A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy
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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DOI：
10.1186/1479-5876-5-45
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发表时间：
2007-09-24
影响因子：
7.4
通讯作者：
Mendell, Jerry R.
中科院分区：
文献类型：
--
作者：
Rodino-Klapac, Louise R.;Janssen, Paul M. L.;Mendell, Jerry R.
背景资料：杜氏肌营养不良症（DMD）是一种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研究，该研究直接涉及以下关键问题，考虑到以下标准，这些问题可以直接适用于使用rAAV的基因治疗临床试验：1）将保护患者免受病毒广泛传播的区域血管递送方法; 2）潜在地促进病毒安全通过以进行有效骨骼肌转导的方法; 3）使用病毒剂量以适应载体生产方法所施加的当前限制; 4）同时，通过转导下肢中的多个肌肉以延长肌张力来实现临床上有意义的结果。AAV 1的能力，AAV 6或AAV 8穿过血管内皮屏障，携带微-在相同的条件下，通过置于mdx小鼠股动脉中的导管递送，比较肌营养不良蛋白cDNA。使用识别微小肌养蛋白的N-末端的抗体（Manexla）通过免疫染色评估转导效率。通过测量强直力和对趾长伸肌（EDL）离心收缩的保护来评估生理矫正的程度。结果：局部血管递送导致rAAV 8.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）。rAAV 6.micro-dystrophin处理的动物显示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。与此形成鲜明对比的是，rAAV 1通过血管递送表现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）]。由rAAV 8和rAAV 6通过股动脉递送的微肌营养不良蛋白显著改善强直力并防止离心收缩。使用类似的血管递送范例将小鼠研究转化为食蟹猴的后肢。携带eGFP的rAAV 8的剂量与小鼠成比例（小鼠5 × 10（12）vg/kg vs猴2 × 10（12）vg/kg）显示广泛的基因表达[内侧腓肠肌-63.8 +/-4.9%，外侧腓肠肌-66.0 +/-4.5%，EDL - 80.2 +/-3.1%，比目鱼肌-86.4 +/-1.9%，结论：这些研究证明了在适用于DMD儿童临床试验的剂量、压力和体积下，用AAV血清型在小鼠和非人灵长类动物中进行区域血管基因递送。
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 (Manexla) 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 x 10(12) vg/kg in mouse vs 2 x 10(12) 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.