Identification of Enhancers of Therapeutic Exon Skipping 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、结缔组织和骨骼罕见遗传性疾病的细胞、分子和基因治疗。杜氏肌营养不良症（DMD）是儿童时期最常见的致命性遗传疾病，每3500名活产男婴中就有1人患有此病。基于肌营养不良蛋白的结构，以及DMD患者的突变特征，这种疾病可能适用于大多数患者的外显子跳过治疗策略。DMD的大多数突变是由44-55外显子之间的DNA缺失引起的。这种缺失通常会导致框外转录本，从而导致肌营养不良蛋白产生的缺乏。最近针对反义寡核苷酸（AON）定向去除抗肌营养不良蛋白转录本加工过程中的特定外显子的策略已经成功地将阅读框恢复到转录本的一小部分，从而导致部分功能的抗肌营养不良蛋白的产生。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给药AON的外显子跳跃的低效率。最好的估计表明，将需要30-60%的野生型（跳过的）肌营养不良蛋白水平在功能上补偿显著水平的肌营养不良蛋白的损失。早期的试验数据虽然很有希望，但表明即使局部IM给药AON也不能达到这一水平，仅产生正常肌营养不良蛋白量的3-35%。预计系统提供AON的效率可能更低。因此，鉴定增加外显子跳变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，以鉴定能够干扰剪接机制的小分子化合物，从而在靶向AON的情况下有利于外显子排除。该项目已经确定了20种化合物，其中大部分已经是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项目，汇集了细胞生物学、基因组学和肌肉萎缩症方面的专家，以确定用于aon介导的外显子跳过临床试验的佐剂药物。最终，该项目将包括寻找新的结构和药物化学，但这超出了本提案的范围，该提案计划在两年内完成。因此，该项目高度响应挑战主题，该主题指出“新的治疗方法提供了恢复缺陷基因功能或补偿基因功能损失的可能性。”这些方法可能非常强大，并可能导致肌肉和其他组织疾病的治疗取得重大进展。这些项目的目标将是找到创造性的方法来克服目前的一些技术障碍....感兴趣的领域包括……在体内编辑基因产物的方法，如外显子跳跃反义寡核苷酸和小rna。”在这里，我们建议创建永活的DMD患者来源的成纤维细胞，它很容易和可重复地诱导到肌管，并开发定量方法来检测这些细胞中的突变和跳过的DMD mRNA产物。一旦开发成功，20个先导化合物将被筛选其促进AON外显子跳跃的功效和特异性。对小鼠DMD模型mdx和mdx的成肌细胞/肌管培养物的活性评估。4Cv将能够进一步评估特异性并确定我们将在mdx或mdx中进行测试的候选药物。体内4Cv模型。这种方法的价值有三个方面：1)创造、发展和永生化诱导肌肉谱系的患者来源细胞将创造一个非常需要的资源，可以被肌肉萎缩症研究人员分配和利用，用于多种潜在治疗方法的临床前评估。由于许多新出现的治疗方法是针对人类肌肉萎缩症突变的，缺乏基于具有相关突变的人类细胞的临床前评估工具的可用性，这代表了推动临床试验向前发展的技术和伦理障碍。2)在小鼠成肌细胞/肌管和小鼠体内模型中筛选这些细胞上的先导化合物将使我们能够验证体外试验对DMD模型中体内结果的预测价值。因此，我们将创建一个筛选和验证从大屏幕或结构活性关系分析（SAR）中出现的化合物的过程。3)在20个先导化合物上实施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。鉴定出一种能够提高AON定向外显子跳脱疗效的化合物，有可能将这种治疗方式从证明肌营养不良蛋白产生的原理转变为导致功能改善的治疗效果，从而使外显子跳脱成为一种实用而有效的治疗DMD的方法。虽然DMD是本应用的靶标，但我们注意到，我们所采用的方法和方法将使鉴定的化合物在其他疾病中普遍有用，适用于外显子跳跃策略。外显子跳跃是一种很有前途的治疗杜氏肌营养不良症（DMD）的新兴疗法，DMD是儿童最常见的致命遗传性疾病。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，但其水平不足以导致功能增加。识别能够提高外显子跳变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，使外显子跳变成为一种实用有效的DMD治疗方法。

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.