RESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. Recently, exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is used as an FDA approved treatment for DMD. Peptide-conjugated PMOs (PPMOs) are used to increase exon skipping efficacy in the heart and are a promising therapy for DMD. Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.
Asunto(s)
Distrofina/genética , Regulación de la Expresión Génica , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Exones , Humanos , Ratones , Ratones Transgénicos , Morfolinos/administración & dosificación , Distrofia Muscular de Duchenne/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a mutation in SMN1 that stops production of SMN (survival of motor neuron) protein. Insufficient levels of SMN results in the loss of motor neurons, which causes muscle weakness, respiratory distress, and paralysis. A nearly identical gene (SMN2) contains a C-to-T transition which excludes exon 7 from 90% of the mature mRNA transcripts, leading to unstable proteins which are targeted for degradation. Although SMN2 cannot fully compensate for a loss of SMN1 due to only 10% functional mRNA produced, the discovery of the intronic splicing silencer (ISS-N1) opened a doorway for therapy. By blocking its function with antisense oligonucleotides manipulated for high specificity and efficiency, exon 7 can be included to produce full-length mRNA, which then compensates for the loss of SMN1. Nusinersen (Spinraza), the first FDA-approved antisense oligonucleotide drug targeting SMA, was designed based on this concept and clinical studies have demonstrated a dramatic improvement in patients. Novel chemistries including phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNAs), as well as peptide conjugates such as Pip that facilitate accurate targeting to the central nervous system, are explored to increase the efficiency of exon 7 inclusion in the appropriate tissues to ameliorate the SMA phenotype. Due to the rapid advancement of treatments for SMA following the discovery of ISS-N1, the future of SMA treatment is highly promising.