Hepatocyte - Based Therapies of Primary Hyperoxaluria 1

原发性高草酸尿症的肝细胞疗法 1

基本信息

批准号：
7651606
负责人：
NAMITA ROY-CHOWDHURY
金额：
$ 32.43万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-16 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7651606
关键词：
Adenovirus Vector Adverse effects Alanine-glyoxylate aminotransferase Allogenic Allografting Autologous Autologous Transplantation Back Biochemical Biological Assay Canis familiaris Cell Cycle Cell Transplants Cells Deposition Deterioration Development Disease Dose Engraftment Ethylene Glycols Excretory function Face Failure Gene Transfer Genes Genetic Models Glycolates Glyoxylates Graft Rejection Growth Hepatic Hepatocyte Hepatocyte Growth Factor Homologous Transplantation Human Hyperoxaluria Immune system Immunocompromised Host Immunosuppression Inherited Kidney Failure Kidney Transplantation Knockout Mice Laboratories LacZ Genes Lentivirus Vector Life Liver Lobe Magnetism Metabolic Diseases Mitosis Mitotic Mus Mutation Nephrectomy Nephrocalcinosis Oral Oxalates Patients Prevention Primary Hyperoxaluria Procedures Production Proteins Rattus Recombinant Hepatocyte Growth Factor Renal function Research Project Grants Resected Residual state Solutions Staining method Stains Suspension substance Suspensions TP53 gene Techniques Testing Therapeutic Tissues Transgenic Mice Transgenic Organisms Transplantation Treatment Protocols Urinary Calculi Western Blotting base design disease-causing mutation ethylene glycol glyoxylate human HGF protein irradiation liver cell proliferation liver transplantation minimally invasive mouse model mutant nanoparticle novel oncoprotein p21 overexpression oxalosis prevent public health relevance pyridoxine small hairpin RNA urinary urolithiasis

项目摘要

DESCRIPTION (provided by applicant): Primary hyperoxaluria type I (PH1) is an autosomal recessive disease caused by excessive oxalate production by hepatocytes due to peroxisomal alanine-glyoxylate aminotransferase (AGT) deficiency, leading to increased conversion of glyoxylate to oxalate. Increased urinary oxalate excretion causes nephrocalcinosis and urolithiasis, leading to renal failure and consequent tissue oxalosis with life-threatening complications. Combined liver-kidney transplantation, the definitive treatment of PH1, is not always successful because of large body oxalate stores. Hepatocyte transplantation, which is much less invasive, could be used potentially as a preemptive treatment. As hepatocytes overproduce oxalate in PH1, a significant fraction of mutant host hepatocytes must be replaced by AGT-competent cells, which is beyond the capacity of currently used hepatocyte transplantation procedures. Also, transplantation of allogeneic hepatocytes requires immunosuppression to prevent graft rejection. Three recent developments in our laboratory offer potential solutions to these problems. (a) We have generated a new Agxt-1 gene-deleted mouse model of PH1 (Agxt-/-) and a new transgenic mouse expressing a mutant human AGXT, which is most common in PH1 patients, in the Agxt-null background. Second, we have devised strategies to massively repopulate the liver by providing proliferative advantage to transplanted hepatocytes, using preparative hepatic X-irradiation (HIR) and expressing hepatocyte growth factor (HGF) as a mitotic stimulant. Third, we found that expressing adenoviral E3 genes or down-regulating Fas in donor hepatocytes prevents their allograft rejection, without suppressing the host immune system. Specific Aim 1 is to determine the minimum effective HIR dose and the optimum temporal relationship between HIR, HGF expression and hepatocyte transplantation using Agxt-/- recipients and congeneic LacZ- transgenic donor mice. We will determine the level of hepatic repopulation needed for therapeutically significant amelioration of hyperoxaluria, and preventing nephrocalcinosis and urolithiasis in the face of oral ethylene glycol challenge or unilateral nephrectomy. We will identify cell cycle regulator proteins that are required for HIR-based hepatic repopulation. In Specific Aim 2 we will evaluate two approaches to abrogate allograft rejection: (a) Autologous primary hepatocytes isolated from a resected liver lobe of Agxt- /- mice will be transduced with human or mouse AGXT using lentiviral vectors, and then transplanted back into the donor to avoid allorejection. (b) Primary hepatocytes isolated from allogeneic wildtype donors will be transduced ex vivo with AdE3 genes or shRNA against Fas, before transplantation into Agxt-/- recipients. Successful completion of these studies will represent a major step toward hepatocyte-based cure of a large number of liver-based inherited metabolic disorders, including PH1. PUBLIC HEALTH RELEVANCE: This research project is aimed at developing novel therapies for primary hyperoxaluria-1 based on extensive repopulation of the liver with transplanted hepatocytes, and designing strategies for circumventing allograft rejection of hepatocytes.

