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

描述(申请人提供)：DMD是一种由于缺乏抗肌营养不良蛋白而引起的进行性肌肉疾病。基因治疗是用腺相关病毒(AAV)替代缺失基因的一种重要的潜在方法。微型肌营养不良蛋白和微型肌营养不良蛋白已被开发用于基因替换，但它们要么缺乏C末端，要么缺乏nNOSm结合位点。为了绕过这些问题，我们产生了双载体，每个载体包含近一半的微型营养不良蛋白转基因，具有372bp的序列同源性，以促进同源重组。重组后，该微型肌营养不良蛋白(ΔH2-R15/ΔR18-19)具有N-末端结构域，10个光谱蛋白重复序列，包括16和17个重复序列，分别与nNOS结合、富含半胱氨酸和C-末端结构域。我们的初步研究表明，在大约50%的转导肌肉纤维中，全长的微型肌营养不良蛋白被重组、表达并定位于肌膜。这项研究的目的是专门针对这种重组转基因的功能结果，为将其应用于临床试验奠定基础。目的1将建立一个原则证明，在肌肉注射后，这种双载体方法的基因表达将恢复特定的力量，并防止收缩引起的损伤，剂量可合理地带到血管交付并最终应用于临床翻译。因此，Aim 2将使用一种血管输送方法，通过隔离肢体灌流将其输送到下肢，我们已经在小鼠和猴子身上广泛使用这种方法，并将在临床试验中替换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。目标1和目标2的结果衡量标准包括抗肌营养不良蛋白基因的表达和比力测量中的功能改善，以及对偏心收缩引起的损伤的保护。12周后将通过RT-PCR、免疫印迹和免疫荧光等方法证实微肌营养不良蛋白的重建和定位。如果在这一实验范式中取得成功，我们的下一步(超出这项拨款的范围)将通过进一步测试在非人类灵长类动物中通过隔离肢体灌流来靶向他们的下肢的重组转基因来将这一研究提高到一个新的水平。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项研究(例如，2013年3月12日针对LGMD2D召开的RAC会议)，并几乎完成了毒理学-生物分布研究。在猴子身上进行组织学分析，使用标志标签作为转导缺陷的标志，将排除任何不利影响，并在必要时进一步改进剂量。在安全方面，我们还将使用干扰素-g ELISpot检测来探索任何免疫问题，就像我们在准备多项临床试验时所做的那样。我们还认为，这种基因产物的折叠将更接近天然的肌营养不良蛋白，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.