RESUMO
BACKGROUND: Microbiomes can have profound impacts on host biology and evolution, but to date, remain vastly understudied in spiders despite their unique and diverse predatory adaptations. This study evaluates closely related species of spiders and their host-microbe relationships in the context of phylosymbiosis, an eco-evolutionary pattern where the microbial community profile parallels the phylogeny of closely related host species. Using 16S rRNA gene amplicon sequencing, we characterized the microbiomes of five species with known phylogenetic relationships from the family Theridiidae, including multiple closely related widow spiders (L. hesperus, L. mactans, L. geometricus, S. grossa, and P. tepidariorum). RESULTS: We compared whole animal and tissue-specific microbiomes (cephalothorax, fat bodies, venom glands, silk glands, and ovary) in the five species to better understand the relationship between spiders and their microbial symbionts. This showed a strong congruence of the microbiome beta-diversity of the whole spiders, cephalothorax, venom glands, and silk glands when compared to their host phylogeny. Our results support phylosymbiosis in these species and across their specialized tissues. The ovary tissue microbial dendrograms also parallel the widow phylogeny, suggesting vertical transfer of species-specific bacterial symbionts. By cross-validating with RNA sequencing data obtained from the venom glands, silk glands and ovaries of L. hesperus, L. geometricus, S. grossa, and P. tepidariorum we confirmed that several microbial symbionts of interest are viably active in the host. CONCLUSION: Together these results provide evidence that supports the importance of host-microbe interactions and the significant role microbial communities may play in the evolution and adaptation of their hosts.
Assuntos
Evolução Biológica , Microbiota , Aranhas/classificação , Aranhas/microbiologia , Simbiose , Animais , Feminino , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Advancement of RNAi-based therapeutics depends on effective delivery to the site of protein synthesis. Although intravenously administered, multi-component delivery vehicles have enabled small interfering RNA (siRNA) delivery and progression into clinical development, advances of single-component, systemic siRNA delivery have been challenging. In pre-clinical models, attachment of a triantennary N-acetylgalactosamine (GalNAc) ligand to an siRNA mediates hepatocyte uptake via the asialoglycoprotein receptor enabling RNAi-mediated gene silencing. In this phase 1 study, we assessed translation of this delivery approach by evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of a GalNAc-siRNA conjugate, revusiran, targeting transthyretin (TTR). Subjects received a placebo or ascending doses of revusiran subcutaneously ranging from 1.25-10 mg/kg in the single and 2.5-10 mg/kg in the multiple ascending dose phases. Revusiran was generally well tolerated, with transient, mild to moderate injection site reactions the most common treatment-emergent adverse events. Doses of 2.5-10 mg/kg revusiran elicited a significant reduction of serum TTR versus the placebo (p < 0.01), with mean TTR reductions of approximately 90% observed with multiple dosing. These results demonstrate translation of this novel delivery platform, enabling clinical development of subcutaneously administered GalNAc-siRNAs for liver-based diseases.
Assuntos
Acetilgalactosamina , Hepatócitos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Acetilgalactosamina/química , Adulto , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Monitoramento de Medicamentos , Feminino , Inativação Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Pré-Albumina/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Adulto JovemRESUMO
Primary hyperoxaluria type 1 (PH1), an inherited rare disease of glyoxylate metabolism, arises from mutations in the enzyme alanine-glyoxylate aminotransferase. The resulting deficiency in this enzyme leads to abnormally high oxalate production resulting in calcium oxalate crystal formation and deposition in the kidney and many other tissues, with systemic oxalosis and ESRD being a common outcome. Although a small subset of patients manages the disease with vitamin B6 treatments, the only effective treatment for most is a combined liver-kidney transplant, which requires life-long immune suppression and carries significant mortality risk. In this report, we discuss the development of ALN-GO1, an investigational RNA interference (RNAi) therapeutic targeting glycolate oxidase, to deplete the substrate for oxalate synthesis. Subcutaneous administration of ALN-GO1 resulted in potent, dose-dependent, and durable silencing of the mRNA encoding glycolate oxidase and increased serum glycolate concentrations in wild-type mice, rats, and nonhuman primates. ALN-GO1 also increased urinary glycolate concentrations in normal nonhuman primates and in a genetic mouse model of PH1. Notably, ALN-GO1 reduced urinary oxalate concentration up to 50% after a single dose in the genetic mouse model of PH1, and up to 98% after multiple doses in a rat model of hyperoxaluria. These data demonstrate the ability of ALN-GO1 to reduce oxalate production in preclinical models of PH1 across multiple species and provide a clear rationale for clinical trials with this compound.
