RESUMO
BACKGROUND: Plasma coagulation Factor XII (FXII) plays a crucial role in contact activation, ultimately regulating both the kinin-kallikrein system and the intrinsic pathway of coagulation. A growing body of evidence suggests that inhibition of FXII can prevent thrombosis. Given FXII does not appear to modulate hemostasis, targeting FXII is a promising strategy for the prevention of pathological thrombus formation without the hemostatic risks typically associated with anticoagulants. To this end, a subcutaneously administered investigational RNAi therapeutic targeting liver F12 mRNA (ALN-F12) was developed. AIM: To investigate the thrombo-protective and hemostatic effects of FXII reduction by ALN-F12 in rodent thrombosis and hemostasis models. METHODS: A single dose of ALN-F12 was subcutaneously administered to C57Bl/6 mice. After reaching steady state FXII reduction, the impact on thrombosis (ferric chloride arterial thrombosis and electrolytic injury induced venous thrombosis models) and hemostasis (saphenous vein injury and tail tip transection bleeding models) was evaluated. RESULT: Administration of ALN-F12 resulted in dose-dependent reductions of both liver F12 mRNA and plasma FXII protein. In mice, ALN-F12 led to dose-dependent reductions in platelet and fibrin accumulation in the venous electrolytic-injury model and in the time to occlusion in the ferric chloride arterial thrombosis model. At 10 mg/kg ALN-F12, the top dose level evaluated, this resulted in >95% reduction of FXII and ~10 fold reduction in fibrin deposition. Finally, hemostasis models showed that >95% reduction of FXII had no impact on bleeding time or blood loss. CONCLUSION: Our findings support that reduction of plasma Factor XII by ALN-F12 provided thrombo-protective effects with no increased bleeding risk in rodent models of thrombosis and hemostasis.
Assuntos
Deficiência do Fator XII , Hemostáticos , Trombose , Animais , Fator XII/genética , Hemostasia , Fígado , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Trombose/genética , Trombose/prevenção & controleAssuntos
Polineuropatias/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Aprovação de Drogas , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Humanos , Nanomedicina , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Pesquisa Translacional BiomédicaRESUMO
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for â¼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
Assuntos
Acetilgalactosamina/efeitos adversos , Acetilgalactosamina/química , Desoxirribonucleotídeos/efeitos adversos , Desoxirribonucleotídeos/química , Flúor/química , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/química , Animais , Feminino , Flúor/efeitos adversos , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Hereditary angioedema (HAE) is a genetic disorder mostly caused by mutations in the C1 esterase inhibitor gene (C1INH) that results in poor control of contact pathway activation and excess bradykinin generation. Bradykinin increases vascular permeability and is ultimately responsible for the episodes of swelling characteristic of HAE. We hypothesized that the use of RNA interference (RNAi) to reduce plasma Factor XII (FXII), which initiates the contact pathway signaling cascade, would reduce contact pathway activation and prevent excessive bradykinin generation. A subcutaneously administered GalNAc-conjugated small-interfering RNA (siRNA) targeting F12 mRNA (ALN-F12) was developed, and potency was evaluated in mice, rats, and cynomolgus monkeys. The effect of FXII reduction by ALN-F12 administration was evaluated in two different vascular leakage mouse models. An ex vivo assay was developed to evaluate the correlation between human plasma FXII levels and high-molecular weight kininogen (HK) cleavage. A single subcutaneous dose of ALN-F12 led to potent, dose-dependent reduction of plasma FXII in mice, rats, and NHP. In cynomolgus monkeys, a single subcutaneous dose of ALN-F12 at 3 mg/kg resulted in >85% reduction of plasma FXII. Administration of ALN-F12 resulted in dose-dependent reduction of vascular permeability in two different mouse models of bradykinin-driven vascular leakage, demonstrating that RNAi-mediated reduction of FXII can potentially mitigate excess bradykinin stimulation. Lastly, ex vivo human plasma HK cleavage assay indicated FXII-dependent bradykinin generation. Together, these data suggest that RNAi-mediated knockdown of FXII by ALN-F12 is a potentially promising approach for the prophylactic treatment of HAE.
