GENETICALLY MODIFIED HEPATOCYTES TO ACHIEVE SUCCESS IN LIVER CELL TRANSPLANTATION

描述(申请人提供)：几年前，我们发现胎肝干/祖细胞(FlsPC)比成人肝细胞具有更高的增殖活性，通过细胞竞争取代了20%-25%的肝脏质量。由于移植的成年肝细胞与宿主肝细胞相比没有增殖优势，它们不会引起细胞竞争，也不会重新填充肝脏，除非移植的肝细胞对宿主肝有广泛的损害。然而，在临床上使用FLSPC进行肝脏再生的主要限制是获得足够的细胞以进行有效治疗的逻辑问题和重大的伦理问题。我们最近发现，FLSPC高表达增殖基因YAP和Survivin，YAP是河马信号通路的效应者，控制肝脏大小，Survivin是YAP的下游靶点，抑制细胞竞争中的关键成分细胞凋亡。因此，我们假设参与细胞竞争的基因和河马信号通路之间存在合作，如果我们将YAP导入成年肝细胞，我们将向它们传授FLSCP独特的(增殖和抗凋亡)特性，使成年肝细胞能够重新填充正常的成年肝细胞。由于在将增殖性基因(如YAP)引入细胞时存在致癌问题，我们将雌激素受体(ERT2)与YAP联系起来，因此他莫昔芬可以用于控制YAP的核功能。有效的细胞治疗的一个主要要求是使细胞在宿主中长期存在，这对于动物模型和人类的移植肝细胞都是有问题的。Lenti病毒载体旨在将转基因永久整合到细胞基因组中，我们将在Gunn大鼠Crigler-Najjar综合征1型高胆红素血症模型中测试Lenti YAP-ERT2转导的肝细胞移植的持久性，以及在初始治疗完成后很长一段时间内再次接受他莫昔芬治疗是否会重新激活移植细胞的增殖，并产生导致血清胆红素降低的治疗反应。其具体目的是：1)用慢病毒YAP ERT2转导的肝细胞通过他莫昔芬调节YAP的增殖功能，重新填充大鼠肝脏，确定再填充发生的细胞和分子机制(S)，并在基本再填充模型中评价肿瘤的发生；2)通过鉴定YAP转导的肝细胞诱导肿瘤发生所必需的遗传因素、步骤和/或组织紊乱来评估肿瘤的危险性；3)应用Lenti病毒载体系统治疗Gunn大鼠的高胆红素血症，证实转导Lenti-Yap ERT2的人肝细胞可以在接受人异种移植的免疫耐受模型中重新填充小鼠肝脏，并在没有AdHGF的情况下加速WT肝细胞在PIZ小鼠体内的肝脏重新填充。通过结合YAP的增殖能力和通过调节YAP的表达/功能来控制细胞扩张的能力，该项目代表了一种新的肝细胞移植方法，作为一种潜在的治疗遗传性肝代谢疾病的方法。

DESCRIPTION (provided by applicant): Several years ago, we discovered that fetal liver stem/progenitor cells (FLSPC), which have much higher proliferative activity than adult hepatocytes, replace 20-25% of liver mass through cell competition. Since transplanted adult hepatocytes do not have a proliferative advantage over host hepatocytes, they do not induce cell competition and do not repopulate the liver, unless there is extensive damage to the host liver to which the transplanted hepatocytes are resistant. However, major limitations in using FLSPC clinically for liver repopulation are logistic problems in obtaining sufficient cells for effective therapy and significant ethical concerns. We recently discovered that FLSPC hyperexpress the proliferative gene Yap, the effector of the Hippo signaling pathway that controls liver size, and survivin, a downstream target of Yap that inhibits apoptosis, a critical component in cell competition. We, therefore, hypothesize that there is cooperation between genes involved in cell competition and the Hippo signaling pathway and that if we introduce Yap into adult hepatocytes, we will impart in them the unique (proliferative and anti-apoptotic) properties of FLSCP that will allow adult hepatocytes to repopulate the normal adult liver. Since there are oncogenicity issues when introducing a proliferative gene, such as Yap, into cells, we have linked the estrogen receptor (ERT2) to Yap, so that tamoxifen can be used to control Yap's nuclear function. A major requirement for effective cell therapy is to maintain the cells in the host long-term and this has been problematic for transplanted hepatocytes in both animal models and in humans. Lenti-viral vectors are designed for permanent incorporation of transgenes into the cellular genome and we will test for durability of lenti Yap-ERT2 transduced hepatocyte transplantation in the Gunn rat hyperbilirubinemia model for Crigler-Najjar Syndrome, type 1 and whether tamoxifen retreatment long after initial therapy has been completed will reactivate transplanted cell proliferation and produce a therapeutic response resulting in decreased serum bilirubin. The Specific Aims are: 1) to repopulate the rat liver with lentivirus Yap ERT2 transduced hepatocytes in which the proliferative function of Yap is regulated by tamoxifen administration, determine the cellular and molecular mechanism(s) by which repopulation occurs and evaluate tumorigenesis in the basic repopulation model, 2) to assess tumor risk by identifying the genetic factors, steps and/or tissue derangements necessary to induce tumorigenesis by Yap transduced hepatocytes; and 3) to use our lenti-viral vector system to treat hyperbilirubinemia in the Gunn rat, to demonstrate that human hepatocytes transduced with lenti-Yap ERT2 can repopulate the mouse liver in an immunotolerant model that accepts human xenografts and to accelerate liver repopulation by WT hepatocytes in PiZ mice in the absence of AdHGF. By combining the proliferate power of Yap with the ability to control cell expansion by regulating Yap expression/function, this project represents a new approach to hepatocyte transplantation as a potential method to treat genetic-based disorders in liver metabolism.