Assuntos
Oxirredutases do Álcool , Hiperoxalúria Primária/enzimologia , Hiperoxalúria Primária/terapia , Oxalatos/metabolismo , Terapêutica com RNAi , Oxirredutases do Álcool/genética , Animais , Modelos Animais de Doenças , Inativação Gênica , Fígado/enzimologia , Masculino , Camundongos , Primatas , RNA Mensageiro , RatosRESUMO
BACKGROUND: Inclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. METHODS: In this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. RESULTS: The most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84). CONCLUSIONS: In this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442 .).
Assuntos
Anticolesterolemiantes/administração & dosagem , Inibidores de PCSK9 , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Acetilgalactosamina/administração & dosagem , Adolescente , Adulto , Idoso , Anticolesterolemiantes/efeitos adversos , LDL-Colesterol/sangue , Feminino , Voluntários Saudáveis , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/biossíntese , Pró-Proteína Convertase 9/sangue , RNA Interferente Pequeno/efeitos adversos , Complexo de Inativação Induzido por RNA/metabolismo , Método Simples-Cego , Adulto JovemRESUMO
ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.
Assuntos
Neuropatias Amiloides Familiares/terapia , Fígado/metabolismo , Pré-Albumina/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Benzoxazóis/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica , Humanos , Fígado/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Pré-Albumina/genética , Pré-Albumina/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinéticaRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) infection in lung transplant (LTx) patients is associated with an increased incidence of bronchiolitis obliterans syndrome (BOS). ALN-RSV01 is a small interfering RNA targeting RSV replication that was shown in an earlier Phase 2a trial to be safe and to reduce the incidence of BOS when compared with placebo. METHODS: We performed a Phase 2b randomized, double-blind, placebo-controlled trial in RSV-infected LTx patients to examine the impact of ALN-RSV01 on the incidence of new or progressive BOS. Subjects were randomized (1:1) to receive aerosolized ALN-RSV01 or placebo daily for 5 days. RESULTS: Of 3,985 symptomatic patients screened, 218 were RSV-positive locally, of whom 87 were randomized to receive ALN-RSV01 or placebo (modified intention-to-treat [mITT] cohort). RSV infection was confirmed by central laboratory in 77 patients (ALN-RSV01, n = 44; placebo, n = 33), which comprised the primary analysis cohort (central mITT [mITTc]). ALN-RSV01 was found to be safe and well-tolerated. At Day 180, in ALN-RSV01-treated patients, compared with placebo, in the mITTc cohort there was a trend toward a decrease in new or progressive BOS (13.6% vs 30.3%, p = 0.058), which was significant in the per-protocol cohort (p = 0.025). Treatment effect was enhanced when ALN-RSV01 was started <5 days from symptom onset, and was observed even without ribavirin treatment. There was no significant impact on viral parameters or symptom scores. CONCLUSIONS: These results confirm findings of the earlier Phase 2a trial and provide further support that ALN-RSV01 reduces the risk of BOS after RSV in LTx recipients.
