RESUMEN
BACKGROUND: Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose. METHODS: In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks. RESULTS: Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%. CONCLUSIONS: In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.).
Asunto(s)
Angioedemas Hereditarios , Sistemas CRISPR-Cas , Edición Génica , Adulto , Humanos , Angioedema , Angioedemas Hereditarios/sangre , Angioedemas Hereditarios/tratamiento farmacológico , Angioedemas Hereditarios/genética , Proteína Inhibidora del Complemento C1/uso terapéutico , Relación Dosis-Respuesta a Droga , Edición Génica/métodos , Calicreína Plasmática/genética , Resultado del TratamientoRESUMEN
BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).
Asunto(s)
Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/terapia , Sistemas CRISPR-Cas , Edición Génica , Liposomas/uso terapéutico , Nanopartículas/uso terapéutico , Prealbúmina/genética , ARN Guía de Kinetoplastida/uso terapéutico , Femenino , Técnicas de Transferencia de Gen , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Prealbúmina/análisis , ARN MensajeroRESUMEN
Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.
Asunto(s)
ARN Interferente Pequeño , Animales , Ratas , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND & AIMS: Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that target all major HBV transcripts. METHODS: We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in humanized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n = 24 and n = 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. RESULTS: In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200 mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. CONCLUSIONS: VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. IMPACT AND IMPLICATIONS: A significant unmet need exists for therapies for chronic HBV (cHBV) infection that achieve functional cure. We report clinical and non-clinical data on two investigational small-interfering RNAs that target HBx, ALN-HBV and VIR-2218, demonstrating that incorporation of enhanced stabilization chemistry plus technology in VIR-2218 reduces its propensity to cause ALT elevations relative to its parent compound, ALN-HBV. We also show that VIR-2218 reduces hepatitis B surface antigen levels in a dose-dependent manner in participants with cHBV infection. These studies support the continued development of VIR-2218 as part of therapeutic regimens for cHBV infection, with the goal of a functional cure, and are important for HBV researchers and physicians.
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Hepatitis B Crónica , Hepatitis B , Humanos , Animales , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Virus de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Tratamiento con ARN de Interferencia , Ensayos Clínicos Controlados Aleatorios como Asunto , Antivirales , ADN Viral , Antígenos e de la Hepatitis B , Hepatitis B/tratamiento farmacológicoRESUMEN
RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.
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Técnicas de Transferencia de Gen , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes erbB-1 , Terapia Genética , Humanos , Ligandos , Ratones , ARN Mensajero , ARN Interferente Pequeño/administración & dosificación , Tenascina/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genéticaRESUMEN
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. METHODS: We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. RESULTS: In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. CONCLUSIONS: In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/genética , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Linfocitos B/inmunología , Portador Sano/inmunología , Portador Sano/virología , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/administración & dosificación , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/virología , Humanos , Inmunización Secundaria , Inmunogenicidad Vacunal , Masculino , Ratones , Linfocitos T Citotóxicos/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
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.
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Acetilgalactosamina/efectos adversos , Acetilgalactosamina/química , Desoxirribonucleótidos/efectos adversos , Desoxirribonucleótidos/química , Flúor/química , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/química , Animales , Femenino , Flúor/efectos adversos , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Acetilgalactosamina/química , Organofosfonatos/química , Interferencia de ARN , ARN Interferente Pequeño/química , Compuestos de Vinilo/química , Animales , Apolipoproteínas B/antagonistas & inhibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas Argonautas/antagonistas & inhibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Cultivadas , Factor IX/antagonistas & inhibidores , Factor IX/genética , Factor IX/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Lipoproteínas LDL/sangre , Ratones , Ratones Endogámicos C57BL , Organofosfonatos/farmacología , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Complejo Silenciador Inducido por ARN/química , Complejo Silenciador Inducido por ARN/metabolismo , Factores de Transcripción/metabolismo , Compuestos de Vinilo/farmacologíaRESUMEN
Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.
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Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , beta Catenina/genética , Células HEK293 , HumanosRESUMEN
The application of small interfering (si)RNAs as potential therapeutic agents requires safe and effective methods for their delivery to the cytoplasm of the target cells and tissues. Recent studies have shown significant progress in the development of targeting reagents that facilitate the recognition of, and siRNA delivery to, specific cell types. Among recently reported delivery approaches, polymers with amphipathic properties have been used to enable endosome escape and cytosolic delivery. Here, we describe a linear amphipathic poly(amido amine) polymer conjugate system for the efficient siRNA delivery in vitro and in vivo. This polymer contains a novel amine bearing bis-acrylamide monomer designed for increasing amine density, which resulted in substantial improvement in liver uptake and RNAi activity compared to our previously reported poly(amido amine disulfide) polymer.1 The activity for this liver targeted delivery system was demonstrated in rodents and nonhuman primates.
