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: 8879230
• 负责人: Jerry Roy Mendell
• 金额: $ 18.31万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8879230
• 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（由申请人提供）：DMD是一种由缺乏肌营养不良蛋白引起的进行性肌肉疾病。利用腺相关病毒（AAV）进行基因治疗是替代缺失基因的一种重要的潜在方法。微肌营养不良蛋白和小型肌营养不良蛋白已被开发用于基因置换，但它们缺乏C-末端或nNOSm结合位点。为了避免这些问题，我们产生了双载体，每个载体含有近一半的微肌营养不良蛋白转基因，具有372 bp的序列同源性，以促进同源重组。重组后，这种微型肌养蛋白（ΔH2-R15/ΔR18-19）具有N-末端结构域、10个血影蛋白重复序列（包括用于nNOS结合的重复序列16和17）、富含半胱氨酸和C-末端结构域。我们的初步研究表明，全长迷你肌营养不良蛋白的重建，表达和本地化的肌膜中约50%的转导的肌纤维。本研究的目的是专门针对这种重组转基因的功能结果，为临床试验奠定基础。目的1将建立以下原理的证据：肌内递送后来自这种双载体方法的基因表达将恢复比力，并在可以合理地用于血管递送并最终用于临床转化的剂量下防止收缩诱导的损伤。因此，Aim 2将通过我们在小鼠和猴中广泛使用的隔离肢体灌注使用血管递送方法至下肢，并将用于临床试验以替代LGMD 2D中的α-肌聚糖基因。血管输送的成功为临床试验奠定了基础。目标1和目标2的结果测量包括肌营养不良蛋白基因表达和比力测量的功能改善以及对离心收缩诱导的损伤的保护。12周后通过RT-PCR、Western免疫印迹和免疫荧光证实微小肌养蛋白的重建和定位。如果在这个实验范例中取得成功，我们的下一步（超出了这项资助的范围）将是通过在非人灵长类动物中进一步测试重组转基因，通过孤立的肢体灌注靶向其下肢，将其提升到一个新的水平。我们已经为许多其他正在进行临床试验的转基因做了这件事（例如，RAC会议于2013年3月12日举行，针对LGMD 2D），并几乎完成了毒理学-生物分布研究。使用FLAG标签作为转导缺陷标志物的猴组织学分析将排除任何不良反应，并在必要时进一步细化剂量。在安全性方面，我们还将使用IFN-g ELISpot检测来探索任何免疫问题，正如我们在准备多项临床试验时所做的那样。我们还相信，这种基因产物的折叠将更接近天然抗肌萎缩蛋白，并且不太可能产生任何可能具有免疫原性的隐藏表位。