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首页> 外文期刊>Journal of Translational Medicine >A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy
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A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

机译：一种无大容量或高压的微型肌营养不良蛋白基因肢体血管输送的平移方法，用于治疗杜氏肌营养不良症

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Background Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. Methods The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. Results Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 1012 vg/kg in mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%. Conclusion These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.
机译：背景Duchenne肌营养不良症（DMD）是X连锁隐性疾病，具有单基因突变，为成功的基因治疗奠定了基础。我们已经完成了一项直接涉及以下关键问题的研究，可以考虑到以下标准，将这些关键问题直接适用于使用rAAV进行基因治疗的临床试验：1）一种区域性血管输送方法，可以保护患者免受病毒的广泛传播; 2）一种潜在促进病毒安全通过以有效进行骨骼肌转导的方法; 3）使用病毒剂量来适应载体生产方法施加的当前限制; 4）同时，通过转导下肢的多条肌肉以延长下肢运动来达到临床上有意义的结果。方法在相同条件下，通过放置在mdx小鼠股动脉中的导管进行比较，比较了AAV1，AAV6或AAV8穿过带有微肌营养不良蛋白cDNA的血管内皮屏障的能力。通过使用识别微量肌营养不良蛋白N末端的抗体（Manex1a）进行免疫染色来评估转导效率。生理矫正的程度是通过测量趾长伸肌（EDL）中的破伤力和防止离心收缩来评估的。在小鼠中发现成功的血管输送范例被带入非人类灵长类动物，以测试其对DMD男孩的潜在翻译。结果局部血管输送导致rAAV8.micro-dystrophin的转导达到94.5±0.9（1个月），91.3±3.1（2个月）和89.6±1.6％（3个月）。 rAAV6。微肌营养不良蛋白治疗的动物表现出87.7±6.8（1个月），78.9±7.4（2个月）和81.2±6.2％（3个月）的转导。与之形成鲜明对比的是，rAAV1通过血管输送显示出非常低的转导效率[0.9±0.3（1个月），2.1±0.8（2个月）和2.1±0.7％（3个月）]。 rAAV8和rAAV6通过股动脉递送的微肌营养不良蛋白可显着改善强直性力量并防止离心收缩。小鼠研究使用类似的血管输送范例将其翻译为猕猴的后肢。携带eGFP的rAAV8的剂量与小鼠成正比（小鼠为5×10 12 vg / kg，而猴为2×10 12 vg / kg）证明了广泛的基因表达[medial腓肠肌– 63.8±4.9％，外侧腓肠肌– 66.0±4.5％，EDL – 80.2±3.1％，比目鱼– 86.4±1.9％，TA – 72.2±4.0％。结论这些研究表明，在小鼠和非人灵长类动物中，具有AAV血清型的区域性血管基因传递具有适用于DMD儿童临床试验的剂量，压力和体积。

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