Assuntos
Antivirais/uso terapêutico , Bronquiolite Obliterante/prevenção & controle , Transplante de Pulmão , RNA Interferente Pequeno/uso terapêutico , Infecções por Vírus Respiratório Sincicial/complicações , Adulto , Bronquiolite Obliterante/etiologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Síndrome , Resultado do TratamentoRESUMO
BACKGROUND: Development of noninvasive imaging modalities to quantify amyloid burden over time is an unmet clinical need. Technetium pyrophosphate (99mTc-PYP) scintigraphy is a simple and widely available radiotracer useful to differentiate transthyretin from light-chain amyloidosis in patients with advanced cardiac amyloidosis. We examined the utility of serial 99mTc-PYP scanning to quantify amyloid burden over time in TTR cardiac amyloidosis (ATTR-CA). METHODS AND RESULTS: Twenty subjects with ATTR-CA (10 wild type, 10 mutant) underwent serial 99mTc-PYP planar cardiac imaging. Cardiac retention was assessed both semiquantitatively (visual score 0, no uptake to 3, uptake greater than bone) and quantitatively (region of interest drawn over the heart, copied, and mirrored over the contralateral chest) to calculate a heart-to-contralateral (H/CL) ratio. Index scan mean visual score and H/CL were 3.0 ± 0.2 and 1.79 ± 0.2, respectively, and after an average 1.5 ± 0.5 years follow-up, did not differ, 3.0 ± 0.2, P = .33 and 1.76 ± 0.2, P = .44. H/CL change was minimal, 0.03 ± 0.17, did not correlate with time between scans, r = 0.19, P = .43, and was observed despite obvious clinical progression (increase in troponin ≥ 0.1 ng/mL, BNP ≥ 400 pg/mL, NYHA class, and/or death). CONCLUSIONS: Serial 99mTc-PYP scanning in subjects with advanced ATTR-CA does not show significant changes over an average 1.5 years of follow-up despite obvious clinical progression.
Assuntos
Amiloidose/diagnóstico por imagem , Cardiomiopatias/diagnóstico por imagem , Aumento da Imagem/métodos , Técnica de Subtração , Pirofosfato de Tecnécio Tc 99m , Idoso , Feminino , Humanos , Estudos Longitudinais , Masculino , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Método Simples-CegoRESUMO
BACKGROUND: Transthyretin-mediated amyloidosis is an inherited, progressively debilitating disease caused by mutations in the transthyretin gene. This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of patisiran (ALN-TTR02), a small interfering RNA encapsulated within lipid nanoparticles, in patients with transthyretin-mediated familial amyloid polyneuropathy (FAP). METHODS: In this phase II study, patients with FAP were administered 2 intravenous infusions of patisiran at one of the following doses: 0.01 (n = 4), 0.05 (n = 3), 0.15 (n = 3), or 0.3 (n = 7) mg/kg every 4 weeks (Q4W), or 0.3 mg/kg (n = 12) every 3 weeks (Q3W). RESULTS: Of 29 patients in the intent-to-treat population, 26 completed the study. Administration of patisiran led to rapid, dose-dependent, and durable knockdown of transthyretin, with the maximum effect seen with patisiran 0.3 mg/kg; levels of mutant and wild-type transthyretin were reduced to a similar extent in Val30Met patients. A mean level of knockdown exceeding 85 % after the second dose, with maximum knockdown of 96 %, was observed for the Q3W dose. The most common treatment-related adverse event (AE) was mild-to-moderate infusion-related reactions in 10.3 % of patients. Four serious AEs (SAEs) were reported in 1 patient administered 0.3 mg/kg Q3W (urinary tract infection, sepsis, nausea, vomiting), and 1 patient administered 0.3 mg/kg Q4W had 1 SAE (extravasation-related cellulitis). CONCLUSIONS: Patisiran was generally well tolerated and resulted in significant dose-dependent knockdown of transthyretin protein in patients with FAP. Patisiran 0.3 mg/kg Q3W is currently in phase III development. TRIAL REGISTRATION NUMBER: NCT01617967 .
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Idoso , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/administração & dosagemRESUMO
OBJECTIVES: To assess the association between severity of neuropathy and disease stage, and estimate the rate of neuropathy progression in a retrospective cross-sectional analysis of a multinational population of patients with familial amyloidotic polyneuropathy (FAP). METHODS: We characterize neuropathy severity and rate of progression in available patients with FAP in France, the United States, Portugal, and Italy. Neuropathy Impairment Scores (NIS), time from symptom onset to NIS measurement, polyneuropathy disability (PND) scores, FAP disease stage, and manual grip strength data were collected. We estimated neuropathy progression using Loess Fit and Gompertz Fit models. RESULTS: For the 283 patients studied (mean age, 56.4 years), intercountry genotypic variation in the transthyretin (TTR) mutation was observed, with the majority of patients in Portugal (92%) having early-onset Val30Met-FAP. There was also marked intercountry variation in PND score, FAP stage, and TTR stabilizer use. NIS was associated with PND score (NIS 10 and 99 for scores I and IV, respectively; p < 0.0001) and FAP stage (NIS 14 and 99 for stages 1 and 3, respectively; p < 0.0001). In addition, there was an association between NIS and TTR genotype. The estimated rate of NIS progression for a population with a median NIS of 32 was 14.3 points/year; the corresponding estimated rate for the modified NIS+7 is 17.8 points/year. CONCLUSIONS: In a multinational population of patients with FAP, rapid neuropathic progression is observed and the severity of neuropathy is associated with functional scales of locomotion.
