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

开发用于治疗 1A 型腓骨肌萎缩症的 PMP22 siRNA 缀合物

基本信息

批准号：
10158135
负责人：
Arthur Thomas Suckow
金额：
$ 38.79万
依托单位：
DTX PHARMA, LLC
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10158135
关键词：
17p11.2 Adult Affect Anatomy Animals Antisense Oligonucleotides Atrophic Automobile Driving Biological Biological Assay Biological Markers Cells Charcot-Marie-Tooth Disease Chromosomes Clinic Clinical Coupled Data Deformity Demyelinations Development Disease Disease Outcome Disease model Distal Dose Drug Delivery Systems Evaluation Fatty Acids Foot Deformities Foundations Functional disorder Future Gene Dosage Gene Targeting Generations Genes Grant Human Hyporeflexia In Vitro Inherited Intrathecal Injections Intravenous Leg Libraries Life Liver Maintenance Measurement Mediating Messenger RNA Modality Modeling Motor Mus Muscle Muscle Weakness Muscular Atrophy Myelin Nerve Neural Conduction Neurons Neuropathy Numbness Occupational Therapy Onset of illness PMP22 gene Patients Peripheral Nerves Peripheral Nervous System Peripheral Nervous System Diseases Pharmaceutical Preparations Pharmacologic Substance Phase Phenotype Physical therapy Property Protein Overexpression Proteins Rattus Repression Risk Rodent Route Safety Schwann Cells Sensory Small Business Innovation Research Grant Small Interfering RNA Structure Technology Testing Therapeutic Thrombocytopenia Tissues Toxic effect Transfection Transgenes Transgenic Mice Treatment Efficacy United States Validation afferent nerve arm base cellular engineering cytotoxicity design efficacy study efficacy trial experimental study hereditary neuropathy improved in vivo in vivo evaluation intravenous injection kidney dysfunction knock-down mRNA Expression mouse model novel therapeutic intervention novel therapeutics overexpression protein expression remyelination sciatic nerve screening siRNA delivery skeletal therapeutic RNA therapeutic siRNA uptake

项目摘要

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.

项目摘要： Charcot-Marie-Tooth（CMT）疾病是影响周围神经的最常见的神经病系统的特征是一组遗传和临床异质性疾病圆盘肌肉，感觉丧失，屈光度和骨骼畸形的进行性无力和萎缩。 CMT 1A型（CMT1A）是最普遍的形式，影响了10,000人中的1人，并且与1.4-Mbp相关在17p11.2染色体中的复制区域，其中包含外周髓蛋白蛋白22（PMP22）基因。 PMP22对于周围神经髓磷脂的结构，开发和维持至关重要。 PMP22 过表达促使脱髓鞘螺旋的循环导致雪旺细胞功能障碍。由于 PMP22基因剂量与神经性表型的关联，治疗策略是主要的专注于抑制PMP22的过表达。 RNA疗法，例如反义寡核苷酸（ASO）和 siRNA很有吸引力，因为它们靶向使者RNA，因此可以调节蛋白质的表达其他治疗方式无法获得的目标。挑战确定安全有效的方法肝脏外部的RNA疗法限制了这种有希望的治疗的临床部署班级。例如，最近的一项研究使用ASO减少受影响神经中的PMP22 mRNA，从而改善 CMT1A的大鼠和小鼠模型中的表型。但是，药物剂量很高（多种100 mg/kg剂量）被要求看到有益的效果。在如此高剂量的情况下，与ASO治疗有关的毒性风险作为血小板减少症和肾功能障碍，无法进一步发展。 DTX Pharma已经确定了脂肪酸基序是在共价耦合到siRNA/ASO时会导致有效地递送到多个细胞和组织时，包括坐骨神经（与CMT相关），导致靶基因mRNA表达的潜在表达。本文中，我们建议探索是否可以将DTX技术应用于pmp22靶向sirnas以纠正其 CMT1A的鼠标模型中的Schwann细胞中的过表达，为设计提供了强大的概念证明未来的治疗功效研究。我们将以2个目标探讨这一点。 AIM 1还将筛选一个库（〜36除了迄今为止，我们已经筛选的siRNA候选者使用两个主要的人类Schwann靶向PMP22 细胞和HEK293细胞设计用于表达人PMP22的细胞，以鉴定潜在的和无毒的siRNA 将其连接到DTX基序并进一步验证体外验证。在AIM 2中，AIM 1的10个最活跃的命中将是通过在C3-PMP22小鼠中通过静脉内或鞘内给药的两项平行研究，是CMT1A的模型，评估剂量途径，安全性和目标参与坐骨神经和雪旺细胞。更多在后续研究中，成功的给药路线将用于探索剂量范围和作用持续时间抑制PMP22表达为野生型水平。这些研究的数据将有助于我们了解DTX PMP22是否 siRNA是一种可行的CMT1A治疗方法，并为以2期SBIR赠款奠定了基础在啮齿动物的有效试验中，非GLP毒性研究和较高物种的验证。