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

描述（由申请人提供）：DMD是一种由肌营养不良蛋白缺乏引起的进行性肌肉疾病。基因治疗是利用腺相关病毒（AAV）替代缺失基因的重要潜在途径。微型和微型肌营养不良蛋白已被开发用于基因替代，但它们要么缺乏c -末端，要么缺乏nNOSm结合位点。为了避免这些问题，我们生成了双载体，每个载体包含近一半的迷你肌营养不良蛋白转基因，序列同源性为372bp，便于同源重组。重组后，该迷你肌营养不良蛋白（ΔH2-R15/ΔR18-19）具有n端结构域、10个谱蛋白重复序列（包括nNOS结合的重复16和17）、富含半胱氨酸和c端结构域。我们的初步研究表明，全长微型肌营养不良蛋白在约50%的转导肌纤维中被重建、表达并定位于肌膜。本研究的目的是专门针对该重组转基因的功能结果，为将其带入临床试验奠定基础。目的1将建立原理证明，即在肌肉内输送后，通过这种双载体方法表达的基因将恢复特定的力量，并防止收缩引起的损伤，其剂量可以合理地用于血管输送并最终用于临床转化。因此，Aim 2将使用血管输送方法，通过分离肢体灌注到达下肢，我们已经在小鼠和猴子身上广泛使用了这种方法，并将用于临床试验，以替代LGMD2D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。Aim 1和Aim 2的结局指标包括肌营养不良蛋白基因表达和比力测量的功能改善，以及对偏心收缩引起的损伤的保护。12周后通过RT-PCR、western免疫印迹和免疫荧光检测证实微肌营养不良蛋白的重构和定位。如果在这个实验范例中取得成功，我们的下一步（超出了本资助的范围）将通过在非人类灵长类动物的下肢中进行分离肢体灌注来进一步测试重组转基因，从而将其提升到一个新的水平。我们已经对许多其他正在进行临床试验的转基因进行了这样的研究（例如，LGMD2D在2013年3月12日的RAC会议上），并几乎完成了毒理学-生物分布研究。对猴子进行组织学分析，使用FLAG标签作为转导缺陷的标记，将排除任何不良反应，并在必要时进一步改进剂量。在安全性方面，我们还将探索使用IFN-g ELISpot检测的任何免疫问题，正如我们在准备多个临床试验时所做的那样。我们还认为，该基因产物的折叠将更接近天然的肌营养不良蛋白，并且不太可能产生任何可能具有免疫原性的隐藏表位。

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.