Preclinic dose and delivery regime optimization and long-term efficacy evaluation of ribitol treatment for FKRP related dystroglycanopathy

项目负责人/主要研究者（末、首、中）：卢启龙 项目摘要 FKRP基因突变导致肌营养不良，其严重程度差异很大， β-DG糖基化缺陷的生化特征。这种疾病的进展性质导致了 肌肉纤维的持续损失和纤维化的增加，最后肌肉功能的丧失， 呼吸和心脏功能衰竭，导致过早死亡。目前尚无治疗方法 尽管几种实验性疗法正在动物模型中进行测试。 最近，FKRP已被鉴定为核糖醇-5-磷酸转移酶，其使用CDP-核糖醇作为底物。 层粘连蛋白结合双糖链（基质糖链）延伸的底物，这是肌肉生长的关键步骤 完整早期的研究表明，核糖醇补充剂可导致细胞中CDP-核糖醇水平的增加。我们 最近也证明突变的FKRPs保留了足够水平的功能，通过AAV使β-DG糖基化， 用突变型P448 L FKRP作为转基因介导的基因治疗。我们现在已经证明， 补充核糖醇增加了我们临床相关的P448 L突变小鼠模型中基质聚糖的水平， 导致心肌和骨骼肌的肌肉病理学和功能的显著改善。 这为治疗大多数FKRP肌源性疾病开辟了一种新的、高度特异性和临床适用的方法。 营养不良 在本研究中，我们将首先优化核糖醇在fkrp P448 L突变体中的剂量和给药方案， 小鼠模型，通过用管饲给药途径和饮用水给药途径测试一系列剂量。的 通过功能性糖基化水平和营养不良病理学的改善来评价效果。我们将 然后检查选择的核糖醇给药和给药方案的长期疗效（12个月）和安全性， 三种FKRP突变小鼠，P448 L、L276 I纯合子和P448 L/L276 I复合物 在超过90%的患者中携带临床相关突变并代表疾病的杂合子 从轻度到严重的营养不良表型。这些突变株已在McColl 洛克伍德实验室。目的是建立至少一种具有长期疗效的剂量和给药方案， 进一步向临床试验发展。 该提案的目标是建立一个实际可交付的剂量和治疗方案， 在IND申请的临床相关小鼠模型中的长期疗效。 PHS 398/2590（Rev.06/09）

Program Director/Principal Investigator (Last, First, Middle): Lu Qi Long Project Summary Mutations in FKRP gene cause muscular dystrophy with wide variation in severity with characteristic biochemical feature of defect in glycosylation of -DG. The progressive nature of the disease leads to a continuous loss of muscle fibers and increase in fibrosis, and finally loss of muscle function and ultimately failure of respiratory and cardiac functions, leading to early death. Currently no treatment is available although several experimental therapies are being tested in animal models. Recently, FKRP has been identified as a ribitol-5-phosphate transferase using CDP-ribitol as the substrate for the extension of the laminin binding biglycan (matriglycan) on -DG, a critical step for muscle integrity. Earlier study suggests that ribitol supplement can lead to an increase of CDP-ribitol levels in cells. We also demonstrated recently that mutant FKRPs retain sufficient levels of function to glycosylate -DG by AAV mediated gene therapy with mutant P448L FKRP as the transgene. We have now demonstrated that supplement of ribitol increases levels of matriglycan in our clinically relevant P448L mutant mouse model, resulting in significant improvement in muscle pathology and functions in both cardiac and skeletal muscles. This opens a novel, highly specific and clinically applicable means to treat the majority of FKRP muscular dystrophy. In this proposal, we will first optimize dosage and administration regime of ribitol in fkrp P448L mutant mouse model by testing a range of doses with both routes of gavage administration and in drinking water. The effects will be evaluated by levels of functional glycosylation and improvement of dystrophic pathology. We will then examine long-term efficacy (12 months) and safety of the selected dosing and delivery regime of ribitol in three strains of FKRP mutant mice, P448L, L276I homozygote, and P448L/L276I compound heterozygotes bearing clinically relevant mutations in more than 90% patients and representing diseases from mild to severe dystrophic phenotypes. These mutant strains have been established in the McColl Lockwood Laboratory. The aim is to establish at least one dose and delivery regime with long-term efficacy for further development towards clinic trials. The goal of this proposal is to establish a practically deliverable dosing and treatment regime with long- term therapeutic efficacy in clinically relevant mouse models for IND application. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page