Dual-vector mediated mini-dystrophin restoration of function in mdx model of DMD

双载体介导的微型肌营养不良蛋白在 DMD mdx 模型中恢复功能

• 批准号: 8770895
• 负责人: Jerry Roy Mendell
• 金额: $ 21.98万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8770895
• 关键词: Adhalin; Adverse effects; Becker Muscular Dystrophy; Binding; Binding Sites; Biodistribution; Biological Assay; Blood Circulation; Blood Vessels; C-terminal; Clinical; Clinical Research; Clinical Trials; Cysteine; Cysteine-Rich Domain; DNA; Data; Defect; Dependovirus; Dose; Dystrophin; Elements; Epitopes; Follistatin; Foundations; Gene Expression; Gene Transfer; Generations; Genes; Genetic Recombination; Goals; Grant; Health; Immune; Immunofluorescence Immunologic; Injury; Interferon Type II; Intramuscular; Intramuscular Injections; Introns; Isolated limb perfusion; Leg; Length; Lower Extremity; Measurement; Mediating; Methods; Modeling; Monkeys; Mus; Muscle; Muscle Fibers; Muscle function; Myopathy; N-terminal; Organ; Outcome Measure; Patients; Physiological; Positioning Attribute; Preparation; Proteins; Quality of life; Recombinant Transgenes; Recombinants; Research Design; Reverse Transcriptase Polymerase Chain Reaction; Safety; Sarcolemma; Sequence Homology; Signal Transduction; Site; Skeletal Muscle; Spectrin; Testing; Toxicology; Transgenes; Translations; Virus; Western Blotting; design; functional improvement; functional outcomes; functional restoration; gene replacement; gene replacement therapy; gene therapy; homologous recombination; immunogenic; improved; meetings; micro-dystrophin; mini-dystrophin; nonhuman primate; prevent; promoter; reconstitution; restoration; retinal rods; success; transduction efficiency; vector

DESCRIPTION (provided by applicant): DMD is a progressive muscle disease caused by lack of dystrophin. Gene therapy is an important potential approach to replace the missing gene using adeno-associated virus (AAV). Micro- and mini-dystrophins have been developed for gene replacement but they either lack the C-terminal or the nNOSm binding site. To circumvent these issues we generated dual vectors, each containing nearly one half of the mini-dystrophin transgene with 372bp sequence homology to facilitate homologuous recombination. Upon reconstitution, this mini-dystrophin (ΔH2-R15/ΔR18-19) has the N-terminal domain, 10 spectrin repeats including repeats 16 and 17 for nNOS binding, cysteine-rich and C-terminal domains. Our preliminary studies indicate that a full-length mini-dystrophin is reconstituted, expressed and localized to the sarcolemma in about 50% of transduced muscle fibers. The aims of this study are specifically directed at functional outcomes for this recombinant transgene to lay the foundation for bringing this to clinical trial. Aim 1 will establish proof of principle that gene expression from this dual vector approach following intramuscular delivery will restore specific force and protect against contraction induced injury at doses that can reasonably be brought to vascular delivery and ultimately to clinical translation. Thus, Aim 2 will use a vascular delivery approach to the lower extremities through isolated limb perfusion that we have used extensively in mouse and monkeys and will be bringing to clinical trial for replacement of the alpha-sarcoglycan gene in LGMD2D. Success in vascular delivery lays the foundation for a clinical trial. Outcome measures for both Aim 1 and Aim 2 include dystrophin gene expression and functional improvements in specific force measurements and protection against eccentric contraction induced damage. Mini-dystrophin reconstitution and localization will be demonstrated by RT-PCR, western immunoblotting and immunofluorescence after 12 weeks. If successful in this experimental paradigm, our next step (beyond the scope of this grant) would be to take this to the next level by further testing the recombinant transgene in the non-human primate targeting their lower limbs by isolated limb perfusion. This we have done for many other transgenes that are being brought to clinical trial (e.g., RAC meeting on March 12, 2013 for LGMD2D) and nearly completed a toxicology-biodistribution study. Histological analysis in the monkey, using a FLAG tag as a marker for transduction deficiency will exclude any adverse effects and further refine the dose if necessary. On the safety front, we will also explore any immune issues using IFN-g ELISpot assays as we have done in preparation for multiple clinical trials. We also believe that folding of this gene product will be closer to the natural dystrophin and less likely to yield any hidden epitopes that could be immunogenic.

描述（由适用提供）：DMD是由于缺乏肌营养不良蛋白引起的一种进行性肌肉疾病。基因治疗是使用腺相关病毒（AAV）替代缺失基因的重要方法。已经开发了用于替代基因的微型和微型肌营养素，但它们要么缺乏C末端或NNOSM结合位点。为了避免这些问题，我们产生了双向载体，每个向量包含近一半的迷你肌营养素转化，具有372bp序列同源性，以促进同源重组。重组后，这种迷你淋巴蛋白（ΔH2-R15/ΔR18-19）具有N末端结构域，10个光谱重复序列，包括重复序列16和17，用于NNOS结合，富含半胱氨酸和C-末端域。我们的初步研究表明，在约50％的转导肌肉纤维中，将全长的迷你肺炎蛋白重组，表达和定位于肌膜。这项研究的目的专门针对这种重组转化的功能结果，为将其带入临床试验奠定了基础。 AIM 1将确定原理证明，肌肉内输送后这种双重载体方法的基因表达将恢复特定的力并防止剂量下的收缩诱导损伤，以合理地将血管递送并最终导致临床翻译。这是AIM 2通过孤立的肢体灌注对下肢使用血管递送方法，我们在小鼠和猴子中广泛使用，并将进行临床试验，以取代LGMD2D中的Alpha-Sarcoglycan基因。血管分娩的成功奠定了临床试验的基础。 AIM 1和AIM 2的结果度量包括肌营养不良蛋白基因的表达和特定力测量的功能改善以及针对偏心收缩造成的损害的保护。 RT-PCR，西部免疫印迹和免疫荧光在12周后将证明微型肺炎的重建和定位。如果在这个实验范式中取得成功，我们的下一步（超出了这笔赠款的范围）将是通过进一步测试非人类灵长类动物中的重组转化，将其通过孤立的肢体灌注靶向其下肢。已经为许多其他正在进行临床试验的翻译（例如，2013年3月12日的LGMD2D会议）进行了做出的做法，几乎完成了毒理学生物学分布研究。猴子中的组织学分析，使用标志标签作为翻译缺陷的标记将排除任何不利影响，并在必要时进一步完善剂量。在安全方面，我们还将使用IFN-G ELISPOT ASSA探索任何免疫问题，就像我们为多次临床试验做准备时所做的那样。我们还认为，该基因产物的折叠将更接近天然肌营养不良蛋白，并且不太可能产生任何可能具有免疫原性的隐藏表位。