Development of novel ASO-based therapeutics for CMT1A

摘要： 本课题的目的是研制一种安全、有效的反义寡核苷酸 能够显著降低在哺乳动物中的PMP 22蛋白产生的吗啉代寡聚体（PMO）骨架化学 施旺细胞，其剂量特性可滴定。对于CMT 1A患者， 在CMT中，PMP 22过度产生这种蛋白质会导致疾病进展。我们之前表演过 PMP 22基因序列的严格分析，这导致了许多ASO的设计， 显示有效地改变PMP 22 mRNA的产生。这些PMO被设计成改变外显子剪接， 导致在翻译过程早期被严重截断的“外显子跳过”产物， 减少PMP 22蛋白的产生。然而，重要的是，PMP 22的产生并没有完全消除，因为 这也是有害的。 CMT 1A是一种非常常见的罕见疾病，由参与CMT 1A的蛋白质的过度表达引起。 周围神经的功能。这种名为PMP 22的蛋白质的作用就像一个绝缘体，允许神经发射它们的信号。 信号正确。虽然太多的PMP 22对细胞有害，但太少的PMP 22也是一个问题，因此 任何治疗策略都不能简单地敲除重要基因的表达。的分子遗传学 CMT 1A的表达使得这种疾病特别适合于基于核酸的治疗， PMP 22表达，因为：1）CMT 1A是单基因的; 2）疾病基因已被鉴定;和3）抑制 PMP 22的表达可以通过多种分子机制来实现。目前，没有药物 可用于有效治疗CMT 1A的疾病进展，因此这种高风险患者类别仍然存在 未经疾病改变疗法治疗。 在第1阶段期间，将在人源化PMP 22小鼠中“体内”筛选许多阿索候选物 活动和紧急安全模型。在目标1中，我们将注射先前在细胞基础上筛选的选定化合物， 模型植入小鼠体内，并监测PMP 22 mRNA的全长和“外显子跳跃”mRNA的总产量。在 周围神经组织周围的许旺细胞（处死后），以确认疗效并监测体内总体情况 效率一旦活性化合物得到确认，在目标2中，我们将把分子注射到另一组小鼠中。 并使用公认的电生理学技术监测神经功能的生物活性。 此前，Shift Pharmaceuticals（与密苏里州大学合作）已成功证明 用另一种候选药物（E1v1.11）改变外显子剪接的阿索方法已被证明是 对脊髓性肌萎缩症的SMN蛋白质的蛋白质产生修饰极其有效， 建议这种方法在CMT的小鼠模型中应该是有效的。我们在以前的研究中也证明了 研究表明，E1v1.11和类似的PMO在极高剂量下是非常安全的，这表明未来 针对CMT开发的分子的安全性研究也应证明可接受的安全性特征。

Abstract: The objective of this project is to develop a safe, effective antisense oligonucleotide with Phosphorodiamidate Morpholino Oligomer (PMO) backbone chemistry capable of significantly reducing PMP22 protein production in Schwann cells, which is titratable in its dosing characteristics. For patients with CMT1A, the most common form of CMT, PMP22 over production of this protein drives disease progression. We have previously performed rigorous analysis of the PMP22 gene sequence, which has led to the design of numerous ASOs that have been shown to effectively alter PMP22 mRNA production. These PMOs have been designed to alter exon splicing, resulting in an “exon-skipped” product that is severely truncated early in the translation process, effectively reducing PMP22 protein production. Importantly, however, PMP22 production is not completely eliminated since this would also be detrimental. CMT1A is a remarkably common rare disease that is caused by the overexpression of a protein involved in the function of nerves in the periphery. The protein, PMP22, acts much like an insulator, allowing nerves to fire their signal properly. While too much of PMP22 is bad for the cell, too little of PMP22 is also a problem and therefore any therapeutic strategy cannot simply knock-out the expression of the important gene. The molecular genetics of CMT1A makes this disease particularly amenable for nucleic acid-based therapeutics designed to modulate PMP22 expression since: 1) CMT1A is monogenic; 2) the disease gene has been identified; and 3) inhibition of PMP22 expression can be accomplished through a variety a molecular mechanisms. Currently, no drugs are available for effectively treating the disease progression of CMT1A, thus this highly at risk patient class remains untreated with disease altering therapies. During Phase 1, a number of the PMO ASO candidates will be screened “in vivo”, in a humanized PMP22 mouse model for activity and acute safety. In Aim 1, we will inject selected compounds previously screened in cell based models into mice and monitor the PMP22 mRNA total for full-length and “exon-skipped” mRNA production. in Schwan cells surrounding peripheral nerve tissues (after sacrifice) to confirm efficacy and monitor overall in vivo efficiency. Once active compounds are confirmed, in Aim 2 we will inject molecules into a different cohort of mice and monitor biological activity of nerve function using accepted electrophysiology techniques. Previously, Shift Pharmaceuticals (collaborating with the University of Missouri) has successfully demonstrated the PMO ASO approach for altering exon splicing with another drug candidate (E1v1.11) that has proven to be extremely effective at modifying protein production of SMN protein for Spinal Muscular Atrophy disease, which suggested this approach should be effective in mouse models for CMT. We have also demonstrated in previous studies that E1v1.11 and similar PMOs are very safe when dosed at extremely high levels, indicating that future safety studies of molecules developed for CMT should also demonstrated acceptable safety profiles.