ABSTRACT
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
Subject(s)
Adenocarcinoma/genetics , Disease Models, Animal , Genes, Tumor Suppressor , Genetic Engineering/methods , Lung Neoplasms/genetics , Oncogenes , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Dendritic Cells/metabolism , Gene Knock-In Techniques , Genetic Vectors , Lentivirus , Mice , Mice, TransgenicABSTRACT
RNA-based gene therapy requires therapeutic RNA to function inside target cells without eliciting unwanted immune responses. RNA can be ferried into cells using non-viral drug delivery systems, which circumvent the limitations of viral delivery vectors. Here, we review the growing number of RNA therapeutic classes, their molecular mechanisms of action, and the design considerations for their respective delivery platforms. We describe polymer-based, lipid-based, and conjugate-based drug delivery systems, differentiating between those that passively and those that actively target specific cell types. Finally, we describe the path from preclinical drug delivery research to clinical approval, highlighting opportunities to improve the efficiency with which new drug delivery systems are discovered.
Subject(s)
Drug Delivery Systems , Genetic Therapy , Genetic Vectors , RNA, Small Interfering/geneticsABSTRACT
The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
Subject(s)
Cells/metabolism , Gene Editing/methods , Genome, Human/genetics , National Institutes of Health (U.S.)/organization & administration , Animals , Genetic Therapy , Goals , Humans , United StatesABSTRACT
Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.
Subject(s)
Liver , Stem Cells , Heart , Cations , Cholesterol , RNA, MessengerABSTRACT
Lipid nanoparticles (LNPs) have delivered RNA to hepatocytes in patients, underscoring the potential impact of nonliver delivery. Scientists can shift LNP tropism to the lung by adding cationic helper lipids; however, the biological response to these LNPs remains understudied. To evaluate the hypothesis that charged LNPs lead to differential cellular responses, we quantified how 137 LNPs delivered mRNA to 19 cell types in vivo. Consistent with previous studies, we observed helper lipid-dependent tropism. After identifying and individually characterizing three LNPs that targeted different tissues, we studied the in vivo transcriptomic response to these using single-cell RNA sequencing. Out of 835 potential pathways, 27 were upregulated in the lung, and of these 27, 19 were related to either RNA or protein metabolism. These data suggest that endogenous cellular RNA and protein machinery affects mRNA delivery to the lung in vivo.
Subject(s)
Lipids , Nanoparticles , Humans , Liposomes/metabolism , Hepatocytes/metabolism , RNA, Messenger/genetics , RNA, Small InterferingABSTRACT
The asialoglycoprotein receptor (ASGPR) is expressed in high density on hepatocytes. Multivalent variants of galactosyl carbohydrates bind ASGPR with high affinity, enabling hepatic delivery of ligand-bound cargo. Virus-like particle (VLP) conjugates of a relatively high-affinity ligand were efficiently endocytosed by ASGPR-expressing cells in a manner strongly dependent on the nature and density of ligand display, with the best formulation using a nanomolar-, but not a picomolar-level, binder. Optimized particles were taken up by HepG2 cells with greater efficiency than competing small molecules or the natural multigalactosylated ligand, asialoorosomucoid. Upon systemic injection in mice, these VLPs were rapidly cleared to the liver and were found in association with sinusoidal endothelial cells, Kupffer cells, hepatocytes, dendritic cells, and other immune cells. Both ASGPR-targeted and nontargeted particles were distributed similarly to endothelial and Kupffer cells, but targeted particles were distributed to a greater number and fraction of hepatocytes. Thus, selective cellular trafficking in the liver is difficult to achieve: even with the most potent ASGPR targeting available, barrier cells take up much of the injected particles and hepatocytes are accessed only approximately twice as efficiently in the best case.
