Development of PMP22 siRNA Conjugates for Treatment of Charcot-Marie-Tooth Disease Type 1A

项目概要： 腓骨肌萎缩症（CMT）是最常见的遗传性周围神经病变， 系统，其特征在于一组遗传和临床异质性疾病，导致 远端肌肉进行性无力和萎缩、感觉丧失、反射减退和骨骼畸形。CMT 1A型（CMT 1A）是最普遍的形式，影响1/10，000的人，并与1.4-Mbp相关。 在染色体17p11.2区域中存在重复，该区域含有外周髓鞘蛋白22（PMP 22）基因。 PMP 22对周围神经髓鞘的结构、发育和维持至关重要。PMP22 过表达促进脱髓鞘-髓鞘再生循环，导致施旺细胞功能障碍。由于 PMP 22基因剂量与神经病表型的关联，治疗策略主要是 专注于抑制PMP 22的过度表达。RNA治疗剂，如反义寡核苷酸（阿索）和 siRNA是有吸引力的，因为它们靶向信使RNA，从而可以调节蛋白质的表达 其他治疗方法无法达到的目标。确定安全有效的交付方式的挑战 RNA治疗剂进入肝外细胞限制了这种有前途的治疗剂的临床部署。 课例如，最近的一项研究使用阿索来减少受影响神经中的PMP 22 mRNA， CMT 1A的大鼠和小鼠模型中的表型。然而，非常高的药物剂量（多次100 mg/kg剂量） 才能看到有益的效果在如此高的剂量下，与阿索治疗相关的毒性风险，如 如血小板减少症和肾功能不全，阻止进一步发展。DTx Pharma发现了一种脂肪 当与siRNA/阿索共价偶联时导致有效递送至多个细胞和组织的酸性基序， 包括坐骨神经（与CMT相关），导致靶基因mRNA表达的有效抑制。 在此，我们建议探索DTx技术是否可以应用于PMP 22靶向siRNA，以纠正其在细胞内的表达。 在CMT 1A小鼠模型中的雪旺细胞中的过表达，为设计 未来的疗效研究。我们将在两个目标中探讨这一点。目标1将筛选一个库（除了 我们迄今为止所筛选的）靶向PMP 22的siRNA候选物在体外使用原代人雪旺氏细胞和 细胞和HEK 293细胞工程化以表达人PMP 22，以鉴定有效且无毒的siRNA， 与DTx基序偶联，并在体外进一步验证。在目标2中，来自目标1的10个最活跃的命中将是 在C3-PMP 22小鼠（CMT 1A模型）中通过静脉内或鞘内给药的两项平行研究中给药， 评估给药途径、安全性和坐骨神经和雪旺细胞中的靶点结合。越 在后续研究中将使用成功的给药途径，以探索剂量范围和作用持续时间， 抑制PMP 22表达至野生型水平。这些研究的数据将帮助我们了解DTx PMP 22是否 siRNA是治疗CMT 1A的一种可行方法，并为第2阶段SBIR拨款提供了基础， 在啮齿类动物中的功效试验、非GLP毒性研究和高等物种中的验证。

PROJECT SUMMARY: Charcot-Marie-Tooth (CMT) disease is the most frequent inherited neuropathy affecting the peripheral nervous system and is characterized by a group of genetically and clinically heterogeneous disorders leading to progressive weakness and atrophy in distal muscles, sensory loss, hyporeflexia and skeletal deformity. CMT type 1A (CMT1A) is the most prevalent form, affecting 1 in 10,000 people, and is associated with a 1.4-Mbp duplication in the chromosome 17p11.2 region, which contains the peripheral myelin protein 22 (PMP22) gene. PMP22 is essential for the structure, development and maintenance of peripheral nerve myelin. PMP22 overexpression prompts cycles of demyelination-remyelination resulting in dysfunction in Schwann cells. Due to the association of PMP22 gene dosage with neuropathic phenotypes, therapeutic strategies are primarily focused on repressing PMP22 overexpression. RNA therapeutics, like antisense oligonucleotides (ASO) and siRNA, are attractive because they target messenger RNA and thus can modulate the expression of protein targets inaccessible to other therapeutic modalities. Challenges identifying safe and effective ways to deliver RNA therapeutics into cells outside the liver have limited the clinical deployment of this promising therapeutic class. For instance, a recent study used an ASO to decrease PMP22 mRNA in affected nerves, improving phenotypes in rat and mouse models of CMT1A. However, very high drug doses (multiple 100 mg/kg doses) were required to see a beneficial effect. At such high doses, the risk of toxicities related to ASO treatment, such as thrombocytopenia and renal dysfunction, preclude further development. DTx Pharma has identified a fatty acid motif that when covalently coupled to siRNA/ASO results in efficient delivery to multiple cells and tissues, including sciatic nerve (relevant for CMT), resulting in potent repression of target gene mRNA expression. Herein, we propose to explore whether DTx technology can be applied to PMP22-targeting siRNAs to correct its overexpression in Schwann cells in a mouse model of CMT1A, providing strong proof of concept for designing future therapeutic efficacy studies. We will explore this in 2 aims. Aim 1 will screen a library (~36 in addition to what we’ve screened to date) of siRNA candidates targeting PMP22 in vitro using both primary human Schwann cells and HEK293 cells engineered to express human PMP22 to identify potent and non-toxic siRNAs that will be conjugated to the DTx motif and further validated in vitro. In Aim 2, the 10 most active hits from aim 1 will be dosed in two parallel studies via intravenous or intrathecal administration in C3-PMP22 mice, a model of CMT1A, to assess dosing route, safety and target engagement in the sciatic nerve and Schwann cells. The more successful dosing route will be utilized in follow-on studies to explore dose range and duration of action for suppressing PMP22 expression to wildtype levels. Data from these studies will help us understand if DTx PMP22 siRNA is a viable approach for treatment of CMT1A and provide the foundation for a phase 2 SBIR grant focused on efficacy trials in rodents, non-GLP toxicity studies and validation in higher species.