A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles.

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

Lipid nanoparticle-mediated silencing of osteogenic suppressor GNAS leads to osteogenic differentiation of mesenchymal stem cells in vivo.

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.

Improved Liver Delivery of Primaquine by Phospholipid-Free Small Unilamellar Vesicles with Reduced Hemolytic Toxicity.

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 invitro and invivo 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.

FAM13A as potential therapeutic target in modulating TGF-ß-induced airway tissue remodeling in COPD.

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-ß1 (TGF-ß1). To determine the role of FAM13A in TGF-ß1 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-ß1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGF-ß1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in ß-catenin (CTNNB1) expression in FAM13A-/- cells compared with WT cells after TGF-ß1 treatment. FAM13A overexpression was partially protective from TGF-ß1-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, ß-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-ß1-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.

Optimized Photoactivatable Lipid Nanoparticles Enable Red Light Triggered Drug Release.

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.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy.

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.

Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics.

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

Poly(Sarcosine) Surface Modification Imparts Stealth-Like Properties to Liposomes.

Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA-pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA-pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.

Zebrafish as a predictive screening model to assess macrophage clearance of liposomes in vivo.

Macrophage recognition of nanoparticles is highly influenced by particle size and surface modification. Due to the lack of appropriate in vivo screening models, it is still challenging and time-consuming to characterize and optimize nanomedicines regarding this undesired clearance mechanism. Therefore, we validate zebrafish embryos as an emerging vertebrate screening tool to assess the macrophage sequestration of surface modified particulate formulations with varying particle size under realistic biological conditions. Liposomes with different PEG molecular weights (PEG350-PEG5000) at different PEG densities (3.0-10.0 mol%) and particle sizes between 60 and 120 nm were used as a well-established reference system showing various degrees of macrophage uptake. The results of in vitro experiments, zebrafish embryos, and in vivo rodent biodistribution studies were consistent, highlighting the validity of the newly introduced zebrafish macrophage clearance model. We hereby present a strategy for efficient, systematic and rapid nanomedicine optimization in order to facilitate the preclinical development of nanotherapeutics.

Secreted Matrix Metalloproteinase-9 of Proliferating Smooth Muscle Cells as a Trigger for Drug Release from Stent Surface Polymers in Coronary Arteries.

Cardiovascular diseases are the leading causes of death in industrialized countries. Atherosclerotic coronary arteries are commonly treated with percutaneous transluminal coronary intervention followed by stent deployment. This treatment has significantly improved the clinical outcome. However, triggered vascular smooth muscle cell (SMC) proliferation leads to in-stent restenosis in bare metal stents. In addition, stent thrombosis is a severe side effect of drug eluting stents due to inhibition of endothelialization. The aim of this study was to develop and test a stent surface polymer, where cytotoxic drugs are covalently conjugated to the surface and released by proteases selectively secreted by proliferating smooth muscle cells. Resting and proliferating human coronary artery smooth muscle cells (HCASMC) and endothelial cells (HCAEC) were screened to identify an enzyme exclusively released by proliferating HCASMC. Expression analyses and enzyme activity assays verified selective and exclusive activity of the matrix metalloproteinase-9 (MMP-9) in proliferating HCASMC. The principle of drug release exclusively triggered by proliferating HCASMC was tested using the biodegradable stent surface polymer poly-l-lactic acid (PLLA) and the MMP-9 cleavable peptide linkers named SRL and AVR. The specific peptide cleavage by MMP-9 was verified by attachment of the model compound fluorescein. Fluorescein release was observed in the presence of MMP-9 secreting HCASMC but not of proliferating HCAEC. Our findings suggest that cytotoxic drug conjugated polymers can be designed to selectively release the attached compound triggered by MMP-9 secreting smooth muscle cells. This novel concept may be beneficial for stent endothelialization thereby reducing the risk of restenosis and thrombosis.

Variable asialoglycoprotein receptor 1 expression in liver disease: Implications for therapeutic intervention.

