Methods in molecular biology
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The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51-53 of the DMD gene.
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Duchenne muscular dystrophy (DMD) is a congenital X-linked disease caused by mutations in the gene encoding the dystrophin protein, which is required for myofiber integrity. Exon skipping therapy is an emerging strategy for restoring the open reading frame of the dystrophin gene to produce functional protein in DMD patients by skipping single or multiple exons. ⋯ Our laboratory previously reported that disrupting the splicing acceptor site in exon 45 by plasmid delivery of the CRISPR-Cas9 system in iPS cells, derived from a DMD patient lacking exon 44, successfully restored dystrophin protein expression in differentiated myoblasts. Herein, we describe an optimized methodology to prepare myoblasts differentiated from iPS cells by mRNA transfection of the CRISPR-Cas9 system to skip exon 45 in myoblasts, and evaluate the restored dystrophin by RT-PCR and Western blotting.
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Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. ⋯ Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.
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Studies in psychoneuroimmunology (PNI) would provide better insights into the "whole mind-body system." Systems biology models of the complex adaptive systems (CASs), such as a conceptual framework of "Yin-Yang dynamics," may be helpful for identifying systems-based biomarkers and targets for more effective prevention and treatment. The disturbances in the Yin-Yang dynamical balance may result in stress, inflammation, and various disorders including insomnia, Alzheimer's disease, obesity, diabetes, cardiovascular diseases, skin disorders, and cancer. At the molecular and cellular levels, the imbalances in the cytokine pathways, mitochondria networks, redox systems, and various signaling pathways may contribute to systemic inflammation. ⋯ The studies of cancer have revealed the importance of the Yin-Yang dynamics in the tumoricidal and tumorigenic activities of the immune system. Stress-induced neuroimmune imbalances are also essential in chronic skin disorders including atopic dermatitis and psoriasis. With the integrative framework, the restoration of the Yin-Yang dynamics can become the objective of dynamical systems medicine.
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Spinal muscular atrophy (SMA) is one of the most common genetic causes of infantile death arising due to mutations in the SMN1 gene and the subsequent loss of motor neurons. With the discovery of the intronic splicing silencer N1 (ISS-N1) as a potential target for antisense therapy, several antisense oligonucleotides (ASOs) are being developed to include exon 7 in the final mRNA transcript of the SMN2 gene and thereby increasing the production of spinal motor neuron (SMN) proteins. Nusinersen (spinraza), a modified 2'-O-methoxyethyl (MOE) antisense oligonucleotide is the first drug to be approved by Food and Drug Agency (FDA) in December of 2016. Here we briefly review the pharmacological relevance of the drug, clinical trials, toxicity, and future directions following the approval of nusinersen.