描述（申请人提供）：原发性高草酸尿症 I 型（PH1）是一种常染色体隐性遗传疾病，是由于过氧化物酶体丙氨酸-乙醛酸转氨酶（AGT）缺陷导致肝细胞产生过多草酸，导致乙醛酸转化为草酸增加所致。尿草酸排泄增加会导致肾钙质沉着症和尿石症，导致肾衰竭和随后的组织草酸盐中毒，并产生危及生命的并发症。肝肾联合移植是 PH1 的最终治疗方法，但由于体内草酸储存量大，并不总是成功。肝细胞移植的侵入性要小得多，有可能用作先发制人的治疗方法。由于肝细胞在PH1中过量产生草酸，很大一部分突变宿主肝细胞必须被AGT感受态细胞取代，这超出了目前使用的肝细胞移植程序的能力。此外，同种异体肝细胞移植需要免疫抑制以防止移植物排斥。我们实验室最近的三项进展为这些问题提供了潜在的解决方案。 (a) 我们生成了一种新的 Agxt-1 基因缺失的 PH1 小鼠模型 (Agxt-/-) 和一种表达突变型人类 AGXT 的新转基因小鼠，这种突变型人类 AGXT 在 Agxt 无效的背景下最常见于 PH1 患者。其次，我们设计了通过为移植的肝细胞提供增殖优势、使用制备型肝脏 X 射线照射 (HIR) 和表达肝细胞生长因子 (HGF) 作为有丝分裂刺激剂来大规模重新填充肝脏的策略。第三，我们发现在供体肝细胞中表达腺病毒E3基因或下调Fas可防止其同种异体移植物排斥，而不抑制宿主免疫系统。具体目标1是使用Agxt-/-受体和同源LacZ-转基因供体小鼠确定最小有效HIR剂量以及HIR、HGF表达和肝细胞移植之间的最佳时间关系。我们将确定在治疗上显着改善高草酸尿症所需的肝脏再生水平，并在面临口服乙二醇挑战或单侧肾切除术时预防肾钙质沉着症和尿石症。我们将鉴定基于 HIR 的肝脏再生所需的细胞周期调节蛋白。在具体目标 2 中，我们将评估消除同种异体移植排斥的两种方法：（a）从 Agxt-/- 小鼠切除的肝叶中分离出的自体原代肝细胞将使用慢病毒载体用人或小鼠 AGXT 转导，然后移植回供体以避免同种异体排斥。 (b)从同种异体野生型供体分离的原代肝细胞将在移植到Agxt-/-受体之前用AdE3基因或针对Fas的shRNA离体转导。这些研究的成功完成将代表着朝着基于肝细胞的方法治疗包括 PH1 在内的大量肝脏遗传性代谢疾病迈出了重要一步。公共健康相关性：该研究项目旨在基于移植肝细胞对肝脏的广泛再增殖，开发原发性高草酸尿症 1 的新疗法，并设计避免肝细胞同种异体移植排斥的策略。