Subject(s)
Endothelial Cells , Liver , Animals , Mice , Asialoglycoprotein Receptor , Ligands , Endothelial Cells/metabolism , Liver/metabolism , Hepatocytes/metabolismABSTRACT
mRNA drugs can preempt infectious disease and treat Mendelian disorders, such as sickle cell anemia, muscular dystrophy, and cystic fibrosis, as well as autoimmunity and cancer. The three major therapeutic areas for which mRNA delivery is currently being explored are antigen production, including the COVID-19 vaccine, protein replacement therapy, and genome engineering. It was demonstrated 30 years ago that introducing in vitro transcribed mRNA intramuscularly results in detectable protein expression for specific antigens protecting against the likes of influenza and cancer. Utilizing mRNA as a therapeutic modality, however, is challenging. mRNA is large and anionic and, as a result, cannot passively diffuse across the negatively charged plasma membrane. In addition, RNases present in the bloodstream and tissues rapidly degrade mRNA, and its administration induces the innate immune response. In consequence, lipid-, polymer-, dendrimer-, and natural membrane-based mRNA drug delivery systems have been developed to deliver mRNA to target cells. Significant efforts and investments have been made to translate some of these systems into the clinic. Specifically, systemically administered lipid nanoparticles (LNPs) have delivered mRNA to the liver, and intramuscularly administered LNPs have delivered mRNA to immune cells to protect against coronavirus disease of 2019. However, clinically relevant delivery in non-liver tissues such as the spleen, lungs, heart, eye, central nervous system, and lymphatics requires improved drug delivery systems.In this Account, we provide an overview of key advances that have led us to Food and Drug Administration approval for the Pfizer/BioNTech mRNA-based vaccine against SARS-CoV-2 and Emergency Use Authorization for the Moderna mRNA-based vaccine against the same disease, and we explain how these developments will contribute to the clinical translation of mRNA therapeutics targeted outside of the liver. We first focus on the chemical modifications and sequence optimization that can improve the potency of mRNA, resulting in greatly improved pharmacokinetics. After detailing what makes an ideal mRNA payload, we review drug delivery systems used to deliver the payload into target cells. We describe efforts to reduce clearance by the liver, a key obstacle to the development of non-liver therapies. We then consider recent examples of nanoparticles that have delivered mRNA to non-liver tissues. Finally, we discuss current clinical mRNA programs, focusing on the COVID vaccines and highlighting lessons that may be applied to future mRNA drugs.
Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , Liposomes , Liver , Nanoparticles , RNA, Messenger/genetics , SARS-CoV-2ABSTRACT
Lipid nanoparticles (LNPs) have delivered therapeutic RNA to hepatocytes in humans. Adsorption of apolipoprotein E (ApoE) onto these clinical LNP-mRNA drugs has been shown to facilitate hepatocyte entry via the low-density lipoprotein receptor (LDLR). Since ApoE-LDLR trafficking is conserved in mice, non-human primates, and humans, characterizing this mechanism eased clinical transition. Recently, LNPs have delivered mRNA to non-hepatocytes in mice and non-human primates, suggesting they can target new cell types via ApoE- and LDLR-independent pathways. To test this hypothesis, we quantified how 60 LNPs delivered mRNA with cell type resolution in wild-type mice and three knockout mouse strains related to lipid trafficking: ApoE-/-, LDLR-/-, and PCSK9-/-. These data suggest that the hydrophobic tail length of diketopiperazine-based lipids can be changed to drive ApoE- and LDLR-independent delivery in vivo. More broadly, the results support the hypothesis that endogenous LNP trafficking can be tuned by modifying lipid chemistry.
Subject(s)
Apolipoproteins E , Lipoproteins, LDL , Nanoparticles , Animals , Mice , Apolipoproteins E/genetics , Lipoproteins, LDL/genetics , Mice, Knockout , Nanoparticles/chemistry , RNA, Messenger/chemistryABSTRACT
To predict whether preclinical lipid nanoparticle (LNP) delivery will translate in humans, it is necessary to understand whether the mechanism used by LNPs to enter cells is conserved across species. In mice, non-human primates, and humans, LNPs deliver RNA to hepatocytes by adsorbing apolipoprotein E (ApoE), which binds low-density lipoprotein receptor (LDLR). A growing number of LNPs can deliver RNA to nonhepatocytes, suggesting that ApoE- and LDLR-independent interactions could affect LNP tropism. To evaluate this hypothesis, we developed a universal DNA barcoding system that quantifies how chemically distinct LNPs deliver small interfering RNA in any mouse model, including genetic knockouts. We quantified how 98 different LNPs targeted 11 cell types in wildtype, LDLR-/-, very low-density lipoprotein receptor, and ApoE-/- mice, studying how these genes, which traffic endogenous lipids, affected LNP delivery. These data identified a novel, stereopure LNP that targets Kupffer cells, endothelial cells, and hepatocytes in an ApoE-independent manner. These results suggest that non-ApoE interactions can affect the tropism of LNP-RNA drugs.
Subject(s)
Lipids , Nanoparticles , Animals , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Endothelial Cells/metabolism , Lipoproteins, LDL , Liposomes , Mice , Nanoparticles/metabolism , RNA, Small Interfering/geneticsABSTRACT
Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.
