GENETICALLY MODIFIED HEPATOCYTES TO ACHIEVE SUCCESS IN LIVER CELL TRANSPLANTATION

描述（由申请人提供）：几年前，我们发现胎儿肝干细胞/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。由于移植的成人肝细胞不具有比宿主肝细胞更强的增殖优势，因此它们不会诱导细胞竞争，也不会重新填充肝脏，除非移植的肝细胞能够抵抗宿主肝脏的广泛损伤。然而，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进行有效治疗的后勤问题和重大的伦理问题。我们最近发现FLSPC超表达增殖基因Yap （Hippo信号通路的效应因子，控制肝脏大小）和survivin （Yap的下游靶点，抑制细胞凋亡，是细胞竞争的关键组成部分）。因此，我们假设参与细胞竞争的基因与Hippo信号通路之间存在合作，如果我们将Yap引入成人肝细胞，我们将赋予它们FLSCP的独特（增殖和抗凋亡）特性，使成人肝细胞能够重新填充正常的成人肝脏。由于在将增生基因（如Yap）引入细胞时存在致癌性问题，我们将雌激素受体（ERT2）与Yap连接起来，这样他莫昔芬就可以用来控制Yap的细胞核功能。有效细胞治疗的一个主要要求是将细胞长期维持在宿主体内，这在动物模型和人类移植肝细胞中都存在问题。lentii病毒载体被设计用于将转基因永久整合到细胞基因组中，我们将在Gunn大鼠高胆红素血症模型中测试lenti Yap-ERT2转导肝细胞移植的持久性，以治疗1型Crigler-Najjar综合征，以及在初始治疗完成后很长时间再治疗他莫昔芬是否会重新激活移植细胞增殖并产生导致血清胆红素降低的治疗反应。具体目的是：1)用慢病毒Yap ERT2转导的肝细胞重建大鼠肝脏，其中Yap的增殖功能受他莫昔芬的调节，确定再生发生的细胞和分子机制，并在基本再生模型中评估肿瘤发生；2)通过确定诱导Yap转导的肝细胞发生肿瘤所需的遗传因素、步骤和/或组织紊乱来评估肿瘤风险；3)利用我们的慢病毒载体系统治疗Gunn大鼠的高胆红素血症，证明用慢病毒ERT2转导的人肝细胞可以在接受人类异种移植物的免疫耐受模型中重新填充小鼠肝脏，并在缺乏AdHGF的情况下加速WT肝细胞在PiZ小鼠的肝脏再生。通过将Yap的增殖能力与通过调节Yap的表达/功能来控制细胞扩增的能力相结合，该项目代表了肝细胞移植作为治疗肝脏代谢遗传性疾病的潜在方法的新途径。

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.