Gene therapy with designed splice factors to correct splicing defects in Rd6 mice

在所有导致遗传疾病的突变中，约有10-20%会干扰剪接。除了少数例外，这一观察结果与疾病基因无关。因此，纠正剪接缺陷的基因治疗方法是许多遗传性疾病（包括视网膜变性）的极具吸引力的治疗选择。由于眼睛是基因治疗干预的理想靶器官，我们开发了基因治疗方法，可以有效地纠正剪接供体部位缺陷，重点关注与视网膜变性相关的基因。我们的疗法应用设计的基于U1和u6 - snrna的剪接因子，改善剪接供体位点突变的影响。该方法的功能和功效已在几种不同的患者来源的细胞系上成功地进行了RNA和蛋白质水平的测试。此外，本研究的初步结果强烈表明，在视网膜下注射和aav介导的体内转导后，基于usrna的治疗方法在视网膜细胞中是有效的。我们的目标是治疗携带Mfrp基因剪接供体位点突变的Rd6小鼠模型。Rd6小鼠的突变导致剪接缺陷（Mfrp外显子4的跳跃），并导致视网膜色素变性样视网膜变性和斑点状视网膜外观。本项目将评估和优化Rd6小鼠模型的基因治疗方法，应用设计的U1-和u6 - snrna来纠正视网膜和体内突变诱导的剪接缺陷。我们将通过组织学、分子和功能测试来评估aav介导的治疗对突变和对照Rd6小鼠视网膜的疗效。此外，我们将安全措施纳入我们治疗方法的评估。本应用中提出的实验将有助于建立基于rna的治疗方法，不仅适用于Rd6，也适用于其他几种视网膜单基因疾病。该项目很好地嵌入了SPP2127项目，并将进一步指导为大型动物模型或人类患者开发极具前景的基因疗法的方式。

Approximately 10-20% of all mutations causing genetic diseases interfere with splicing. With few exceptions, this observation is made irrespective of the disease gene. Consequently, gene therapeutic approaches that correct splice defects are highly attractive treatment options for many inherited diseases, including retinal degeneration. As the eye constitutes an ideal target organ for gene therapeutic interventions, we developed gene therapeutic approaches that are efficient in correcting splice donor site defects focusing on genes associated with retinal degeneration.Our therapies apply designed U1- and U6-snRNA-based splice factors that ameliorate the effect of splice donor site mutations. The functionality and efficacy of the approach was successfully tested in several different patient-derived cell lines on RNA and protein level. Furthermore, the preliminary results presented in this proposal strongly suggest that the UsnRNA-based therapeutic approach is efficient in retinal cells after subretinal injection and AAV-mediated transduction in vivo.We aim to treat the Rd6 mouse model which carries a splice donor site mutation in the Mfrp gene. The mutation in the Rd6 mouse causes a splice defect (skipping of Mfrp exon 4) and leads to retinitis pigmentosa-like retinal degeneration and flecked retinal appearance. The proposed project will evaluate and optimize gene therapeutic treatments of the Rd6 mouse model applying designed U1- and U6-snRNAs to correct the mutation-induced splice defect in the retina and in vivo. We will evaluate the efficacy of AAV-mediated therapies in the retina of mutant and control Rd6 mice using histological, molecular and functional testing. In addition, we include safety measures into the evaluation of our therapeutic approach.The experiments proposed in this application will help to establish RNA-based treatments suitable not only in the Rd6 context, but also for several other monogenetic diseases of the retina. The project is well-embedded in the SPP2127 program and will further guide the way to develop highly promising gene therapies for large animal models or human patients.