RESUMEN
Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.
Asunto(s)
Acetilgalactosamina , Porfirias Hepáticas , Acetilgalactosamina/farmacocinética , Receptor de Asialoglicoproteína/metabolismo , Hepatocitos/metabolismo , Humanos , Porfirias Hepáticas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
Asunto(s)
Acetilgalactosamina/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Portadores de Fármacos , Silenciador del Gen , Prealbúmina/genética , ARN Interferente Pequeño/metabolismo , Acetilgalactosamina/química , Animales , Proteínas Argonautas/genética , Receptor de Asialoglicoproteína/genética , Transporte Biológico , Estabilidad de Medicamentos , Femenino , Glicoconjugados/química , Glicoconjugados/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Nanopartículas/metabolismo , Prealbúmina/antagonistas & inhibidores , Prealbúmina/metabolismo , ARN Interferente Pequeño/genética , Factores de TiempoRESUMEN
Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.
Asunto(s)
Glicoles/química , Ácidos Nucleicos/química , ARN Interferente Pequeño/química , Animales , Cristalografía por Rayos X , Silenciador del Gen/efectos de los fármacos , Concentración 50 Inhibidora , Ratones , Modelos Biológicos , Ácidos Nucleicos Heterodúplex/química , ARN Interferente Pequeño/farmacología , Receptores de Albúmina/efectos de los fármacos , TemperaturaRESUMEN
Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.
Asunto(s)
Exorribonucleasas/química , Oligonucleótidos/química , Pirimidinas/química , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Estereoisomerismo , TermodinámicaRESUMEN
We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.
Asunto(s)
Acetilgalactosamina/química , Portadores de Fármacos/química , Silenciador del Gen , Hepatocitos/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Animales , Ratones , Prealbúmina/deficiencia , Prealbúmina/genéticaRESUMEN
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.
Asunto(s)
Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , Lípidos/química , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Línea Celular , Factor VII/genética , Factor VII/metabolismo , Silenciador del Gen , Terapia Genética , Humanos , Lípidos/farmacocinética , Macaca fascicularis , Masculino , Ratones , Nanopartículas/química , Nanopartículas/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/química , RatasRESUMEN
Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005 mg kg(-1) in mice and less than 0.03 mg kg(-1) in non-human primates.
Asunto(s)
Silenciador del Gen , Lípidos/administración & dosificación , Hígado/fisiología , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Aminas/química , Animales , Femenino , Terapia Genética/métodos , Humanos , Cinética , Lípidos/química , Liposomas/administración & dosificación , Liposomas/química , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , ARN Interferente Pequeño/químicaRESUMEN
Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.
Asunto(s)
Interferencia de ARN/fisiología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Femenino , Células HeLa , Hepatocitos/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.
Asunto(s)
LDL-Colesterol/sangre , Primates/metabolismo , ARN Interferente Pequeño/genética , Serina Endopeptidasas/metabolismo , Animales , Humanos , Hígado/enzimología , Ratones , Ratones Noqueados , Estructura Molecular , Primates/genética , ARN Mensajero/genética , Ratas , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Factores de TiempoRESUMEN
RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.
Asunto(s)
Lípidos/química , Hígado/metabolismo , ARN Interferente Pequeño/química , Animales , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Factor VII/genética , Factor VII/metabolismo , Lípidos/síntesis química , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN/fisiología , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.
Asunto(s)
Técnicas de Transferencia de Gen , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Colesterol , Cricetinae , Lipoproteínas HDL , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Mesocricetus , Ratones , Receptores de Lipoproteína/metabolismoRESUMEN
Camptothecin (CPT) and its derivatives target mammalian DNA topoisomerase I (top1) and are among the most effective novel anticancer drugs. However, the activity of CPTs is limited by several factors, including drug inactivation by lactone ring opening, tumor drug resistance, and toxicity in patients. Novel top1 inhibitors are being searched to overcome such limitations and expand the anticancer spectrum of camptothecins. MJ-III-65 (NSC 706744) is among the most promising indenoisoquinolines to date. In this study, we show that MJ-III-65 enhances top1 cleavage complexes by both inhibiting their reversal (religation) more efficiently than CPT and by enhancing their formation. The top1 DNA cleavage complexes induced by MJ-III-65 exhibit a different distribution pattern compared with CPT and exhibit different base sequence preferences immediately around the top1 cleavage sites. Although CPTs have a preference for thymine at the (-1) position and guanine at the (+1) position of the top1-mediated DNA cleavage sites, MJ-III-65 can accommodate different base pairs at the (-1), (+1), or (+2) position, with a preference for a cytosine at the (-1) position on the scissile strand. Another difference with CPTs is the activity of MJ-III-65 against CPT-resistant top1 enzymes, implying that the amino acid residue interactions with top1 are different for MJ-III-65 and CPTs. As with CPT, MJ-III-65 is inactive against vaccinia top1. This study shows the specific molecular interactions of MJ-III-65 with top1 and demonstrates that MJ-III-65 is a potentially useful top1 inhibitor that enhances and traps top1 cleavage sites not sensitive to CPTs.
