RESUMEN
Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.
Asunto(s)
Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Oligonucleótidos Antisentido/farmacología , Alelos , Línea Celular , Expresión Génica/efectos de los fármacos , Humanos , Proteína Huntingtina , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Oligonucleótidos/genética , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/farmacología , Ribonucleasa H/metabolismo , Relación Estructura-Actividad , Expansión de Repetición de Trinucleótido/efectos de los fármacosRESUMEN
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.
RESUMEN
This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-methyl-2'-deoxyadenosine from 2'-deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2'-deoxyadenosine followed by methylation of 5'-O-protected nucleoside at N-1. Subsequent N-chloroacetylation leads to N(6)-chloroacetyl-N(1)-methyl-5'-O-(p-anisyldiphenylmethyl)-2'-deoxyadenosine, which is finally converted to its 3' phosphoramidite derivative. This phosphoramidite is used to incorporate N(1)-methyl-2'-deoxyadenosine into synthetic oligonucleotides. N-Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement.
Asunto(s)
Química/métodos , Desoxiadenosinas/química , Oligodesoxirribonucleótidos/química , Cromatografía Líquida de Alta Presión , Desoxiadenosinas/aislamiento & purificación , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/aislamiento & purificaciónRESUMEN
BACKGROUND: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. METHODS: MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. RESULTS: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. CONCLUSIONS: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.
Asunto(s)
Análisis Mutacional de ADN/métodos , Tamizaje Neonatal/métodos , Reacción en Cadena de la Polimerasa/métodos , Autoanálisis , Fibrosis Quística/diagnóstico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genotipo , Humanos , Recién Nacido , Mutación , Estudios Prospectivos , Estudios Retrospectivos , Robótica , Programas InformáticosRESUMEN
BACKGROUND: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. METHODS: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RESULTS: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). CONCLUSION: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.