carbonate to adjust the exterior pH to 8-10 for creating a transmembrane pH gradient. Weak-base drugs, such as chloroquine (CQ) and hydroxychloroquine (HCQ), could be actively and completely loaded into the MLVs at a drug-to-lipid ratio of 15-20 wt%. This technique formulated weak-base drugs from the powder or tablet form into a liquid preparation, and the complete drug encapsulation would prevent contact between the drug molecules and the taste buds. The gradient MLV formulation could be preserved by lyophilization and stored at room temperature for at least 8 weeks. Upon reconstitution with water, the MLV formulation could completely encapsulate CQ at 20 wt%, which was comparable to the freshly prepared MLVs. The CQ-loaded MLV formulation could be stored at 4 °C for 2 weeks without drug leakage. In vitro release studies indicated that MLV could retain CQ in the simulated saliva, but released up to 50% and 30% of the drug in the simulated gastric and intestinal fluids, respectively. The orally delivered MLV-CQ formulation displayed higher CQ absorption in mice, with a 2-fold increase in the area under the curve (AUC) of the plasma profile compared to CQ solution. Our data suggest that the new MLV method could serve as a platform to prepare child-friendly oral formulation for weak-base drugs.

In this brief perspective, we describe key events in the history of the lipid-based nanomedicine field, highlight Canadian contributions, and outline areas where lipid nanoparticle technology is poised to have a transformative effect on the future of medicine.

Type 2 diabetes is routinely identified in clinical practice by tests that rely on a hyperglycemic index. However, people at risk for developing type 2 diabetes may not present with hyperglycemia. We identified several underlying risks for type 2 diabetes, insulin resistance, and associated co-morbidities, using a liquid chromatography mass spectrometry–based analysis of blood metabolites, in participants with normoglycemia and no clinical symptoms. Personalized lifestyle recommendations, including diet, exercise, and nutritional supplement recommendations, were conveyed to these participants by a web-based platform, and after 100 days of following their recommendations, these participants reported reductions in the health risks associated with type 2 diabetes and associated diseases. Our comprehensive metabolite-based assay can be used for type 2 diabetes risk stratification, and our personalized lifestyle recommendation system could be deployed as a preventative treatment option to improve health outcomes, reduce the incidence of chronic disease, and live healthier lives in an evidence-based way.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2′O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

Contains fulltext : 282460.pdf (Publisher’s version ) (Open Access) Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

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.

Hemolytic toxicity caused by primaquine (PQ) is a high-risk condition that hampers the wide use of PQ to treat liver-stage malaria. This study demonstrated that phospholipid-free small unilamellar vesicles (PFSUVs) composed of Tween80 and cholesterol could encapsulate and deliver PQ to the hepatocytes with reduced exposure to the red blood cells (RBCs). Nonionic surfactant (Tween80) and cholesterol-forming SUVs with a mean diameter of 50 nm were fabricated for delivering PQ. Drug release/retention, drug uptake by RBCs, pharmacokinetics, and liver uptake of PFSUVs-PQ were evaluated in in vitro and in vivo models in comparison to free drugs. Additionally, the stress effect on RBCs induced by free PQ and PFSUVs-PQ was evaluated by examining RBC morphology. PFSUVs provided >95% encapsulation efficiency for PQ at a drug-to-lipid ratio of 1:20 (w/w) and stably retained the drug in the presence of serum. When incubated with RBCs, PQ uptake in the PFSUVs group was reduced by 4- to 8-folds compared to free PQ. As a result, free PQ induced significant RBC morphology changes, while PFSUVs-PQ showed no such adverse effect. Intravenously (i.v.) delivered PFSUVs-PQ produced a comparable plasma profile as free PQ, given i.v. and orally, while the liver uptake was increased by 4.8 and 1.6-folds, respectively, in mice. Within the liver, PFSUVs selectively targeted the hepatocytes, with no significant blood or liver toxicity in mice. PFSUVs effectively targeted PQ to the liver and reduced RBC uptake compared to free PQ, leading to reduced RBC toxicity. PFSUVs exhibited potential in improving the efficacy of PQ for treating liver-stage malaria.

Successfully employing therapeutic nucleic acids, such as small interfering RNA (siRNA), requires chemical modifications or the use of nanocarrier technology to prevent their degradation in the circulation and to facilitate intracellular delivery. Lipid nanoparticles (LNP) are among the most advanced nanocarriers culminating in the first siRNA therapeutic’s clinical translation and approval. At the same time, their applicability as modular platform technology due to the interchangeable building blocks and siRNA payload hallmarks one of LNPs’ major advantages. In addition, drug derivatization approaches to synthesize lipophilic small molecule prodrugs enable stable incorporation in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, we describe how the modular LNP platform can be applied for combined gene silencing and chemotherapy to induce additive anti-cancer effects. We show 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. In addition, we demonstrate that prodrug incorporation does not affect LNPs’ gene silencing ability and that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, we quantitively determined the LNPs’ and prodrugs’ pharmacokinetic properties and biodistribution following systemic administration in tumor-bearing mice. Our results indicate that co-encapsulation of siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward approach for combination therapy development.GRAPHICAL ABSTRACT

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.

Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs s holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene (editing) therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation., Graphical abstract Unlabelled Image

The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clinical development of many nucleic acid-based therapies enabled by nanoparticle delivery.