Assuntos
Angioedemas Hereditários/tratamento farmacológico , Bradicinina/biossíntese , Fator XII/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Permeabilidade Capilar/efeitos dos fármacos , Proteína Inibidora do Complemento C1/genética , Fator XII/análise , Feminino , Humanos , Cininogênios/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RatosRESUMO
We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.
Assuntos
Inativação Gênica , RNA Interferente Pequeno/genética , Acetilgalactosamina/genética , Animais , Sequência de Bases , Biotecnologia , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/química , Oligonucleotídeos/genética , Interferência de RNA , RNA Interferente Pequeno/químicaRESUMO
Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.
Assuntos
Acetilgalactosamina/farmacocinética , Rim/metabolismo , Fígado/metabolismo , RNA Interferente Pequeno/farmacocinética , Acetilgalactosamina/administração & dosagem , Acetilgalactosamina/metabolismo , Animais , Área Sob a Curva , Sistemas de Liberação de Medicamentos/métodos , Humanos , Fígado/citologia , Masculino , Taxa de Depuração Metabólica , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. METHODS: In this phase 1 dose-escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. RESULTS: No thromboembolic events were observed during the study. The most common adverse events were mild injection-site reactions. Plasma levels of fitusiran increased in a dose-dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose-dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. CONCLUSIONS: Once-monthly subcutaneous administration of fitusiran resulted in dose-dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605 .).
Assuntos
Antitrombina III/antagonistas & inibidores , Hemofilia A/terapia , Hemofilia B/terapia , Terapêutica com RNAi , Adulto , Antitrombinas/sangue , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Hemofilia A/sangue , Hemofilia B/sangue , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Método Simples-Cego , Trombina/biossíntese , Adulto JovemRESUMO
Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
Assuntos
Acetilgalactosamina/química , Organofosfonatos/química , Interferência de RNA , RNA Interferente Pequeno/química , Compostos de Vinila/química , Animais , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Cultivadas , Fator IX/antagonistas & inibidores , Fator IX/genética , Fator IX/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipoproteínas LDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Organofosfonatos/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de Transcrição/metabolismo , Compostos de Vinila/farmacologiaRESUMO
BACKGROUND & AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.
Assuntos
Genômica/métodos , Fígado/crescimento & desenvolvimento , Nanopartículas , Interferência de RNA , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular , Expressão Gênica , Genes da Neurofibromatose 2 , Fator de Crescimento de Hepatócito/genética , Hepatomegalia/etiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Serina-Treonina Quinase 3 , Proteína Supressora de Tumor p53/fisiologia , Proteínas de Sinalização YAPRESUMO
Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.
Assuntos
RNA Interferente Pequeno/administração & dosagem , Animais , Células Cultivadas , Colesterol , Endossomos/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Células HeLa , Hepatócitos/metabolismo , Humanos , Lipídeos , Camundongos , Nanopartículas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.
Assuntos
Antitrombinas/química , Coagulação Sanguínea/efeitos dos fármacos , Fator IX/química , Fator VIII/química , Hemofilia A/tratamento farmacológico , Interferência de RNA , Animais , Relação Dose-Resposta a Droga , Feminino , Hemofilia A/genética , Hemostasia/efeitos dos fármacos , Homozigoto , Humanos , Masculino , Camundongos , MutaçãoRESUMO
INTRODUCTION: A requisite step in developing a therapeutic to modulate the levels of hepcidin is the development of a quantitative method for measuring the concentration of serum hepcidin. METHODS: To this end, an LC-MS method, based on selected reaction monitoring (SRM) with a triple quadrupole MS and an isotopically labeled hepcidin as internal standard, was developed to measure hepcidin in mouse and monkey sera. RESULTS: Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/mL in the mice. Next, experiments were conducted where an siRNA, targeting hepcidin, was administered to cynomolgus monkeys, resulting in effective hepcidin reduction (inhibition rate) of 87% after 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin. CONCLUSIONS: For better sensitivity, especially for the low volumes available for mouse sera, a second LC-MS method, based on parallel reaction monitoring (PRM) using a Orbitrap MS was developed and shown to be at least 10 fold lower in detection limits (or consumption of serum volume) than the SRM approach.
