GENETICALLY MODIFIED HEPATOCYTES TO ACHIEVE SUCCESS IN LIVER CELL TRANSPLANTATION

基因改造肝细胞实现肝细胞移植的成功

• 批准号: 8762032
• 负责人: DAVID A SHAFRITZ
• 金额: $ 67.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8762032
• 关键词: Adult; Animal Model; Apoptosis; Apoptotic; Automobile Driving; Bilirubin; Biological Models; Cell Count; Cell Proliferation; Cell Therapy; Cell Transplantation; Cell Transplants; Cells; Chronic; Crigler-Najjar Syndrome; Dependence; Disease; Engineering; Estrogen Receptors; Ethics; Exhibits; Fetal Liver; Genes; Genetic; Genome; Goals; Growth Factor; Gunn Rats; Hepatic; Hepatic Mass; Hepatocyte; Histologic; Human; Hyperbilirubinemia; Inborn Genetic Diseases; Kinetics; Knockout Mice; Lasers; Link; Liver; Liver diseases; Liver neoplasms; Location; Logistics; Mediating; Messenger RNA; Metabolism; Methods; Modeling; Molecular; Mus; Nuclear; Oncogenic; Production; Proliferating; Property; Protein C Inhibitor; Protocols documentation; RNA; Rattus; Resistance; Retreatment; Risk; Role; Serum; Signal Pathway; Signal Transduction; Staining method; Stains; Stem cells; Subfamily lentivirinae; System; Tamoxifen; Testing; Therapeutic; Time; Tissues; Transgenes; Transgenic Mice; Transplantation; Viral Vector; Xenograft procedure; base; deep sequencing; design; effective therapy; liver injury; liver metabolism; novel strategies; preconditioning; public health relevance; response; retrorsine; stem; success; survivin; tumor; tumorigenesis; tumorigenic

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.

描述（由申请人提供）：几年前，我们发现具有比成人肝细胞高得多的增殖活性的胎肝干/祖细胞（FLSPC）通过细胞竞争替代20-25%的肝脏质量。由于移植的成体肝细胞不具有超过宿主肝细胞的增殖优势，因此它们不诱导细胞竞争并且不重新填充肝脏，除非对移植的肝细胞具有抗性的宿主肝脏存在广泛损伤。然而，在临床上使用FLSPC用于肝脏再生的主要限制是在获得足够的细胞用于有效治疗方面的后勤问题和重大的伦理问题。我们最近发现，FLSPC高表达增殖基因雅普和生存素，前者是控制肝脏大小的Hippo信号通路的效应子，后者是抑制细胞凋亡的雅普下游靶点，细胞凋亡是细胞竞争的关键成分。因此，我们假设参与细胞竞争和Hippo信号通路的基因之间存在合作，并且如果我们将雅普引入成年肝细胞，我们将赋予它们FLSCP的独特（增殖和抗凋亡）特性，这将允许成年肝细胞重新填充正常的成年肝脏。由于当将增殖基因如雅普引入细胞时存在致癌性问题，我们将雌激素受体（ERT 2）与雅普连接，使得他莫昔芬可用于控制雅普的核功能。有效的细胞治疗的主要要求是将细胞长期维持在宿主中，这对于动物模型和人类中的移植肝细胞都是有问题的。慢病毒载体设计用于将转基因永久掺入细胞基因组中，并且我们将在Crigler-Najjar综合征1型的古恩大鼠高胆红素血症模型中测试慢病毒Yap-ERT 2转导的肝细胞移植的耐久性，以及在初始治疗已经完成很长时间后的他莫昔芬再治疗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。具体目标是：1）用慢病毒雅普ERT 2转导的肝细胞（其中雅普的增殖功能受他莫昔芬给药调节）重建大鼠肝脏，确定重建发生的细胞和分子机制，并评估基本重建模型中的肿瘤发生，2）通过鉴定遗传因子评估肿瘤风险，通过雅普转导的肝细胞诱导肿瘤发生所必需的步骤和/或组织紊乱;和3）使用我们的慢病毒载体系统治疗古恩大鼠的高胆红素血症，证明用lenti-Yap ERT 2转导的人肝细胞可以在接受人异种移植物的免疫耐受模型中重新填充小鼠肝脏，并在不存在AdHGF的情况下加速PiZ小鼠中WT肝细胞的肝脏重新填充。通过将雅普的增殖能力与通过调节雅普表达/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中基于遗传的疾病的潜在方法的新方法。