Use of Gene Therapy to Induce Transplantation Tolerance

可用于终末期疾病患者移植的人体器官严重短缺，导致越来越多的注意力集中在开发允许使用猪作为供体器官替代来源的方法上。然而，从未接触过猪细胞的人类有能够迅速排斥这些器官的抗体。这些抗体主要与猪细胞上表达的一种叫做半乳糖tx1,3半乳糖碳水化合物表位（gal表位）的特定糖反应。这种糖被人类或东半球灵长类动物认定为外来糖，因为在进化过程中发生了一种突变，阻止了这种碳水化合物在人类和特定类型的猴子中表达。因此，当人类暴露于来自其他物种（异种移植）的器官时，会产生gal抗体。一种特殊的小鼠品种最近被开发出来，作为一种很好的异种移植排斥的小动物模型，因为它们产生的抗半乳糖抗体与人类排斥猪异种移植的抗体相似。在这个模型中，在小鼠暴露于猪细胞之前，可以通过骨髓移植引入单个基因来防止产生排斥猪细胞的抗体。在临床前灵长类动物模型中重现这一实验的能力将为人类患者在异种移植前进行治疗提供一种手段，以防止猪器官的排斥反应，并将对移植领域产生重大影响。该实验尚未在灵长类动物中复制，因为与使用常规方法的小鼠模型相比，在灵长类动物骨髓干细胞中引入新基因更为困难。我们最近建立了一种新的方法，利用一种新型的基因传递载体（慢病毒载体）更有效地将基因引入灵长类动物和人类骨髓干细胞。在这项应用中，我们建议在灵长类干细胞中引入负责半乳糖碳水化合物表达的基因，并确定这一过程是否会阻止灵长类动物产生抗半乳糖抗体。我们的假设是，这种新的载体将允许我们将负责编码半乳糖碳水化合物的基因引入灵长类干细胞，其水平足以阻止移植的恒河猴对猪细胞的免疫反应。

The critical shortage of human organs available for transplantation to patients with end-stage diseases has led to an increasing amount of attention directed at developing methods to allow for the use of pigs as an alternative source of donor organs. Humans that have never been exposed to pig cells, however, have antibodies that are capable of rapidly rejecting these organs. These antibodies react predominantly with a specific sugar called the galactose tx1,3 galactose carbohydrate epitope (gal epitope) expressed on pig cells. This sugar is identified as foreign by humans or Old World primates because a mutation has occurred during evolution that prevents this carbohydrate from being expressed in humans and specific types of monkeys. Consequently, humans produce antibodies to gal when exposed to organs derived from other species (xenografts). A special strain of mice has recently been developed that serves as a good small animal model of xenograft rejection because they produce anti-gal antibodies similar to the antibodies that reject pig xenografts in humans. In this model, production of antibodies that reject pig cells can be prevented by introducing a single gene by bone marrow transplantation before the mouse is exposed to the pig cells. The ability to reproduce this experiment in a preclinical primate model will provide a means by which human patients may be treated prior to xenotransplantation to prevent rejection of pig organs, and will have a significant impact on the field of transplantation. This experiment has not yet been reproduced in primates because introduction of the new gene in primate stem cells of the bone marrow is more difficult compared with the mouse model using conventional methods. We have recently established a new method to introduce genes more efficiently into primate and human stem cells of the bone marrow using a new type of gene delivery vehicle (lentiviral vector). In this application, we propose to introduce the gene responsible for gal carbohydrate expression in primate stem cells and determine whether this procedure prevents anti-gal antibody production in primates. Our hypothesis is that this new vector will allow us to introduce the gene responsible for encoding the gal carbohydrate into primate stem cells at levels that are sufficient to prevent the immune response to pig cells in transplanted rhesus monkeys.