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.

项目概要：腓骨肌萎缩症 (CMT) 病是影响周围神经系统的最常见的遗传性神经病系统，其特征是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。 1A 型 (CMT1A) 是最常见的形式，影响万分之一的人，并与 1.4-Mbp 相关染色体 17p11.2 区域出现重复，该区域包含外周髓磷脂蛋白 22 (PMP22) 基因。 PMP22 对于周围神经髓磷脂的结构、发育和维护至关重要。过度表达会促进脱髓鞘-髓鞘再生循环，导致雪旺细胞功能障碍。 PMP22 基因剂量与神经病表型的关系，治疗策略主要是专注于抑制 PMP22 过度表达的 RNA 疗法，如反义寡核苷酸 (ASO) 和 siRNA 很有吸引力，因为它们靶向信使 RNA，因此可以调节蛋白质的表达其他治疗方式无法达到的目标确定安全有效的治疗方法的挑战。 RNA 疗法进入肝外细胞限制了这种有前途的疗法的临床部署例如，最近的一项研究使用 ASO 来减少受影响神经中的 PMP22 mRNA，从而改善症状。 CMT1A 大鼠和小鼠模型中的表型然而，药物剂量非常高（多次 100 mg/kg 剂量）。需要在如此高的剂量下观察到与 ASO 治疗相关的毒性风险，例如由于血小板减少和肾功能障碍，DTx Pharma已确定了脂肪的进一步发展。酸性基序与 siRNA/ASO 共价偶联时可有效递送至多个细胞和组织，包括坐骨神经（与 CMT 相关），从而有效抑制靶基因 mRNA 表达。在此，我们建议探索DTx技术是否可以应用于PMP22靶向siRNA以纠正其 CMT1A 小鼠模型中施万细胞的过度表达，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 2 个目标中对此进行探索。目标 1 将筛选一个文库（除了 36 个）。使用原代人雪旺体外靶向 PMP22 的 siRNA 候选物细胞和 HEK293 细胞被设计为表达人 PMP22，以鉴定有效且无毒的 siRNA，从而与 DTx 基序缀合并在体外进一步验证，目标 1 中的 10 个最活跃的命中将被标记。在两项平行研究中通过静脉内或鞘内给药对 C3-PMP22 小鼠（CMT1A 模型）进行给药，评估坐骨神经和雪旺细胞的给药途径、安全性和靶点参与度。成功的给药途径将用于后续研究，以探索剂量范围和作用持续时间将 PMP22 表达抑制至野生型水平，这些研究的数据将帮助我们了解 DTx PMP22 是否存在。 siRNA 是治疗 CMT1A 的可行方法，并为 2 期 SBIR 资助重点提供基础啮齿动物功效试验、非 GLP 毒性研究和高等物种验证。