Subject(s)
CRISPR-Cas Systems , Endothelial Cells/metabolism , Gene Editing , Gene Transfer Techniques , Lipids/chemistry , Nanoparticles/administration & dosage , RNA, Guide, Kinetoplastida/genetics , RNA, Messenger/genetics , Animals , Cells, Cultured , Endothelial Cells/cytology , HEK293 Cells , Hepatocytes/cytology , Hepatocytes/metabolism , High-Throughput Screening Assays , Humans , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , RNA, Guide, Kinetoplastida/chemistry , RNA, Messenger/chemistryABSTRACT
BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.
Subject(s)
Endothelium, Vascular/physiology , Glycine/metabolism , Hypertension, Pulmonary/genetics , Mitochondrial Proteins/metabolism , Adolescent , Adult , Animals , Cell Respiration , Cells, Cultured , Child , Child, Preschool , Disease Models, Animal , Female , Humans , Hypertension, Pulmonary/metabolism , Infant , Iron-Sulfur Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/genetics , Mutation/genetics , Oxidation-Reduction , RNA, Small Interfering/genetics , Young AdultABSTRACT
Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.
Subject(s)
DNA Barcoding, Taxonomic/methods , Drug Discovery/methods , Nanoparticles/chemistry , Nucleic Acids/administration & dosage , RNA, Small Interfering/administration & dosage , Animals , Cell Separation , Drug Delivery Systems/methods , Factor VII/genetics , Female , Flow Cytometry , Liver/cytology , Liver/drug effects , Lung/cytology , Lung/drug effects , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Nucleic Acids/therapeutic use , Pharmaceutical Preparations/administration & dosage , RNA Interference , RNA, Small Interfering/therapeutic use , Tissue DistributionABSTRACT
Nanoparticles are often targeted to receptors expressed on specific cells, but few receptors are (i) highly expressed on one cell type and (ii) involved in endocytosis. One unexplored alternative is manipulating an endocytic gene expressed on multiple cell types; an ideal gene would inhibit delivery to cell type A more than cell type B, promoting delivery to cell type B. This would require a commonly expressed endocytic gene to alter nanoparticle delivery in a cell type-dependent manner in vivo; whether this can occur is unknown. Based on its microenvironmental regulation, we hypothesized Caveolin 1 (Cav1) would exert cell type-specific effects on nanoparticle delivery. Fluorescence was not sensitive enough to investigate this question, and as a result, we designed a platform named QUANT to study nanoparticle biodistribution. QUANT is 108× more sensitive than fluorescence and can be multiplexed. By measuring how 226 lipid nanoparticles (LNPs) delivered nucleic acids to multiple cell types in vivo in wild-type and Cav1 knockout mice, we found Cav1 altered delivery in a cell-type specific manner. Cav1 knockout did not alter LNP delivery to lung and kidney macrophages but substantially reduced LNP delivery to Kupffer cells, which are liver-resident macrophages. These data suggest caveolin-mediated endocytosis of nanomedicines by macrophages varies with tissue type. These results suggest manipulating receptors expressed on multiple cell types can tune drug delivery.
Subject(s)
Caveolin 1/metabolism , Drug Carriers/metabolism , Nanoparticles/metabolism , Nucleic Acids/administration & dosage , Animals , Caveolin 1/genetics , Cell Line , Cells, Cultured , Drug Carriers/chemistry , Drug Delivery Systems , Endocytosis , Kupffer Cells/metabolism , Lipid Metabolism , Lipids/chemistry , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nanoparticles/chemistry , Nucleic Acids/pharmacokinetics , Tissue DistributionABSTRACT
Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.
Subject(s)
Drug Delivery Systems , Lipids/chemistry , Nanoparticles/chemistry , Nucleic Acids/administration & dosage , Animals , Cell Line , Cells, Cultured , Endothelial Cells/metabolism , Female , Humans , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Nanotechnology , Nucleic Acids/pharmacokineticsABSTRACT
Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the siRNA drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.