Assuntos
Hepcidinas/biossíntese , Hepcidinas/sangue , RNA Interferente Pequeno/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Haplorrinos , Hepcidinas/genética , Espectrometria de Massas , Camundongos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genéticaRESUMO
Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
Assuntos
Acetilgalactosamina/química , Inativação Gênica , Hepatócitos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
Formulation of short interfering RNA (siRNA) into multicomponent lipid nanoparticles (LNP) is an effective strategy for hepatic delivery and therapeutic gene silencing. This study systematically evaluated the effect of polyethylene glycol (PEG) density on LNP physicochemical properties, innate immune response stimulation, and in vivo efficacy. Increased PEG density not only shielded LNP surface charge but also reduced hemolytic activity, suggesting the formation of a steric barrier. In addition, increasing the PEG density reduced LNP immunostimulatory potential as reflected in cytokine induction both in vivo and in vitro. Higher PEG density also hindered in vivo efficacy, presumably due to reduced association with apolipoprotein E (ApoE), a protein which serves as an endogenous targeting ligand to hepatocytes. This effect could be overcome by incorporating an exogenous targeting ligand into the highly shielded LNPs, thereby circumventing the requirement for ApoE association. Therefore, these studies provide useful information for the rational design of LNP-based siRNA delivery systems with an optimal safety and efficacy profile.
RESUMO
Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.
Assuntos
Proteínas Argonautas/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Fibroblastos/citologia , Fígado/metabolismo , RNA Mensageiro/genética , Animais , Proteínas Argonautas/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Interferência de RNARESUMO
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.
Assuntos
Células Endoteliais/metabolismo , Nanopartículas/química , Polímeros/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular , Humanos , Camundongos , Nanopartículas/ultraestrutura , Neoplasias/genética , Neoplasias/terapia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 â¼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipopeptídeos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Apolipoproteínas E/metabolismo , Microscopia Crioeletrônica , Inativação Gênica , Hepatócitos/metabolismo , Macaca fascicularis , Camundongos , RNA Interferente Pequeno/uso terapêutico , RatosRESUMO
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e139; doi:10.1038/mtna.2013.66; published online 17 December 2013.
RESUMO
In this article, we briefly review the endocytic pathways used by cells, pointing out their defining characteristics and highlighting physical limitations that may direct the internalization of nanoparticles to a subset of these pathways. A more detailed description of these pathways is presented in the literature. We then focus on the endocytosis of nanomedicines and present how various nanomaterial parameters impact these endocytic processes. This topic is an area of active research, motivated by the recognition that an improved understanding of how nanomaterials interact at the molecular, cellular, and whole-organism level will lead to the design of better nanomedicines in the future. Next, we briefly review some of the important nanomedicines already on the market or in clinical development that serve to exemplify how endocytosis can be exploited for medical benefit. Finally, we present some key unanswered questions and remaining challenges to be addressed by the field.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endocitose , Nanomedicina/métodos , Nanoestruturas/química , Animais , Transporte Biológico , Endossomos/metabolismo , Humanos , Nanotecnologia/métodos , Oligonucleotídeos Antissenso/química , Fagocitose , Interferência de RNA , Propriedades de Superfície , Tecnologia Farmacêutica/tendênciasRESUMO
In recent years, RNA interference (RNAi) therapeutics, most notably with lipid nanoparticle-based delivery systems, have advanced into human clinical trials. The results from these early clinical trials suggest that lipid nanoparticles (LNPs), and the novel ionizable lipids that comprise them, will be important materials in this emerging field of medicine. A persistent theme in the use of materials for biomedical applications has been the incorporation of biodegradability as a means to improve biocompatibility and/or to facilitate elimination. Therefore, the aim of this work was to further advance the LNP platform through the development of novel, next-generation lipids that combine the excellent potency of the most advanced lipids currently available with biodegradable functionality. As a representative example of this novel class of biodegradable lipids, the lipid evaluated in this work displays rapid elimination from plasma and tissues, substantially improved tolerability in preclinical studies, while maintaining in vivo potency on par with that of the most advanced lipids currently available.