Optimization of gene therapy approaches for hemophilia

评价G AAV血清型的实用性和组织特异性。在肌肉和肝脏中，AAV1和AAV8的表达最高。一年多来，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。除了这些组织外，脂肪组织也被认为是基因转移的目标；它是体内最丰富的组织之一，旨在生理上分泌各种蛋白质，使其适合向体循环供应转基因产品。值得注意的是，在发生意外事件的情况下，可以安全地取出被转导的组织，而不会有任何后遗症。然而，传统的方法并不能有效地转导脂肪组织。为了开发一种实用的方法，我们测试了辅料对基因转移的促进作用。首先，将编码LacZ的AAV载体注射到DB/DB小鼠的脂肪组织中。在生物复合…中观察到广泛的β-半乳糖表达载体溶液中含有较易溶出的表面活性剂。为了验证该方法的有效性，利用编码小鼠促红细胞生成素(EPO)的载体。载体注射两周后，观察到显著的血浆EPO浓度，其水平几乎与肌肉或肝脏介导的基因转移相当。在此后的观察期内，血浆促红细胞生成素浓度保持在相似的水平。血液中高水平的血红蛋白保证了脂肪细胞来源的促红细胞生成素的生物活性。用抗EPO抗体和RT-PCR对脂肪组织进行免疫荧光染色也证实了高效表达。此外，在完全去除转导的脂肪组织后，升高的血浆EPO浓度恢复正常，表明其他组织不是通过这种方法转导的。虽然这一现象的确切机制尚待分析，但我们的方法能够有效地将基因转移到脂肪细胞中，为补充基因治疗方法增加了新的选择。较少

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