Identification of Enhancers of Therapeutic Exon Skipping for DMD

DMD 治疗性外显子跳跃增强子的鉴定

• 批准号: 7938694
• 负责人: M CARRIE MICELI
• 金额: $ 49.77万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938694
• 关键词: Address; Adjuvant; Affect; Antisense Oligonucleotides; Applications Grants; Area; Biological Assay; Birth; Cells; Cellular biology; Childhood; Clinical; Clinical Research; Clinical Trials; Connective Tissue; DNA; Data; Detection; Development; Disease; Drug usage; Duchenne muscular dystrophy; Dystrophin; Enhancers; Ethics; Excision; Exclusion; Exons; FDA approved; Fibroblasts; Genes; Genomics; Goals; Hereditary Disease; Human; In Vitro; Inherited; Intramuscular; Lead; Libraries; Life; Mediating; Messenger RNA; Methods; Modality; Modeling; Molecular Genetics; Mus; Muscle; Muscle Fibers; Muscle function; Muscular Dystrophies; Mutation; Myoblasts; Myopathy; Outcome; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase I Clinical Trials; Population; Predictive Value; Process; Production; Proteins; RNA Splicing; Reading Frames; Relative (related person); Reporter; Research; Research Personnel; Resources; Rest; Screening procedure; Skin; Small RNA; Specificity; Structure; Structure-Activity Relationship; Testing; Therapeutic; Tissues; Transcript; Treatment Efficacy; base; bone; effective therapy; falls; functional gain; functional improvement; functional restoration; gene function; gene therapy; high throughput screening; improved; in vitro Assay; in vivo; in vivo Model; interest; male; mouse model; muscular dystrophy mouse model; mutant; novel; novel therapeutic intervention; novel therapeutics; pre-clinical; programs; small molecule; success; tool

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04): Clinical Research, and specific topic: 04-AR-106 Cellular, Molecular and Genetic Therapies for Rare Inherited Diseases of Muscle, Skin and Connective Tissue and Bone. Duchenne Muscular Dystrophy (DMD) is the most common lethal genetic disease of childhood, occurring in 1 in every 3500 live male births. Based on the structure of dystrophin, as well as the mutational profile of patients with DMD, this disease is potentially amenable to an exon skipping therapeutic strategy for the majority of those affected. Most of the mutations in DMD result from DNA deletions between exons 44-55. Such deletions usually lead to out of frame transcripts, which result in lack of dystrophin protein production. Recent strategies aimed at anti-sense oligonucleotide (AON) directed removal of specific exons during processing of the dystrophin transcript have succeeded in restoring reading frame to a fraction of the transcripts, leading to some production of partially functional dystrophin protein. While early stage clinical trials are underway, the ultimate success of this therapeutic approach rests on overcoming the inefficiencies of exon skipping from systemically administered AON. Best estimates indicate that 30-60% of wild-type levels of (skipped) dystrophin will be required to functionally compensate for loss of dystrophin at a significant level. Early trial data, while promising, indicate that even local IM delivery of AON falls short of inducing such