RESUMEN
Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2'-Fluoro (2'-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2'-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2'-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2'-O-methyl (2'-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2'-F nucleotides induced efficient exon-23 skipping, with LNA/2'-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2'-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2'-OMe/2'-F chimeras. Overall, our findings certainly expand the scope of constructing 2'-F modified AOs in splice modulation by incorporating 2'-OMe and LNA modifications.
Asunto(s)
Fibras Musculares Esqueléticas/efectos de los fármacos , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/farmacología , Empalme del ARN/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Química Sintética/economía , Técnicas de Química Sintética/métodos , Química Farmacéutica/economía , Química Farmacéutica/métodos , Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofina/metabolismo , Exones/efectos de los fármacos , Exones/genética , Terapia Genética/economía , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos mdx , Morfolinos/economía , Morfolinos/uso terapéutico , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Oligonucleótidos/química , Oligonucleótidos/economía , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/economía , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
Familial dysautonomia (FD) is an autonomic and sensory neuropathy caused by a mutation in the splice donor site of intron 20 of the ELP1 gene. Variable skipping of exon 20 leads to a tissue-specific reduction in the level of ELP1 protein. We have shown that the plant cytokinin kinetin is able to increase cellular ELP1 protein levels in vivo and in vitro through correction of ELP1 splicing. Studies in FD patients determined that kinetin is not a practical therapy due to low potency and rapid elimination. To identify molecules with improved potency and efficacy, we developed a cell-based luciferase splicing assay by inserting renilla (Rluc) and firefly (Fluc) luciferase reporters into our previously well-characterized ELP1 minigene construct. Evaluation of the Fluc/Rluc signal ratio enables a fast and accurate way to measure exon 20 inclusion. Further, we developed a secondary assay that measures ELP1 splicing in FD patient-derived fibroblasts. Here we demonstrate the quality and reproducibility of our screening method. Development and implementation of this screening platform has allowed us to efficiently screen for new compounds that robustly and specifically enhance ELP1 pre-mRNA splicing.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Disautonomía Familiar/genética , Precursores del ARN/genética , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Elongación Transcripcional/genética , Línea Celular , Citocininas/farmacología , Exones/efectos de los fármacos , Exones/genética , Células HEK293 , Humanos , Cinetina/farmacología , Empalme del ARN/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.
Asunto(s)
Compuestos Azo/farmacocinética , Atrofia Muscular Espinal/tratamiento farmacológico , Fármacos Neuromusculares/farmacocinética , Pirimidinas/farmacocinética , Empalme del ARN/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Compuestos Azo/líquido cefalorraquídeo , Compuestos Azo/farmacología , Compuestos Azo/uso terapéutico , Encéfalo/metabolismo , Encéfalo/patología , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Exones/efectos de los fármacos , Exones/genética , Femenino , Humanos , Macaca fascicularis , Células de Riñón Canino Madin Darby , Masculino , Ratones , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Fármacos Neuromusculares/líquido cefalorraquídeo , Fármacos Neuromusculares/farmacología , Fármacos Neuromusculares/uso terapéutico , Pirimidinas/líquido cefalorraquídeo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas , Ratas Wistar , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Porcinos , Distribución TisularRESUMEN
INTRODUCTION: Antisense nucleic acid analogues can interact with pre-mRNA motifs and influence exon or splice site selection and thereby alter gene expression. Design of antisense molecules to target specific motifs can result in either exon exclusion or exon inclusion during splicing. Novel drugs exploiting the antisense concept are targeting rare, life-limiting diseases; however, the potential exists to treat a wide range of conditions by antisense-mediated splice intervention. Areas covered: In this review, the authors discuss the clinical translation of novel molecular therapeutics to address the fatal neuromuscular disorders Duchenne muscular dystrophy and spinal muscular atrophy. The review also highlights difficulties posed by issues pertaining to restricted participant numbers, variable phenotype and disease progression, and the identification and validation of study endpoints. Expert opinion: Translation of novel therapeutics for Duchenne muscular dystrophy and spinal muscular atrophy has been greatly advanced by multidisciplinary research, academic-industry partnerships and in particular, the engagement and support of the patient community. Sponsors, supporters and regulators are cooperating to deliver new drugs and identify and define meaningful outcome measures. Non-conventional and adaptive trial design could be particularly suited to clinical evaluation of novel therapeutics and strategies to treat serious, rare diseases that may be problematic to study using more conventional clinical trial structures.
Asunto(s)
Exones/genética , Terapia Genética/tendencias , Distrofia Muscular de Duchenne/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Empalme del ARN/genética , Investigación Biomédica Traslacional/métodos , Animales , Terapia Biológica/métodos , Terapia Biológica/tendencias , Distrofina/genética , Exones/efectos de los fármacos , Expresión Génica , Regulación de la Expresión Génica , Terapia Genética/métodos , Humanos , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Empalme del ARN/efectos de los fármacos , Investigación Biomédica Traslacional/tendenciasRESUMEN
Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and regulates the survival, differentiation, and maintenance of function in different neuronal populations. BDNF is strongly expressed in hypothalamic neurons, where it exerts long- or short-lasting actions. Because glutamate has been associated with regulations of hypothalamic hormones, we examined the regulation of the four promoters of the BDNF gene by glutamate in fetal hypothalamic neurons. The expression levels of BDNF transcripts were investigated using semiquantitative RT-PCR. BDNF protein was determined by enzyme immunoassay, and BDNF and Trk B (BDNF receptor) gene variations were determined by RNAse protection assay. By RT-PCR, we showed that, under basal conditions, BDNF transcripts from exons I, II, and III but not from IV were expressed in the hypothalamic neurons. Glutamate increased expression of both the protein and the four transcripts via N-methyl-D-aspartate receptors, with maximal stimulations after 3 hr of application for exon I and II mRNAs and after 1 hr for exon III and IV mRNAs. Actinomycin D blocked the increase of all transcripts, whereas cycloheximide treatment inhibited stimulation only of exon I and II mRNAs. Trk B mRNA was rapidly and transiently reduced after glutamate application. Our results demonstrate that glutamate 1) regulates BDNF mRNA expression at an early developmental stage in hypothalamic neurons and 2) exerts a differential regulation of BDNF transcripts.