AIM: One of the most promising strategies for the treatment of liver diseases is targeted drug delivery via the asialoglycoprotein receptor (ASGPR). The success of this approach heavily depends on the ASGPR expression level on parenchymal liver cells. In this study, we assessed the mRNA and protein expression levels of the major receptor subunit, ASGR1, in hepatocytes both in vitro and in vivo. METHODS: In vitro, various liver cancer-derived cell lines were evaluated. In vivo, we screened the ASGR1 mRNA on 59 hepatocellular carcinoma and matched non-neoplastic tissue using RNA microarray. In addition, 350 human liver specimens of patients with hepatocellular carcinoma or non-neoplastic liver diseases were screened for ASGR1 protein level using tissue microarray analysis. RESULTS: Our data reveal that the ASGR1 mRNA expression directly correlates with the protein level. We demonstrate that the ASGR1 expression is upregulated in cirrhotic specimens and is significantly decreased with increasing hepatocellular carcinoma grade. CONCLUSION: Because the ASGR1 expression levels are variable between patients, our findings suggest that ASGPR-based targeting strategies should be combined with ASGPR-companion diagnostics to maximize clinical benefit.

Loop-miRs: active microRNAs generated from single-stranded loop regions.

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene regulation. The miRNA precursors are processed by the endonucleases Drosha and Dicer into a duplex, bound to an Argonaute protein and unwound into two single-stranded miRNAs. Although alternative ways to generate miRNAs have been discovered, e.g. pre-miRNA cleavage by Ago2 or cleavage products of snoRNAs or tRNAs, all known pathways converge on a double-stranded RNA duplex. Exogenous single-stranded siRNAs (ss-siRNAs) can elicit an effective RNA interference reaction; recent studies have identified chemical modifications increasing their stability and activity. Here, we provide first evidence that endogenous, unmodified, single-stranded RNA sequences are generated from single-stranded loop regions of human pre-miRNA hairpins, the so called loop-miRs. Luciferase assays and immunoprecipitation validate loop-miR activity and incorporation into RNA-induced silencing complexes. This study identifies endogenous miRNAs that are generated from single-stranded regions; hence, it provides evidence that precursor-miRNAs can give rise to three distinct endogenous miRNAs: the guide strand, the passenger strand and the loop-miR.

Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues.

Lipid nanoparticles (LNPs) for delivery of mRNA usually contain ionizable lipid/helper lipid/cholesterol/PEG-lipid in molar ratios of 50:10:38.5:1.5, respectively. These LNPs are rapidly cleared from the circulation following intravenous (i.v.) administration, limiting uptake into other tissues. Here, we investigate the properties of LNP mRNA systems prepared with high levels of "helper" lipids such as 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) or N-(hexadecanoyl)-sphing-4-enine-1-phosphocholine (egg sphingomyelin [ESM]). We show that LNP mRNAs containing 40 mol % DSPC or ESM have a unique morphology with a small interior "solid" core situated in an aqueous compartment that is bounded by a lipid bilayer. The encapsulated mRNA exhibits enhanced stability in the presence of serum. LNP mRNA systems containing 40 mol % DSPC or ESM exhibit significantly improved transfection properties in vitro compared with systems containing 10 mol % DSPC or ESM. When injected i.v., LNP mRNAs containing 40 mol % ESM exhibit extended circulation lifetimes compared with LNP mRNA systems containing 10 mol % DSPC, resulting in improved accumulation in extrahepatic tissues. Systems containing 40 mol % ESM result in significantly improved gene expression in spleen and bone marrow as well as liver post i.v. injection compared with 10 mol % DSPC LNP mRNAs. We conclude that LNP mRNAs containing high levels of helper lipid provide a new approach for transfecting hepatic and extrahepatic tissues.

Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepared using 300 mm sodium citrate buffer displaying maximum transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

The role of lipid components in lipid nanoparticles for vaccines and gene therapy.

Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases.

Hepatocyte-targeted delivery of imiquimod reduces hepatitis B virus surface antigen.

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 provided >95% 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.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

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.

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

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.