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: 7821508
• 负责人: M CARRIE MICELI
• 金额: $ 49.78万
• 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/7821508
• 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）：此申请涉及广泛的挑战领域（04）：临床研究和特定主题：106-ar-106细胞，分子和基因疗法，用于罕见的肌肉，皮肤和结缔组织和结缔组织和骨骼的罕见遗传疾病。 Duchenne肌肉营养不良（DMD）是最常见的儿童致命遗传疾病，每3500名活着的男性出生中有1个。基于肌营养不良蛋白的结构以及DMD患者的突变特征，该疾病可能适合大多数受影响的外显子跳过治疗策略。 DMD中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框架转录本，从而导致缺乏肌营养不良蛋白的产生。旨在抗敏感性寡核苷酸（AON）在处理肌营养不良蛋白转录本期间指示特定外显子的最新策略已成功地将阅读框恢复到了一小部分转录本，从而导致某些功能性障碍性障碍蛋白的产生。尽管正在进行早期临床试验，但这种治疗方法的最终成功取决于克服从系统地管理的AON跳过外显子的效率低下。最佳估计表明，将需要30-60％的野生型（跳过）肌营养不良蛋白水平在功能上弥补显着水平的肌营养不良蛋白的损失。早期的试验数据虽然有希望，但表明即使是局部的AON递送也没有诱导此类水平，仅产生正常肌营养不良蛋白量的3-35％。预计AON的全身交付可能更效率更低。因此，鉴定增加外显子跳动功效的化合物代表了一种可行的方法，可以将肌营养不良蛋白替代为功能相关的水平。我们已经实施了高吞吐量屏幕，以识别能够在靶向AON的背景下扰动剪接机械的小分子化合物。该程序已经确定了20种化合物，其中大多数已经是FDA批准的药物，这些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，以将细胞生物学，基因组学和肌营养不良症的专家汇集在一起​​，以识别药物作为AN介导的外显子跳过临床试验的辅助药物。最终，该计划将包括寻找新颖的结构和药物化学反应，但这超出了该提案的范围，该提案旨在在两年内完成。因此，该项目对挑战主题具有很高的反应，该主题说：“新颖的治疗方法为恢复功能恢复功能或补偿基因功能丧失的可能性。这些方法可能相当强大。可能会很强大，并可能导致肌肉疾病和其他组织的疾病的治疗方面的疾病治疗，以构建创造性的方法。体内，例如外显子的反义寡核苷酸和小RNA。”在这里，我们建议创建永生的DMD患者衍生的成纤维细胞，这些成纤维细胞容易且可重复地诱导肌管，并开发用于检测这些细胞中突变体和跳过DMD mRNA产物的定量方法。一旦开发，将筛选20种铅化合物，以促进Aon Exon跳过的功效和特异性。评估这些相同化合物对小鼠DMD模型MDX和MDX的成肌细胞/成肌管培养物的活性。4CV可以进一步评估特异性并确定我们将在MDX或MDX.4CV模型中测试的候选者。这种方法的价值是三个方面：1）肌肉谱系诱导的患者衍生细胞的创造，发育和永生化将创造出急需的资源，可以通过肌肉营养不良研究人员进行分配和利用，以评估多种潜在治疗剂的临床前评估。由于许多新兴治疗是针对人类肌肉营养不良突变的特异性，因此缺乏基于具有相关突变的人类细胞中的临床前评估工具，并且代表了向前临床试验的技术和道德障碍。 2）在这些细胞上与小鼠成肌细胞/肌动物以及体内小鼠模型一起筛选铅化合物将使我们能够在DMD模型中验证体内体内结果的体外测定的预测值。因此，我们将创建一个过程，用于筛选和验证从较大的屏幕或结构活动关系分析（SAR）出现的化合物。 3）在20种铅化合物上实施这些筛选有可能验证其活动，确定其特异性并确定潜在的目标DMD种群。鉴定可以提高AON定向外显子跳过功效的化合物有可能将这种治疗方式从肌营养不良蛋白产生原理证明转变为治疗功效，从而改善功能，从而使外显子跳过实用和有效的DMD治疗。虽然DMD是本应用程序中的目标，但我们注意到我们正在采用的方法和方法将使化合物通常在外显子跳过策略的其他疾病中有用。外显子跳过是一种有前途的新兴疗法，用于杜钦肌营养不良（DMD），这是最常见的儿童致命遗传疾病。早期的临床试验结果预测，这种方法可以恢复DMD患者的肌肉缺失的肌营养素蛋白，但水平不足以导致功能增长。鉴定可以提高外显子跳动功效的化合物具有将这种治疗方式从原理证明转移到治疗功效的潜力，从而使外显子跳过了对DMD的实用有效治疗。