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

项目总结： Charcot-Marie-Tooth(CMT)病是影响周围神经的最常见的遗传性神经病 系统，特点是一组遗传和临床上不同的疾病导致 远端肌肉进行性虚弱和萎缩，感觉丧失，反射减退和骨骼畸形。CMT A型(CMT1A型)是最常见的形式，每10,000人中就有1人受到影响，并与1.4-MBP有关 染色体17p11.2区域的重复，该区域包含外周髓鞘蛋白22(PMP22)基因。 PMP22对周围神经髓鞘的结构、发育和维持是必不可少的。PMP22 过表达促使脱髓鞘-再髓鞘循环，导致雪旺细胞功能障碍。由于 PMP22基因剂量与神经病变表型的关系，治疗策略主要是 专注于抑制PMP22的过度表达。RNA疗法，如反义寡核苷酸(ASO)和 SiRNA是有吸引力的，因为它们以信使rna为靶标，因此可以调节蛋白质的表达。 其他治疗方式无法达到的靶点。确定安全有效的交付方式面临的挑战 进入肝脏外细胞的rna疗法限制了这种有希望的疗法的临床应用。 班级。例如，最近的一项研究使用ASO来降低受影响神经中的PMP22 mRNA，从而改善 CMT1a大鼠和小鼠模型的表型。然而，很高的药物剂量(100毫克/千克的倍数) 都需要看到有益的效果。在如此高的剂量下，与ASO治疗相关的毒性风险，如 由于血小板减少和肾功能不全，阻碍了进一步的发展。DTX Pharma发现了一名胖子 当与siRNA/ASO共价偶联时导致高效地递送到多个细胞和组织的酸性基序， 包括坐骨神经(与CMT相关)，导致对靶基因mRNA表达的有效抑制。 在这里，我们建议探索DTX技术是否可以应用于PMP22靶向siRNAs以纠正其 CMT1a小鼠模型中雪旺细胞的高表达，为设计提供了强有力的概念证明 未来的治疗效果研究。我们将在两个目标中探索这一点。目标1将筛选一个图书馆(除了~36个 到目前为止我们已经筛选的)体外靶向PMP22的siRNA候选者使用原代人类Schwann 经过改造的细胞和HEK293细胞可以表达人PMP22，以识别有效且无毒的siRNA，从而 与DTX基序结合，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 在两个平行的研究中，通过静脉或鞘内给药，C3-PMP22小鼠，CMT1A模型， 评估坐骨神经和雪旺细胞的给药途径、安全性和靶向接触。越多 成功的给药途径将用于后续研究，以探索治疗以下疾病的剂量范围和持续时间 将PMP22表达式抑制到通配类型级别。这些研究的数据将帮助我们了解DTX PMP22 SiRNA是治疗CMT1A的一种可行方法，并为重点关注SBIR的第二阶段赠款奠定了基础 在啮齿动物上进行药效试验，在高等物种中进行非GLP毒性研究和验证。

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.