Subject(s)
Bone Marrow/metabolism , Drug Carriers/chemistry , Nanoparticles/chemistry , RNA, Guide, Kinetoplastida/pharmacology , RNA, Small Interfering/pharmacology , Animals , Antigens, CD/genetics , Cell Adhesion Molecules/genetics , Computational Biology , Directed Molecular Evolution , Drug Carriers/metabolism , Endothelial Cells/metabolism , Gene Silencing , Mice , Nanoparticles/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Small Interfering/geneticsABSTRACT
RATIONALE: Macrophages reside in the healthy myocardium, participate in ischemic heart disease, and modulate myocardial infarction (MI) healing. Their origin and roles in post-MI remodeling of nonischemic remote myocardium, however, remain unclear. OBJECTIVE: This study investigated the number, origin, phenotype, and function of remote cardiac macrophages residing in the nonischemic myocardium in mice with chronic heart failure after coronary ligation. METHODS AND RESULTS: Eight weeks post MI, fate mapping and flow cytometry revealed that a 2.9-fold increase in remote macrophages results from both increased local macrophage proliferation and monocyte recruitment. Heart failure produced by extensive MI, through activation of the sympathetic nervous system, expanded medullary and extramedullary hematopoiesis. Circulating Ly6C(high) monocytes rose from 64±5 to 108±9 per microliter of blood (P<0.05). Cardiac monocyte recruitment declined in Ccr2(-/-) mice, reducing macrophage numbers in the failing myocardium. Mechanical strain of primary murine and human macrophage cultures promoted cell cycle entry, suggesting that the increased wall tension in post-MI heart failure stimulates local macrophage proliferation. Strained cells activated the mitogen-activated protein kinase pathway, whereas specific inhibitors of this pathway reduced macrophage proliferation in strained cell cultures and in the failing myocardium (P<0.05). Steady-state cardiac macrophages, monocyte-derived macrophages, and locally sourced macrophages isolated from failing myocardium expressed different genes in a pattern distinct from the M1/M2 macrophage polarization paradigm. In vivo silencing of endothelial cell adhesion molecules curbed post-MI monocyte recruitment to the remote myocardium and preserved ejection fraction (27.4±2.4 versus 19.1±2%; P<0.05). CONCLUSIONS: Myocardial failure is influenced by an altered myeloid cell repertoire.
Subject(s)
Biomechanical Phenomena/physiology , Cell Proliferation/physiology , Heart Failure/pathology , Macrophages/physiology , Myocardium/cytology , Animals , Cells, Cultured , Chronic Disease , Female , Heart Failure/physiopathology , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer.
Subject(s)
Lung Neoplasms/therapy , MicroRNAs/therapeutic use , RNA, Small Interfering/therapeutic use , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/therapy , Animals , Antineoplastic Agents/administration & dosage , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/therapy , Cell Line, Tumor , Cisplatin/administration & dosage , Combined Modality Therapy , Gene Expression , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , MAP Kinase Signaling System , Mice , Mice, Knockout , Mice, Transgenic , MicroRNAs/administration & dosage , MicroRNAs/genetics , Mutation , Nanoparticles/administration & dosage , Nanoparticles/therapeutic use , Nanotechnology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , ras Proteins/geneticsABSTRACT
Medicine relies on the use of pharmacologically active agents (drugs) to manage and treat disease. However, drugs are not inherently effective; the benefit of a drug is directly related to the manner by which it is administered or delivered. Drug delivery can affect drug pharmacokinetics, absorption, distribution, metabolism, duration of therapeutic effect, excretion, and toxicity. As new therapeutics (e.g., biologics) are being developed, there is an accompanying need for improved chemistries and materials to deliver them to the target site in the body, at a therapeutic concentration, and for the required period of time. In this Perspective, we provide an historical overview of drug delivery and controlled release followed by highlights of four emerging areas in the field of drug delivery: systemic RNA delivery, drug delivery for localized therapy, oral drug delivery systems, and biologic drug delivery systems. In each case, we present the barriers to effective drug delivery as well as chemical and materials advances that are enabling the field to overcome these hurdles for clinical impact.
Subject(s)
Drug Delivery Systems , Pharmaceutical Preparations/administration & dosage , Pharmaceutical Preparations/metabolism , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Humans , Molecular StructureABSTRACT
OBJECTIVE: Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease. APPROACH AND RESULTS: We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage- or endothelial cell (EC)-targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion development in low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, and suppressed macrophage accumulation and macrophage expression of proinflammatory M1 genes. Dll4 antibody treatment for 7 days after grafting also reduced macrophage burden at day 28. Dll4 silencing via macrophage-targeted lipid nanoparticles reduced lesion development and macrophage accumulation, whereas EC-targeted Dll4 small-interfering RNA produced no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces proinflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell proliferation and migration and suppressed their differentiation. CONCLUSIONS: These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4-Notch axis as a novel therapeutic target.