Development of novel ASO-based therapeutics for CMT1A

摘要： 本项目的目标是开发一种安全、有效的含磷二酰胺的反义寡核苷酸。 吗啉齐聚物(PMO)主链化学能显著减少PMP22蛋白的产生 雪旺细胞，它的剂量特性是可滴定的。对于患有CMT1a的患者，最常见的形式 CMT、PMP22这种蛋白质的过度生产会推动疾病的进展。我们之前曾表演过 对PMP22基因序列的严格分析，导致了许多ASO的设计 显示能有效改变PMP22基因的表达。这些PMO被设计成改变外显子剪接， 从而导致在翻译过程的早期被严重截断、有效地跳过外显子的产物 减少PMP22蛋白的产生。然而，重要的是，PMP22的生产并未完全消除，因为 这也将是有害的。 CMT1a是一种非常常见的罕见疾病，它是由参与 外周神经的功能。这种名为PMP22的蛋白质的作用很像绝缘体，允许神经释放 正确地发出信号。虽然过多的PMP22对细胞有害，但过少的PMP22也是一个问题，因此 任何治疗策略都不能简单地敲除这一重要基因的表达。分子遗传学 使这种疾病特别适合于基于核酸的治疗方法，旨在调节 PMP22的表达是因为：1)CMT1A是单基因；2)疾病基因已被鉴定；以及3)抑制 PMP22的表达可通过多种分子机制完成。目前，没有药物是 可用于有效治疗CMT1a的疾病进展，因此这一高风险患者类别仍然存在 没有用疾病改变疗法治疗的。 在第一阶段，一些PMO ASO候选者将在人源化的PMP22小鼠中进行体内筛选 活跃性和急性安全性的模型。在目标1中，我们将注入先前在细胞基础上筛选的选定化合物 并监测PMP22的全长和“外显子跳过”的信使核糖核酸的产生。在……里面 周围周围神经组织周围的Schwan细胞(牺牲后)以确认疗效并在体内整体监测 效率。一旦活性化合物被确认，在目标2中，我们将向不同的小鼠队列中注射分子 并使用公认的电生理学技术监测神经功能的生物学活动。 此前，Shift制药公司(与密苏里大学合作)已成功演示 PMO ASO方法改变与另一个候选药物(E1v1.11)的外显子剪接，该药物已被证明是 对脊髓性肌萎缩症患者SMN蛋白的蛋白生产有极其有效的修饰作用， 提示这种方法在CMT小鼠模型中应该是有效的。我们还在前面的演示中演示了 研究表明，E1v1.11和类似的PMO在剂量极高时非常安全，表明未来 为CMT开发的分子的安全性研究也应证明可接受的安全性。

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.