Intravenous Protein Therapy for Myotonic Dystrophy Type 1

描述（由申请人提供）：我们的目标是开发用于1型肌强直性营养不良（DM1）患者的静脉输送重组肌盲1 （MBNL1）。DM1是成人最常见的肌肉萎缩症，是由DMPK基因3'非翻译区CTG大量扩增引起的（1-3）。虽然DMPK和邻近的Six5基因的功能受到CTG扩增的负面影响，但DMPK活性的缺乏并不能完全解释DM1中观察到的表型；包括肌肉萎缩和肌强直、胰岛素抵抗、睾丸萎缩、皮肤肿瘤、心律失常和认知缺陷（4- 8）。随后的研究表明，在转录的DMPK mRNA中，CUG的大量扩增会迅速结合、隔离和失活MBNL1蛋白，MBNL1蛋白是一种mRNA剪接因子，可从mRNA模板中去除胎儿外显子（9- 13）。通过polyCUG扩增或基因消融MBNL1使MBNL1失活，会导致成人分化组织中胎儿蛋白的不适当表达（9,14 - 17）。虽然MBNL1的隔离显然不能为DM1提供一个统一的解释，但来自转基因小鼠和果蝇模型以及患者来源细胞的研究的证据支持这样的观点，即DM1的症状部分取决于与环境中MBNL1蛋白供应相关的CUG扩增RNA的化学计量学（18,19,20）。DM1学界的共识是，任何恢复MBNL1蛋白mRNA剪接可用性的方法都将构成DM1的治疗方法（21,20）。目前考虑的DM1治疗方案包括[1]小分子诱导内源性MBNL1过表达，[2]通过小分子和核苷酸治疗破坏polyCUG- MBNL1关联，[3]通过基因治疗转基因MBNL1过表达，[4]直接静脉注射外源性MBNL1。3E10是一种鼠源性单克隆抗体，可穿透活细胞并定位到细胞核，而不会对靶细胞造成明显损伤（22,23）。3E10的单链Fv片段（Fv3E10）具有原始单克隆抗体的所有细胞穿透能力，并且过氧化氢酶、肌营养不良蛋白、HSP70和p53等蛋白在与Fv3E10偶联后保持其活性（24-27）。ENT2核苷酸清除物转运体在骨骼肌和癌细胞中富集，并介导Fv3E10和Fv3E10偶联物的细胞穿透能力（28）。鉴于Fv3E10对骨骼肌的亲和力以及Fv3E10偶联物维持其各自活性的能力，基于Fv3E10的疗法将代表一种治疗多种肌病的通用方法，包括DM1、DM2、杜氏肌营养不良症和埃莫里-德雷弗斯综合征。我们寻求两年的资金，将3E10和MBNL1转化为DM1的商业上可行的产品。我们将化学或基因偶联3E10到MBNL1，在DM1细胞系中测试纯化的材料，将纯化的材料注射到DM1小鼠模型中，并评估对疾病终点的任何纠正。为了执行这一建议，我们收集了适当的技术，生物技术行业的承诺，以及DM1科学和患者倡导社区的专业知识和资源。该提案的成功结论将证明进一步的产品优化，包括检查截断和/或人性化的3E10-MBNL1以及确定最佳制造工艺。最终的产品概念将进行进一步的疗效、药理学和毒理学研究、扩大GLP的生产、额外的新药开发前药理学和毒理学研究，并在FDA批准后进行1期和2期临床试验。

DESCRIPTION (provided by applicant): Our objective is to develop intravenously delivered recombinant Muscle Blind 1 (MBNL1) for patients with Myotonic Dystrophy Type 1 (DM1). DM1 is the most common muscular dystrophy of adulthood and is caused by a large CTG expansion in the 3' untranslated region of the DMPK gene (1-3). Although the function of DMPK and the neighboring Six5 gene are negatively affected by the CTG expansion, the lack of DMPK activity does not fully account for the observed phenotype in DM1; including muscle wasting and myotonia, insulin resistance, testicular atrophy, cutaneous tumors cardiac arrhythmia and cognition defects (4- 8). Subsequent studies have shown that the large CUG expansion within the transcribed DMPK mRNA avidly binds, sequesters and inactivates the MBNL1 protein, an mRNA splicing factor that removes fetal exons from mRNA templates (9- 13). The inactivation of MBNL1 by polyCUG expansions or through genetic ablation of MBNL1 results in the inappropriate expression of fetal proteins in adult differentiated tissues (9, 14- 17). Though sequestration of MBNL1 clearly cannot provide a unitary explanation for DM1, evidence from transgenic mouse and fly models, and studies of patient-derived cells, supports the idea that symptoms of DM1 are partly determined by the stoichiometry of CUG expansion RNA in relation to ambient supplies of MBNL1 protein (18, 19, 20). The consensus in the DM1 community is that any approach that restores the availability of MBNL1 proteins for mRNA splicing would constitute a therapy for DM1 (21, 20). Treatment options being considered for DM1 include [1] small-molecule induced overexpression of endogenous MBNL1, [2] disruption of the polyCUG- MBNL1 association through small-molecule and nucleotide-based therapies, [3] transgenic overexpression of MBNL1 via gene therapy, and [4] direct intravenous application of exogenous MBNL1. 3E10 is a murine-derived monoclonal antibody that penetrates living cells and localizes to the cell nucleus without apparent injury to target cells (22, 23). A single chain Fv fragment of 3E10 (Fv3E10) possesses all the cell penetrating capabilities of the original monoclonal antibody and proteins such as catalase, dystrophin, HSP70 and p53 retain their activity following conjugation to Fv3E10 (24-27). The ENT2 nucleotide scavenger transporter is enriched in skeletal muscle and cancer cells and mediates the cell-penetrating ability of Fv3E10 and Fv3E10 conjugates (28). Given the affinity of Fv3E10 for skeletal muscle and the ability of Fv3E10 conjugates to maintain their respective activities, Fv3E10-based therapies would represent a versatile approach to treat many myopathies, including DM1, DM2, Duchenne muscular dystrophy and Emery-Dreifuss syndrome. We seek funding for two years to translate 3E10 and MBNL1 into a commercially viable product for DM1. We will chemically or genetically conjugate 3E10 to MBNL1, test the purified material in DM1 cell lines, inject the purified material into DM1 mouse models and evaluate any correction of the disease endpoints. To execute this proposal we have gathered the appropriate technology, the commitment from the biotechnology industry, and the expertise and resources of the DM1 scientific and patient advocacy community. Successful conclusion of this proposal will justify further product optimization, including examination of truncated and/or humanized 3E10-MBNL1 and determination of the optimal manufacturing process. The final product concept will undergo further efficacy, pharmacology and toxicology studies, scaled-up GLP production, additional pre- IND pharmacology and toxicology studies, and upon FDA approval the development of phase 1 and 2 clinical trials. PUBLIC HEALTH RELEVANCE: Myotonic dystrophy is the most common muscular dystrophy of adults for which there are no effective therapies. We will test if a muscle-targeted Muscleblind protein therapy will alleviate the spliceopathy in DM1 mouse models.