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Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Poliaminas/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacocinética , Animales , Endosomas/química , Femenino , Silenciador del Gen , Células Hep G2 , Hepatocitos/citología , Humanos , Hígado/citología , Macaca mulatta , Ratones , Estructura Molecular , Poliaminas/síntesis química , Poliaminas/metabolismo , ARN Interferente Pequeño/química , Ratas , Ratas Sprague-DawleyRESUMEN
Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.
Asunto(s)
Concentración de Iones de Hidrógeno , Polímeros/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Animales , Polímeros/química , Polímeros/farmacocinética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacocinética , RatasRESUMEN
Efficient siRNA delivery is dependent not only on the ability of the delivery vehicle to target a specific organ but also on its ability to enable siRNA entry into the cytoplasm of the target cells. Polymers with endosomolytic properties are increasingly being used as siRNA delivery vehicles due to their potential to facilitate endosomal escape and intracellular delivery. Addition of disulfide bonds in the backbone of these polymers was expected to provide degradability through reduction by glutathione in cytosol. This paper describes the synthesis of new endosomolytic bioreducible poly(amido amine disulfide) polymers whose lytic potential can be masked at physiological pH, but can be restored at acidic endosomal pH. These polymer conjugates gave good in vitro knockdown (KD) and did not demonstrate cytotoxicity in a MTS assay. Efficient mRNA KD for apolipoprotein B in mouse liver was observed with these polyconjugates following intravenous dosing.
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Disulfuros/química , Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Poliaminas/química , ARN Interferente Pequeño/administración & dosificación , Animales , Apolipoproteínas B/deficiencia , Apolipoproteínas B/genética , Eritrocitos/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Hemólisis/efectos de los fármacos , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Estructura Molecular , Oxidación-Reducción , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.
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Apoptosis , Genes cdc/fisiología , Cinesinas/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Huso Acromático/fisiología , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Estructura Molecular , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Pirroles/farmacología , Proteína X Asociada a bcl-2RESUMEN
Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% ± 6.2%), diaphragm (32.7% ± 5.8%), and liver (41.3% ± 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.
Asunto(s)
Distrofia Muscular de Duchenne , Ratones , Animales , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Ratones Endogámicos mdx , Distrofina/genética , Regulación hacia Abajo , ARN Interferente Pequeño/uso terapéutico , Músculo Esquelético/patología , ARN Mensajero/metabolismo , Modelos Animales de EnfermedadRESUMEN
Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.
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Técnicas de Transferencia de Gen , ARN Interferente Pequeño/genética , Animales , Animales Modificados Genéticamente , Anticuerpos/aislamiento & purificación , Anticuerpos/metabolismo , Anticuerpos/farmacología , Especificidad de Anticuerpos , Proteínas Argonautas , Células Cultivadas , Factor 2 Eucariótico de Iniciación/inmunología , Factor 2 Eucariótico de Iniciación/metabolismo , Estudios de Evaluación como Asunto , Femenino , Silenciador del Gen/fisiología , Marcación de Gen/métodos , Técnicas de Transferencia de Gen/normas , Humanos , Inmunoprecipitación/métodos , Inmunoprecipitación/normas , Macaca mulatta , Ratones , Ratones Endogámicos ICR , Unión Proteica , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , RoedoresRESUMEN
Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.
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Antineoplásicos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/química , Piridinas/síntesis química , Tiazoles/síntesis química , Amidas/química , Antineoplásicos/farmacología , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Diseño de Fármacos , Etilenodiaminas/química , Humanos , Enlace de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología , Agua/químicaRESUMEN
Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.
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Antineoplásicos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/química , Piridinas/síntesis química , Tiazoles/síntesis química , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/química , Diseño de Fármacos , Halogenación , Humanos , Cinética , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Tiazoles/farmacología , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.
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Diferenciación Celular/genética , Inhibidores de Crecimiento/análisis , MicroARNs/análisis , Regulación hacia Arriba , Apoptosis , Butiratos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ciclo Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Inhibidores de Crecimiento/farmacología , Humanos , Cloruro de Litio/farmacología , MicroARNs/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.
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Inhibidores Enzimáticos/metabolismo , Quinasas Janus/antagonistas & inhibidores , Lípidos , Nanopartículas , ARN Interferente Pequeño/toxicidad , Animales , Citocinas/sangre , Dexametasona/metabolismo , Etoricoxib , Femenino , Técnicas de Inactivación de Genes , Quinasa I-kappa B/antagonistas & inhibidores , Interferón gamma/genética , Interleucina-6/genética , Quinasas Janus/genética , Lípidos/química , Lípidos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Sulfonas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Tacrolimus/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.