Use of Gene Therapy to Induce Transplantation Tolerance

可用于移植给终末期疾病患者的人体器官严重短缺，导致人们越来越关注开发允许使用猪作为供体器官替代来源的方法。然而，从未接触过猪细胞的人类具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称为半乳糖tx1，3半乳糖碳水化合物表位（gal表位）的特定糖反应。这种糖被人类或旧世界灵长类动物鉴定为外来的，因为在进化过程中发生了突变，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，当人类接触来自其他物种的器官（异种移植物）时，会产生半乳糖苷抗体。最近开发了一种特殊的小鼠品系，作为异种移植排斥反应的良好小动物模型，因为它们产生的抗gal抗体类似于人类排斥猪异种移植物的抗体。在该模型中，在小鼠接触猪细胞之前，通过骨髓移植导入单个基因，可以防止排斥猪细胞的抗体的产生。在临床前灵长类动物模型中重现该实验的能力将提供一种方法，通过该方法可以在异种移植之前治疗人类患者以防止猪器官的排斥反应，并且将对移植领域产生重大影响。该实验尚未在灵长类动物中重现，因为与使用常规方法的小鼠模型相比，将新基因导入灵长类动物骨髓干细胞中更加困难。我们最近建立了一种新的方法，更有效地引入基因到灵长类动物和人类骨髓干细胞使用一种新型的基因传递载体（慢病毒载体）。在本申请中，我们建议将负责半乳糖碳水化合物表达的基因引入灵长类干细胞中，并确定该过程是否会阻止灵长类动物中的抗半乳糖抗体产生。我们的假设是，这种新载体将允许我们将负责编码半乳糖碳水化合物的基因导入灵长类干细胞，其水平足以阻止移植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.