levels, yielding only 3-35% of normal dystrophin amount. It is anticipated that systemic delivery of AON may be even more inefficient. Therefore, identification of compounds that increase the efficacy of exon skipping represents a viable approach toward increasing replacement of dystrophin to functionally relevant levels. We have implemented high throughput screens to identify small molecule compounds capable of perturbing the splicing machinery to favor exonic exclusion in the context of targeted AON. The program has identified 20 compounds, most of which are already FDA approved drugs that now need to be assessed in the context of various relevant human mutations and in mouse models. Here we set up a multi-PI program to bring together experts in cell biology, genomics, and muscular dystrophy to identify drugs for use as adjuvants to AON-mediated exon skipping clinical trials. Ultimately, the program will include the search for novel structures and medicinal chemistry, but this is outside the scope of this proposal, which is intended to be completed within two years. Thus, the project is highly responsive to the Challenge Topic, which states "novel therapeutic approaches offer the possibility of restoring function to a defective gene or compensating for the loss of gene function. These approaches are potentially quite powerful and could lead to significant advances in the treatment of diseases of muscle and other tissues. The goal of the projects will be to find creative approaches to overcome some of the current technical obstacles.... Areas of interest include ..., methods for editing gene products in vivo, such as exon-skipping antisense oligonucleotides and small RNAs." Here we propose to create immortalized DMD patient derived fibroblasts, which are readily and reproducibly inducible to myotubes and to develop quantitative methods for detecting mutant and skipped DMD mRNA products in these cells. Once developed, 20 lead compounds will be screened for their efficacy and specificity in facilitating AON exon skipping. Assessment of the activity of these same compounds on myoblast/myotube cultures from mouse DMD models mdx and mdx.4Cv will enable a further assessment of specificity and identify candidates which we will test in the mdx or mdx.4Cv models in vivo. The value of such an approach is threefold: 1) Creation, development and immortalization of patient derived cells inducible to muscle lineage will create a much needed resource that can be distributed and utilized by muscular dystrophy researchers for preclinical assessment of multiple potential therapeutics. Because many emerging treatments are specific to human muscular dystrophy mutations, availability of preclinical assessment tools based in human cells with relevant mutations are lacking, and represent a technical and ethical barrier toward moving clinical trials forward. 2) Screening lead compounds on these cells alongside mouse myoblast/myotubes and in mouse models in vivo will enable us to validate the predictive value of the in vitro assays on in vivo outcome in DMD models. Thus, we will have created a process for screening and validating compounds emerging from larger screens or Structure Activity Relationship analysis (SAR). 