ConspectusDelivering 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.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

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.

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.

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.

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.

Liposomes containing ionizable cationic lipids have been widely used for the delivery of nucleic acids such as small-interfering RNA and mRNA. The utility of cationic lipids with a permanent positive charge, however, is limited to in vitro transfection of cultured cells due to its dose-limiting toxic side effects observed in animals. Several reports have suggested that the permanently charged cationic lipids induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. We therefore hypothesized that the concomitant use of ROS inhibitor could reduce toxicity and improve drug efficacy. In this study, suppression of the cationic toxicity was evaluated using an ROS scavenger, edaravone, which is a low-molecular-weight antioxidant drug clinically approved for acute-phase cerebral infarction and amyotrophic lateral sclerosis. Cell viability assay in the mouse macrophage-like cell line RAW264 indicated that the concomitant use of edaravone were not able to suppress the cytotoxicity induced by cationic liposomes comprised of monovalent cationic lipid N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA) over a short period of time. Cationic lipids-induced necrosis was assumed to be involved in the cytotoxicity upon short-term exposure to cationic liposomes. On the other hand, the significant improvement of cell viability was observed when the short treatment with cationic liposomes was followed by exposure to edaravone for 24 h. It was also confirmed that apoptosis inhibition by ROS elimination might have contributed to this effect. These results suggest the utility of continuous administration with edaravone as concomitant drug for suppression of adverse reactions in therapeutic treatment using cationic liposomes.

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.

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.

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., Highlights • Gcgr siRNA improves glucose metabolism but not lipid metabolism in STZ diabetic mice. • Leptin improves both glucose and lipid metabolism in STZ diabetic mice. • Gcgr siRNA improves glucose metabolism in HFD/STZ diabetic mice. • Leptin does not improve glucose metabolism in HFD/STZ diabetic mice.

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.

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.

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 ( 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 AUC0-24H 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 IC50 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.

Curcumin exhibits potent anticancer activity via various mechanisms, but its in vivo efficacy has been hampered by poor solubility. Nanotechnology has been employed to deliver curcumin, but most of the reported systems suffered from low drug loading capacity and poor stability. Here, we report the development and optimization of a liposomal formulation for curcumin (Lipo-Cur) using an automated microfluidic technology. Lipo-Cur exhibited a mean diameter of 120 nm with a low polydispersity index (

Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.

Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.

Background Radiotherapy is commonly used for treating cancer. Novel sensitizers, such as gold nanoparticles (GNPs), are being used to enhance the local radiation dose. It is not known how the uptake and radiation dose enhancement of GNPs vary in synchronized vs unsynchronized (control) tumor cell populations. Successful application of GNPs in radiation therapy requires NPs to be accumulated within individual tumor cells at clinically feasible NP concentrations. Use of small GNPs as a radiation dose enhancer in the past required very high NP concentration, since the driving force for the uptake of smaller GNPs is low. We used a novel lipid-based NP of 50 nm diameter system as a Trojan horse to deliver smaller GNPs of size 5 nm (LNP–GNP) at 0.2 nM concentration. We investigated the changes in GNP uptake and survival fraction with the LNP delivery at different cell stages using human breast cancer as our tumor model and choosing the triple-negative MDA-MB-231 cell line. Results Using the LNP–GNP system resulted in a 39- and 73-fold enhancement in uptake of 5 nm GNPs in unsynchronized and synchronized tumor cell populations, respectively. The NP uptake per cell increased from 800 to 1200 and from 30,841 to 88,477 for individual 5 nm GNPs and 5 nm GNPs incorporated in LNPs, respectively. After a radiation dose of 2 Gy with 6 MeV photons, synchronized tumor cell populations incorporated with LNP–GNPs produced a 27% enhancement in tumor cell death compared to the control (unsynchronized; no GNPs; 2 Gy). The findings of our experimental results were supported by modeling predictions based on Monte Carlo calculations. Conclusions This study clearly shows that the cell cycle, GNPs, and radiation therapy can be combined to improve outcome of cancer therapy. Using the experimental data, we estimated the predicted improvement for a clinical treatment plan where 30 fractions of 2 Gy radiation dose were given over a period of time. Enhanced uptake and radiation sensitivity of a synchronous tumor cell population would produce a significant improvement in cell killing. For example, synchronizing cells and the addition of LNP–GNPs into tumor cells produced a 1000-fold enhancement in cell killing. Because the agents used for cell synchronization are in clinical practice, this approach may be a simple and cost-effective way to further enhance local radiation dose. Finally, this study provides a novel lipid-based NP platform to further improve GNP-mediated radiation therapy through synchronization of breast cancer cell population.

Gene therapy is a promising strategy for the treatment of monogenic disorders. Non-viral gene delivery systems including lipid-based DNA therapeutics offer the opportunity to deliver an encoding gene sequence specifically to the target tissue and thus enable the expression of therapeutic proteins in diseased cells. Currently, available gene delivery approaches based on DNA are inefficient and require improvements to achieve clinical utility. In this Review, we discuss state-of-the-art lipid-based DNA delivery systems that have been investigated in a preclinical setting. We emphasize factors influencing the delivery and subsequent gene expression in vitro, ex vivo, and in vivo. In addition, we cover aspects of nanoparticle engineering and optimization for DNA therapeutics. Finally, we highlight achievements of lipid-based DNA therapies in clinical trials.