RÉSUMÉ
We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA2 compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA2-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA2, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA2-based liposomes demonstrate a linear dose-response with an ED50 of 0.1 mg/kg against liver-specific target genes in BALB/c mice.
Sujet(s)
Acides aminés/composition chimique , Liposomes/composition chimique , Petit ARN interférent/génétique , Animaux , Femelle , Cellules HepG2 , Humains , Souris , Souris de lignée BALB CRÉSUMÉ
Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.
Sujet(s)
Protéines du cycle cellulaire/métabolisme , Protéines IAP/génétique , Protein-Serine-Threonine Kinases/métabolisme , Protéines proto-oncogènes/métabolisme , Petit ARN interférent/génétique , Protéines de répression/génétique , Tumeurs de la vessie urinaire/génétique , Tumeurs de la vessie urinaire/thérapie , Administration par voie vésicale , Animaux , Technique de Western , Protéines du cycle cellulaire/génétique , Lignée cellulaire tumorale , Cholestérol/administration et posologie , Cholestérol ester/administration et posologie , Modèles animaux de maladie humaine , Femelle , Expression des gènes , Humains , Liposomes/administration et posologie , Souris , Souris nude , Phosphatidyléthanolamine/administration et posologie , Réaction de polymérisation en chaîne , Protein-Serine-Threonine Kinases/génétique , Protéines proto-oncogènes/génétique , Interférence par ARN , ARN messager/génétique , Petit ARN interférent/administration et posologie , Petit ARN interférent/usage thérapeutique , Survivine , Tumeurs de la vessie urinaire/anatomopathologie ,RÉSUMÉ
siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.
Sujet(s)
Interférence par ARN , Petit ARN interférent/composition chimique , Lignée cellulaire , Analyse de profil d'expression de gènes , Humains , microARN/métabolismeRÉSUMÉ
The use of short interfering RNAs (siRNA) in animals for target validation or as potential therapeutics is hindered by the short physical half-life when delivered as unencapsulated material and in turn the short active half-life of siRNAs in vivo. Here we demonstrate that the character of the two 3'-overhang nucleotides of the guide strand of siRNAs is a determinant of the duration of silencing by siRNAs both in vivo and in tissue culture cells. We demonstrate that deoxyribonucleotides in the guide strand overhang of siRNAs have a negative impact on maintenance of both the in vitro and in vivo activity of siRNAs over time. Overhangs that contain ribonucleotides or 2'-O-methyl modified nucleotides do not demonstrate this same impairment. We also demonstrate that the sequence of an siRNA is a determinant of the duration of silencing of siRNAs directed against the same target even when those siRNAs have equivalent activities in vitro. Our experiments have determined that a measurable duration parameter exists, distinct from both maximum silencing ability and the potency of siRNAs. Our findings provide information on incorporating chemically modified nucleotides into siRNAs for potent, durable therapeutics and also inform on methods used to select siRNAs for therapeutic and research purposes.
Sujet(s)
Interférence par ARN , Petit ARN interférent/composition chimique , Animaux , Lignée cellulaire tumorale , Cytokines/métabolisme , Cinétique , Souris , Souris de lignée C57BL , Petit ARN non traduitRÉSUMÉ
The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1-3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes.
Sujet(s)
Diabète de type 2/traitement médicamenteux , Diabète de type 2/métabolisme , Hyperglycémie/traitement médicamenteux , Foie/métabolisme , Petit ARN interférent/pharmacologie , Transactivateurs/physiologie , Facteurs de transcription/physiologie , Animaux , Glycémie/métabolisme , Technique de Western , Cellules cultivées , Chimie pharmaceutique , Néoglucogenèse/effets des médicaments et des substances chimiques , Technique du clamp glycémique , Hépatocytes/métabolisme , Homéostasie/effets des médicaments et des substances chimiques , Insuline/sang , Insulinorésistance/physiologie , Mâle , Souris , Souris de lignée C57BL , Réaction de polymérisation en chaîne , Acide pyruvique/métabolisme , Petit ARN interférent/effets indésirables , Petit ARN interférent/usage thérapeutique , Rats , Rat Zucker , Transactivateurs/antagonistes et inhibiteurs , Transactivateurs/biosynthèse , Facteurs de transcription/antagonistes et inhibiteurs , Facteurs de transcription/biosynthèseRÉSUMÉ
Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.
