Gene therapy
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The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. ⋯ CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.
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The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. ⋯ Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.
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After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. ⋯ The combination of AAV2. PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.
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Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. ⋯ We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.
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Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. ⋯ Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.