A novel exon-specific U1 snRNA strategy to correct splicing in Familial Dysautonomia

家族性自主神经异常(FD)是一种隐性神经退行性疾病，由剪接突变引起。 IKBKAP基因导致组织特异性变量跳过外显子20。这反过来又导致急剧减少 IKAP/ELP1蛋白在中枢和外周神经系统中的表达。尽管FD患者患有多发性骨髓瘤 神经症状，进行性失明极大地降低了生活质量，是 病人和他们的家人。在这项建议中，我们概述了一种新的策略，以靶向视网膜中的mrna剪接为 一种潜在的治疗FD视网膜变性的方法。佛朗哥·帕加尼博士的实验室已经开发出一种新型的改进型 剪接体U1SnRNAs(ExSpeU1s)的版本，可以被特定地靶向来改善包括 跳过外显子。这一策略已被证明在其他几种剪接障碍中是成功的，包括脊椎肌肉 萎缩和囊性纤维化。最近，他的实验室创造了IKBKAP特定的U1，具有精确的靶向碱基- 配对和合作，我们已经证明了它们纠正了剪接并增加了FD细胞和 活体通过使用AAV9的腹膜腔给药。在过去的几年里，我们一直在努力生成新的FD 同时概括人类疾病表型和组织特异性剪接缺陷的小鼠模型。我们的 新小鼠是唯一可以用来评估IKBKAP剪接调节治疗效果的模型 关于疾病表型。改变信使核糖核酸剪接的突变占人类全部基因的很大一部分 疾病，以及纠正剪接的方法，包括小分子和寡核苷酸，已经在临床试验中。 MRNA剪接是一个复杂的过程，因此不可能预测这些方法的效果。 在各种突变中，事实上，联合疗法最终可能是必要的。在这 建议我们将研究一种新的治疗策略，即外显子靶向U1单链RNA的传递，我们 考虑到AAV指导视网膜基因治疗的速度，将专门在视网膜上测试它们的有效性 疾病正在向诊所转移。我们已经组建了一个专家团队，我们的研究不仅将解决 FD中未得到满足的关键医疗需求，也将使我们能够揭示这些外显子 靶向分子可以纠正剪接。所述目标的成功完成肯定会对 治疗FD以外的基因剪接疾病，进一步增加了这一提议的意义。

Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in the IKBKAP gene that leads to tissue-specific variable skipping of exon 20. This, in turn, leads to a drastic reduction of IKAP/ELP1 protein in the central and peripheral nervous system. Although FD patients suffer from multiple neurological symptoms, progressive blindness drastically reduces quality of life and is a major concern of patients and their families. In this proposal, we outline a novel strategy to target mRNA splicing in the retina as a potential therapy for retinal degeneration in FD. Dr. Franco Pagani's laboratory has developed novel modified versions of the spliceosomal U1snRNAs (ExSpeU1s) that can be specifically targeted to improve inclusion of skipped exons. This strategy has proven successful in several other splicing disorders, including spinal muscular atrophy and cystic fibrosis. Recently, his lab has created IKBKAP specific U1s with precisely targeted base- pairing and together we have shown that they correct splicing and increase IKAP protein both in FD cells and in vivo by intraperitoneal delivery using AAV9. For the past several years, we have worked to generate a new FD mouse model that both recapitulates the human disease phenotype and the tissue-specific splice defect. Our new mouse is the only model that can be used to evaluate the therapeutic effect of IKBKAP splicing modulation on disease phenotype. Mutations that alter mRNA splicing account for a large fraction of all human genetic disease, and methods to correct splicing, including small molecules and oligos, are already in clinical trials. mRNA splicing is a complex process, and therefore it is impossible to predict the efficacy of these methods across various mutations, and in fact it is likely that combination therapies will ultimately be necessary. In this proposal we will study a novel therapeutic strategy, namely the delivery of exon-targeted U1 snRNAs, and we will test their efficacy specifically in the retina given the speed with which AAV directed gene therapy for retinal disease is moving into the clinic. We have assembled an expert team and our studies will not only address a critical unmet medical need in FD but will also allow us to uncover the precise mechanism by which these exon targeted molecules correct splicing. Successful completion of the stated Aims will certainly have implications for treating genetic splicing disease beyond FD, further increasing the significance of this proposal.