Sujet(s)
microARN/pharmacologie , Syndromes neurotoxiques/traitement médicamenteux , Interférence par ARN , Petit ARN interférent/effets indésirables , Animaux , Encéphale/effets des médicaments et des substances chimiques , Corps strié , Extinction de l'expression des gènes , Thérapie génétique/méthodes , Humains , Maladie de Huntington/thérapie , Souris , microARN/synthèse chimique , microARN/usage thérapeutique , Syndromes neurotoxiques/étiologieRÉSUMÉ
BACKGROUND: RNA interference is an endogenous cellular mechanism in which short interfering RNAs (siRNAs) direct the sequence specific degradation of a target mRNA. siRNAs can be synthesized with chemical modifications to increase stability and reduce double-stranded RNA-induced immune responses without affecting their ability to elicit degradation of target mRNA. OBJECTIVES: This study examined the use of chemically modified siRNAs in a mouse model of allergen-induced airway hyperresponsiveness. METHODS: Chemically modified siRNAs were designed and screened in a cell-based reporter assay. The most potent siRNAs were then screened in bone marrow-derived mast cells to demonstrate efficacy in primary cells. RESULTS: A candidate siRNA was formulated and administered to sensitized mice just before airway challenge with allergen. Administration of the siRNA was shown to reduce airway resistance significantly in sensitized and challenged mice by 60%, whereas a control siRNA had no effect. CONCLUSION: These data demonstrate the effectiveness of introducing targeted siRNAs to prevent induction of allergen-induced airway dysfunction and suggest potential therapeutic applications.
Sujet(s)
Hyperréactivité bronchique/thérapie , Interleukine-13/métabolisme , Interférence par ARN , Petit ARN interférent/usage thérapeutique , Animaux , Cellules de la moelle osseuse , Hyperréactivité bronchique/étiologie , Modèles animaux de maladie humaine , Femelle , Gènes rapporteurs , Humains , Interleukine-13/génétique , Mastocytes , Souris , Souris de lignée BALB C , Petit ARN interférent/synthèse chimique , Petit ARN interférent/génétique , Petit ARN interférent/métabolisme , Organismes exempts d'organismes pathogènes spécifiques , Résultat thérapeutiqueRÉSUMÉ
The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
Sujet(s)
Systèmes de délivrance de médicaments/méthodes , Virus de l'hépatite B/génétique , Hépatite B/thérapie , Hépatite B/virologie , Liposomes/composition chimique , Petit ARN interférent/administration et posologie , Petit ARN interférent/composition chimique , Animaux , Antiviraux/administration et posologie , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/métabolisme , Carcinome hépatocellulaire/virologie , Lignée cellulaire tumorale , Matériaux revêtus, biocompatibles/administration et posologie , Matériaux revêtus, biocompatibles/composition chimique , Femelle , Ciblage de gène/méthodes , Thérapie génétique/méthodes , Hépatite B/génétique , Hépatite B/métabolisme , Virus de l'hépatite B/effets des médicaments et des substances chimiques , Humains , Liposomes/pharmacocinétique , Foie/effets des médicaments et des substances chimiques , Foie/métabolisme , Mâle , Taux de clairance métabolique , Souris , Petit ARN interférent/génétique , Petit ARN interférent/pharmacocinétique , Distribution tissulaire , Résultat thérapeutiqueRÉSUMÉ
To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.