3) Implementing these screens on 20 lead compounds has the potential to validate their activity, determine their specificity, and identify potential target DMD populations. Identification of a compound that can improve efficacy of AON directed exon skipping has the potential to move this therapeutic modality from proof of principle of dystrophin production to therapeutic efficacy resulting in functional improvement, thus rendering exon skipping a practical and effective treatment for DMD. While DMD is the target in this application, we note that the methods and the approach that we are taking will make the compounds identified generally useful in other disorders amenable to an exon skipping strategy. Exon skipping is a promising emerging therapy for Duchene Muscular Dystrophy (DMD), the most common lethal genetic disease of childhood. Early clinical trial results predict that this approach can restore the missing dystrophin protein to muscle in DMD patients, but at levels insufficient to result in functional gain. Identification of compounds that can improve the efficacy of exon skipping has the potential to move this therapeutic modality from proof of principle to therapeutic efficacy, rendering exon skipping a practical and effective treatment for DMD.

描述（由申请人提供）：本申请涉及广泛的挑战领域 (04)：临床研究，具体主题：04-AR-106 肌肉、皮肤、结缔组织和骨骼的罕见遗传性疾病的细胞、分子和基因疗法。杜氏肌营养不良症 (DMD) 是儿童期最常见的致命遗传性疾病，每 3500 名活产男性新生儿中就有 1 人患有杜氏肌营养不良症 (DMD)。根据肌营养不良蛋白的结构以及 DMD 患者的突变特征，对于大多数受影响的患者来说，这种疾病可能适合外显子跳跃治疗策略。 DMD 的大多数突变是由外显子 44-55 之间的 DNA 缺失引起的。这种缺失通常会导致转录物超出框架，从而导致抗肌营养不良蛋白产生不足。最近针对抗肌营养不良蛋白转录物加工过程中反义寡核苷酸（AON）定向去除特定外显子的策略已成功恢复部分转录物的阅读框，从而产生部分功能性肌营养不良蛋白。虽然早期临床试验正在进行中，但这种治疗方法的最终成功取决于克服系统施用 AON 时外显子跳跃的低效性。最佳估计表明，需要野生型水平（跳过的）肌营养不良蛋白的 30-60% 才能在功能上补偿显着水平的肌营养不良蛋白的损失。早期试验数据虽然有希望，但表明即使局部 IM 递送 AON 也无法诱导这种水平，仅产生正常肌营养不良蛋白量的 3-35%。预计 AON 的全身给药可能效率更低。因此，鉴定提高外显子跳跃功效的化合物代表了将肌营养不良蛋白的替代增加到功能相关水平的可行方法。我们已经实施了高通量筛选来识别能够扰乱剪接机制的小分子化合物，从而有利于在靶向 AON 的背景下排除外显子。该计划已鉴定出 20 种化合物，其中大部分已是 FDA 批准的药物，现在需要在各种相关的人类突变和小鼠模型中进行评估。在这里，我们建立了一个多 PI 计划，汇集细胞生物学、基因组学和肌营养不良症方面的专家，以确定可用作 AON 介导的外显子跳跃临床试验佐剂的药物。最终，该计划将包括寻找新颖的结构和药物化学，但​​这超出了本提案的范围，该提案计划在两年内完成。因此，该项目对挑战主题高度敏感，该主题指出“新颖的治疗方法提供了恢复缺陷基因功能或补偿基因功能丧失的可能性。这些方法可能非常强大，并可能导致重大进展这些项目的目标是寻找创造性的方法来克服当前的一些技术障碍……感兴趣的领域包括……体内基因产物的编辑方法，例如作为外显子跳跃反义寡核苷酸和小RNA。”在这里，我们建议创建永生化的 DMD 患者来源的成纤维细胞，这些成纤维细胞易于且可重复地诱导肌管，并开发用于检测这些细胞中突变和跳过的 DMD mRNA 产物的定量方法。一旦开发完成，将筛选 20 种先导化合物，以确定它们在促进 AON 外显子跳跃方面的功效和特异性。评估这些相同化合物对小鼠 DMD 模型 mdx 和 mdx.4Cv 的成肌细胞/肌管培养物的活性，将能够进一步评估特异性并确定我们将在体内 mdx 或 mdx.4Cv 模型中测试的候选化合物。这种方法的价值有三重：1）可诱导肌肉谱系的患者来源细胞的创建、发育和永生化将创造一种急需的资源，可供肌营养不良研究人员分配和利用，以对多种潜在疗法进行临床前评估。由于许多新兴治疗方法都是针对人类肌营养不良症突变的，因此缺乏基于具有相关突变的人类细胞的临床前评估工具，这对推进临床试验构成了技术和伦理障碍。 2) 在这些细胞上与小鼠成肌细胞/肌管以及小鼠体内模型中筛选先导化合物将使我们能够验证体外测定对 DMD 模型体内结果的预测价值。因此，我们将创建一个筛选和验证从较大屏幕或结构活性关系分析 (SAR) 中出现的化合物的流程。 3) 对 20 种先导化合物进行这些筛选有可能验证它们的活性、确定它们的特异性并确定潜在的目标 DMD 群体。鉴定出能够提高 AON 定向外显子跳跃功效的化合物有可能将这种治疗方式从抗肌营养不良蛋白产生原理证明转向导致功能改善的治疗效果，从而使外显子跳跃成为一种实用且有效的 DMD 治疗方法。虽然 DMD 是本申请的目标，但我们注意到我们正在采取的方法和途径将使所鉴定的化合物普遍适用于其他适合外显子跳跃策略的疾病。外显子跳跃是治疗杜氏肌营养不良症 (DMD) 的一种有前景的新兴疗法，DMD 是儿童最常见的致命遗传病。早期临床试验结果预测，这种方法可以恢复 DMD 患者肌肉中缺失的肌营养不良蛋白，但水平不足以实现功能增益。鉴定出能够提高外显子跳跃疗效的化合物有可能将这种治疗方式从原理证明转变为治疗效果，使外显子跳跃成为一种实用且有效的 DMD 治疗方法。