Optimization of gene therapy approaches for hemophilia

评价了g型AAV的实用性和组织特异性。对于肌肉和肝脏，AAV 1和AAV 8显示最高表达。通过肌肉介导的基因转移证实了人凝血因子IX的治疗水平超过一年。除了这些组织之外，还检查了脂肪组织作为基因转移的靶标;它是体内最丰富的组织之一，并且被设计为生理性地分泌各种蛋白质，使其适合于将转基因产物供应到体循环中。值得注意的是，在发生意外事件的情况下，转导的组织可以安全地移除而没有任何后遗症。然而，通过常规方法有效转导脂肪组织尚不可行。为了开发实用的方法，我们测试了赋形剂对基因转移的增强作用。首先，将编码LacZ的AAV载体施用到Db/Db小鼠的脂肪组织中。当一个生物相容性良好的细胞中， ...更多信息 载体溶液中包括活性表面活性剂。为了验证该方法的功效，利用编码小鼠促红细胞生成素（Epo）的载体。载体注射后两周，观察到血浆Epo的显著浓度，并且该水平几乎与肌肉或肝脏介导的基因转移的水平相当。在此后的观察期内，血浆Epo浓度保持相似水平。增加的血液血红蛋白水平保证了脂肪细胞衍生的Epo的生物活性。通过使用抗Epo抗体的脂肪组织的免疫荧光染色和RT-PCR也证实了有效的表达。此外，在完全去除转导的脂肪组织后，增加的血浆Epo浓度恢复正常，表明其他组织未被该方法转导。虽然这种现象的确切机制还有待分析，但我们的方法导致了有效的基因转移到脂肪细胞中，为补充基因治疗方法增加了一种新的选择。少

The utility and tissue specificity ofg AAV serotypes were evaluated. For the muscle and liver, AAV1 and AAV8 showed the highest expression. Therapeutic levels of human coagulation factor IX was demonstrated for more than one year by muscle mediated gene transfer. In addition to these tissues, adipose tissue was examined as a target of gene transfer ; it is one of the most abundant tissue in the body, and are designed to secrete various proteins physiologically, making it suitable to supply transgene product into systemic circulation. Of note, the transduced tissue can be safely removed without any sequelae in case of unexpected events. However, efficient transduction of adipose tissue has not been feasible by conventional methods. In order to develop a practical method, we tested the enhancement effect of the excipients upon gene transfer. At first, AAV vectors encoding LacZ were administered into adipose tissue of Db/Db mice. Widespread beta-Gal expression was observed when a bio-comp … More atible surfactant was included in the vector solution. To validate the efficacy of this method, vectors encoding mouse erythropoietin (Epo) were utilized. Two weeks following vector injection, significant concentrations of plasma Epo were observed and the levels were almost comparable to that of muscle- or liver- mediated gene transfer. Plasma Epo concentrations kept similar levels during the observation period thereafter. Increased blood hemoglobin levels warrant biological activity of adipocyte-derived Epo. Efficient expression was also confirmed by both immunofluorescence staining of the adipose tissue using anti-Epo antibody and RT-PCR. In addition, following complete removal of transduced adipose tissue, increased plasma Epo concentrations returned normal, suggesting that other tissues were not transduced by this method. Although the precise mechanisms of this phenomenon are yet to be analyzed, our method leads to efficient gene transfer into adipocytes, adding a novel choice for the supplemental gene therapy approaches. Less