Assuntos
Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/epidemiologia , Progressão da Doença , Índice de Gravidade de Doença , Adulto , Idade de Início , Idoso , Neuropatias Amiloides Familiares/genética , Estudos Transversais , Feminino , França/epidemiologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Portugal/epidemiologia , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. METHODS: We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. FINDINGS: Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0·0001). INTERPRETATION: Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. FUNDING: Alnylam Pharmaceuticals.
Assuntos
LDL-Colesterol/sangue , Terapia Genética/métodos , Pró-Proteína Convertases/biossíntese , RNA Interferente Pequeno/farmacologia , Serina Endopeptidases/biossíntese , Adulto , LDL-Colesterol/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Terapia Genética/efeitos adversos , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/sangue , Pró-Proteína Convertases/genética , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/efeitos adversos , Serina Endopeptidases/sangue , Serina Endopeptidases/genética , Método Simples-CegoRESUMO
BACKGROUND: Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS: We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS: Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS: ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).
Assuntos
Neuropatias Amiloides Familiares/terapia , Pré-Albumina/genética , RNA Interferente Pequeno/uso terapêutico , Adolescente , Adulto , Neuropatias Amiloides Familiares/genética , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipossomos , Macaca fascicularis , Masculino , Nanocápsulas , Pré-Albumina/metabolismo , RNA Interferente Pequeno/administração & dosagem , Adulto JovemRESUMO
Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.
Assuntos
Anticorpos Monoclonais/metabolismo , Engenharia Metabólica/métodos , RNA Interferente Pequeno/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Inativação Gênica , Humanos , Engenharia Metabólica/instrumentação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismoRESUMO
Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. -thalassemia is a congenital anemia caused by partial or complete loss of -globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe(-/-)) and -thalassemia intermedia (Hbb(th3/+)) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe(-/-) and Hbb(th3/+) mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbb(th3/+) mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of -thalassemia intermedia.
Assuntos
Sobrecarga de Ferro/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Talassemia beta/terapia , Anemia/genética , Anemia/metabolismo , Anemia/terapia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Sequência de Bases , Modelos Animais de Doenças , Envelhecimento Eritrocítico , Eritropoese , Feminino , Hemocromatose/genética , Hemocromatose/metabolismo , Hemocromatose/terapia , Proteína da Hemocromatose , Hepcidinas , Antígenos de Histocompatibilidade Classe I/genética , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Serina Endopeptidases/genética , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
BACKGROUND: Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. RESULTS: Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. CONCLUSIONS: The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process.
Assuntos
Dípteros/genética , Ecossistema , Evolução Molecular , Proteínas de Choque Térmico HSP70/genética , Família Multigênica , Animais , Genes de Insetos , Biblioteca Genômica , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , TemperaturaRESUMO
The bioisosteric replacement of the indole core of CRTH2 antagonists using thienopyrroles was investigated, resulting in potent antagonists with good selectivity over DP1. Early ADME/PK assessment of this chemotype demonstrated bioavailability in mice.
Assuntos
Acetatos/farmacologia , Pirróis/química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Acetatos/química , Acetatos/farmacocinética , Animais , Disponibilidade Biológica , Camundongos , Microssomos Hepáticos/metabolismo , RatosRESUMO
BACKGROUND: While increasing numbers of small interfering RNA (siRNA) therapeutics enter into clinical trials, the quantification of siRNA from clinical samples for pharmacokinetic studies remains a challenge. This challenge is even more acute for the quantification of chemically modified and formulated siRNAs such as those typically required for systemic delivery. RESULTS: Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction. CONCLUSIONS: HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs.