Sujet(s)
Virus de l'hépatite B/physiologie , Petit ARN interférent/physiologie , Réplication virale/génétique , Animaux , Lignée cellulaire tumorale , Séquence conservée , ADN viral/sang , Dimérisation , Relation dose-effet des médicaments , Stabilité de médicament , Extinction de l'expression des gènes , Ciblage de gène , Génome viral , Antigènes de surface du virus de l'hépatite B/sang , Virus de l'hépatite B/génétique , Humains , Souris , Petit ARN interférent/administration et posologie , Petit ARN interférent/synthèse chimique , Petit ARN interférent/composition chimique , ARN viral/sangRÉSUMÉ
Human rhinoviruses (HRV) are the main cause of the common cold. Viral replication utilizes the activity of the HRV3C protease (3CP) enzyme [Antimicrob. Agents Chemother. 43 (1999) 2444; Antimicrob. Agents Chemother. 44 (2000) 1236]. Therefore, 3CP is an attractive target for antiviral drug development, and a new class of orally bioavailable irreversible 3CP inhibitors has been designed [J. Med. Chem. 45 (2002) 1607]. We have used related inhibitors to develop a rapid test for rhinovirus. The optical immuno assay (OIA) thin film detection technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. The purpose of this study was to develop a rapid assay for the determination of 3CP combining the Thermo Electron Bio Star OIA technology and the newly designed inhibitor compounds. The advantage of this assay was in its approach, in which therapeutic and diagnostic targets are the same thus allowing patients with detected rhinoviruses to receive optimal treatment. Three different biotinylated inhibitor compounds were synthesized. The length of the spacer between the inhibitor and biotin core was 5, 10, and 15 atoms. These compounds were incorporated into the OIA format for the HRV assay development. A rapid (20 min) OIA test was developed using a 15 atom spacer biotinylated inhibitor (4). Forty different HRV serotypes were studied and thirty three serotypes of these 40 were detected (80%).
Sujet(s)
Cysteine endopeptidases/analyse , Dosage immunologique , Inhibiteurs de protéases , Rhinovirus/isolement et purification , Protéines virales/antagonistes et inhibiteurs , Protéines virales/analyse , Protéases virales 3C , Anticorps antiviraux , Protéines bactériennes/métabolisme , Biotinylation , Rhume banal/diagnostic , Rhume banal/virologie , Cysteine endopeptidases/immunologie , Cellules HeLa , Horseradish peroxidase/métabolisme , Humains , Structure moléculaire , Protéines virales/immunologieRÉSUMÉ
Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.
Sujet(s)
Hépatite C/diagnostic , ARN catalytique/métabolisme , ARN viral/analyse , Séquence nucléotidique , Test ELISA/méthodes , Hépatite C/virologie , Dosage immunologique/méthodes , Cinétique , Données de séquences moléculaires , Conformation d'acide nucléique , Sensibilité et spécificitéRÉSUMÉ
BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.
Sujet(s)
Protéine CFTR/génétique , Séquençage par oligonucléotides en batterie/méthodes , Analyse automatique , Sondes d'ADN , Dépistage génétique/méthodes , Génotype , Humains , Mutation , Polymorphisme de nucléotide simple , RobotiqueRÉSUMÉ
We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.
Sujet(s)
Hepacivirus/génétique , ARN catalytique/métabolisme , ARN viral/analyse , Concentration en ions d'hydrogène , ARN catalytique/génétique , Facteurs tempsRÉSUMÉ
We have developed a rapid and sensitive thin film assay for in-process monitoring of target protein purification. This novel biosensor method provides rapid (5-min) visual evaluation of column purification fractions. The method can be used to monitor the efficiency of purification and potential loss of protein if the column binding capacity is exceeded. The eluted fractions containing the highest yield of target protein can be quickly identified, pooled, and processed. This convenient platform, known as the SILAS product, is a thin-film detection technology in which specific molecular interactions are transduced into visible color changes based on changes in the optical thickness of layers on a silicon surface. The results are interpreted without instrumentation. Proteins eluted from a purification column are adsorbed to the assay surface, and the ligand of interest (target) can be identified with specific binding reagents. Here we demonstrate two protein purification applications for the SILAS technology product: monitoring antibody elution from a Protein G column and evaluating the efficiency of purification of a glutathione-S-transferase (GST)-tagged recombinant protein through each step of the purification process.