Hepatocyte - Based Therapies of Primary Hyperoxaluria 1

描述（由申请人提供）：原发性高草酸尿I型（PH1）是一种常染色体隐性遗传病，由过氧化物体丙氨酸-乙醛酸氨基转移酶（AGT）缺乏导致肝细胞产生过量草酸引起，导致乙醛酸转化为草酸增加。尿草酸盐排泄增加可引起肾钙化症和尿石症，导致肾功能衰竭和随之而来的组织草酸盐化，并发危及生命的并发症。肝肾联合移植，PH1的最终治疗方法，并不总是成功的，因为体内大量的草酸储存。肝细胞移植的侵入性小得多，有可能成为一种先发制人的治疗方法。由于肝细胞在PH1中过量产生草酸盐，很大一部分突变宿主肝细胞必须被agt - capable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)我们建立了一个新的Agxt-1基因缺失的PH1小鼠模型（Agxt-/-）和一个新的转基因小鼠，在Agxt-null背景下表达突变的人类Agxt，这在PH1患者中最常见。其次，我们设计了大量的策略，通过为移植的肝细胞提供增殖优势，使用肝x射线辐照（HIR）和表达肝细胞生长因子（HGF）作为有丝分裂刺激物来重新填充肝脏。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可防止其异体移植排斥，而不会抑制宿主免疫系统。特异性目的1是确定HIR的最小有效剂量以及HIR、HGF表达与Agxt-/-受体和同源LacZ-转基因供鼠肝细胞移植之间的最佳时间关系。我们将确定在治疗上显著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化症和尿石症。我们将鉴定基于hir的肝脏再生所需的细胞周期调节蛋白。在特异性目标2中，我们将评估消除同种异体移植排斥反应的两种方法：(a)从切除的Agxt- /-小鼠肝叶中分离的自体原代肝细胞将使用慢病毒载体与人或小鼠Agxt进行转导，然后移植回供体以避免同种异体排斥反应。(b)从同种异体野生型供体分离的原代肝细胞将在体外用AdE3基因或抗Fas的shRNA转导，然后移植到Agxt-/-受体中。这些研究的成功完成将代表着以肝细胞为基础治疗包括PH1在内的大量肝脏遗传性代谢疾病的重要一步。公共卫生相关性：本研究项目旨在基于移植肝细胞在肝脏的广泛再生，以及设计规避同种异体肝细胞排斥反应的策略，开发治疗原发性高草酸血症-1的新疗法。

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.