Asunto(s)
Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Indenos/farmacología , Isoquinolinas/farmacología , Inhibidores de Topoisomerasa I , Animales , ADN-Topoisomerasas de Tipo I/genética , Humanos , Oligonucleótidos/metabolismo , Especificidad por Sustrato , Vaccinia/enzimología , Vaccinia/genéticaRESUMEN
A number of novel dihydroindenoisoquinolines and indenoisoquinolinium salts were synthesized and examined for cytotoxicity in cancer cell cultures and for inhibition of topoisomerase I (top1). The top1-mediated DNA cleavage patterns produced in the presence of several of the new analogues were also investigated, and a few of the more potent compounds were examined for activity in hollow fiber animal models. Very cytotoxic dihydroindenoisoquinoline and isoquinolinium salts were obtained with mean graph midpoints (MGMs) for growth inhibition in the low submicromolar range. Two of the new dihydroindenoisoquinolines were found to be weaker top1 inhibitors than the lead compound 1, while two of the indenoisoquinolinium salts were more potent. The top1-mediated DNA cleavage patterns of the indenoisoquinolines examined were found to be similar to each other but different from that of camptothecin. Several of the more potent indenoisoquinolines displayed promising anticancer activities in hollow fiber animal models.
Asunto(s)
Antineoplásicos/síntesis química , Isoquinolinas/síntesis química , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoquinolinas/química , Isoquinolinas/farmacología , Ratones , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales CultivadasAsunto(s)
Polineuropatías/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Animales , Aprobación de Drogas , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos , Humanos , Nanomedicina , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Investigación Biomédica TraslacionalRESUMEN
Chelation of Cu(I) ions to an immobilized hydrophilic tris(triazolylmethyl)amine chelator on a solid support allowed synthesis of RNA oligonucleotide conjugates from completely deprotected alkyne-oligonucleotides. No oligonucleotide strand degradation or metal ion contamination was observed. Furthermore, use of the immobilized copper(I) ion overcame regioselectivity issues associated with strain-promoted copper-free azide-alkyne cycloaddition.
Asunto(s)
Cobre/química , Oligonucleótidos/química , Alquinos/química , Azidas/química , Catálisis , Quelantes/química , Química Clic , Iones/química , Ligandos , Oligonucleótidos/síntesis química , Poliestirenos/química , EstereoisomerismoRESUMEN
We evaluated the abilities of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), and a single-stranded siRNA (ss-siRNA) to inhibit expression from the PTEN gene in mice when formulated identically with lipid nanoparticles (LNPs). Significantly greater reductions in levels of PTEN mRNA were observed for LNP-formulated agents compared to unformulated drugs when gene silencing was evaluated after a single dose in the livers of mice. An unformulated ss-siRNA modified with a metabolically stable phosphate mimic 5'-(E)-vinylphosphonate showed dose-dependent reduction of PTEN mRNA in mice, albeit at doses significantly higher than those observed for formulated ss-siRNA. These results demonstrate that LNPs can be used to deliver functional antisense and ss-siRNA therapeutics to the liver, indicating that progress in the field of siRNA delivery is transferable to other classes of nucleic acid-based drugs.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lípidos/química , Nanopartículas/química , Oligonucleótidos Antisentido , Fosfohidrolasa PTEN/genética , ARN Interferente Pequeño , Animales , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Concentración 50 Inhibidora , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacologíaRESUMEN
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.
RESUMEN
Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t(1/2) ≈ 24.2 min) than the parent siRNA (t(1/2) ≈ 6 min).
Asunto(s)
ADN , Sistemas de Liberación de Medicamentos/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Nanopartículas , Neoplasias Experimentales/tratamiento farmacológico , ARN Interferente Pequeño , Animales , ADN/química , ADN/genética , ADN/farmacología , Femenino , Ácido Fólico/química , Ácido Fólico/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
RESUMEN
Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.