RESUMO
The Drosophila argonaute2 (ago2) gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs). We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop) mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex), is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Glutamina/química , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/genética , Alelos , Animais , Proteínas Argonautas , Feminino , Dosagem de Genes , Inativação Gênica , Variação Genética , Heterozigoto , Mutação , Filogenia , Isoformas de Proteínas , Estrutura Terciária de Proteína , Interferência de RNARESUMO
Hsc/Hsp70-interacting protein (HIP) is a rapidly evolving Hsp70 cofactor. Analyses of multiple Drosophila species indicate that the HIP gene is duplicated only in D. melanogaster. The HIP region, in fact, contains seven distinctly evolving duplicated genes. The regional duplication occurred in two steps, fixed rapidly, and illustrates multiple modes of duplicate gene evolution. HIP and its duplicate HIP-R are adaptively evolving in a manner unique to the region: they exhibit elevated divergence from other drosophilids and low polymorphism within D. melanogaster. HIP and HIP-R are virtually identical, share polymorphisms, and are subject to gene conversion. In contrast, two other duplicate genes in the region, CG33221 and GP-CG32779, are pseudogenes, and the chimeric gene Crg1 is subject to balancing selection. HIP and HIP-R are evolving rapidly and adaptively; however, positive selection is not sufficient to explain the molecular evolution of the region as a whole.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular , Genes Duplicados , Animais , Genes de Insetos , Variação Genética , Proteínas de Choque Térmico HSP70/genética , Método de Monte Carlo , Reação em Cadeia da Polimerase , Pseudogenes , Fatores de Transcrição/genéticaRESUMO
Thermotolerance involves more than life or death. Investigating the complexity of this trait will aid identification of its genetic contributors. We examined variation in thermally stressed walking behavior and performance in natural Drosophila melanogaster strains and strains mutant for the heat shock protein Hsp70, to determine which aspects of locomotion are affected by heat shock and genotype. We developed software for the large-scale capture, analysis, and visualization of locomotion, and determined: (1) Heat shock and thermal pretreatment significantly and differentially impact fly locomotor behavior and performance. (2) Stressed locomotion traits vary extensively among natural strains. (3) Interactions among treatments, strains, and traits are substantial and often counterintuitive. (4) Hsp70 overexpressing flies are faster and more basally thermoprotected in performance than Hsp70 null flies, but null flies are more unidirectional. (5) Natural variation in most stressed locomotion traits exceeds that caused by Hsp70 mutation, reveals uncoupling between thermoprotection of behavior and performance, and suggests significant genetic variation for trait-specific modifiers of thermotolerance.
Assuntos
Variação Genética/genética , Proteínas de Choque Térmico HSP70/genética , Locomoção/genética , Estresse Fisiológico/genética , Sensação Térmica/genética , Animais , Animais Geneticamente Modificados , Análise Mutacional de DNA , Drosophila melanogaster , Feminino , Expressão Gênica/genética , Genótipo , Habituação Psicofisiológica/genética , Mutação INDEL , Processamento de Imagem Assistida por Computador , Masculino , Tempo de Reação/genética , Software , Análise de Sobrevida , Gravação de VideoteipeRESUMO
A series of 2-aminopyrimidines was synthesized as ligands of the histamine H4 receptor (H4R). Working in part from a pyrimidine hit that was identified in an HTS campaign, SAR studies were carried out to optimize the potency, which led to compound 3, 4- tert-butyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-ylamine. We further studied this compound by systematically modifying the core pyrimidine moiety, the methylpiperazine at position 4, the NH2 at position 2, and positions 5 and 6 of the pyrimidine ring. The pyrimidine 6 position benefited the most from this optimization, especially in analogs in which the 6- tert-butyl was replaced with aromatic and secondary amine moieties. The highlight of the optimization campaign was compound 4, 4-[2-amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]benzonitrile, which was potent in vitro and was active as an anti-inflammatory agent in an animal model and had antinociceptive activity in a pain model, which supports the potential of H 4R antagonists in pain.