Hepatocyte - Based Therapies of Primary Hyperoxaluria 1

描述(由申请人提供)：原发性高草酸尿症I型(PH1)是一种常染色体隐性遗传病，由于过氧酶体丙氨酸-乙醛转氨酶(AGT)缺乏导致肝细胞过度产生草酸，导致乙醛转化为草酸增加。尿草酸排泄增加会导致肾钙化和尿石症，导致肾功能衰竭和随后的组织草酸中毒，危及生命的并发症。肝肾联合移植是PH1的最终治疗方法，但由于体内草酸盐储存较多，并不总是成功。肝细胞移植侵袭性小得多，可以作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，很大一部分突变宿主肝细胞必须被AGT活性细胞取代，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反应。我们实验室的三项最新发展为这些问题提供了潜在的解决方案。(A)我们已经建立了一种新的PH1的Agxt-1基因缺失的小鼠模型(Agxt-/-)，并在Agxt缺失的背景下产生了一种新的表达突变的人AGXT的转基因小鼠，这在PH1患者中最常见。其次，我们设计了通过为移植的肝细胞提供增殖优势，使用制备性肝X-射线(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可以防止其同种异体移植排斥反应，而不会抑制宿主免疫系统。具体目标1是利用Agxt/-受体和同基因LacZ转基因供体小鼠，确定HIR、HGF表达与肝细胞移植之间的最小有效剂量和最佳时间关系。我们将确定在口服乙二醇或单侧肾切除的情况下，在治疗上显著改善高草酸尿、预防肾钙化和尿石症所需的肝脏再充血水平。我们将确定基于HIR的肝脏再繁殖所需的细胞周期调节蛋白。在特定的目标2中，我们将评估两种消除同种异体移植排斥反应的方法：(A)从切除的Agxt-/-小鼠肝叶分离的自体原代肝细胞将通过慢病毒载体与人或小鼠AGXT转导，然后移植回供者体内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在移植到Agxt/-受体之前，将在体外转导AdE3基因或抗Fas的shRNA。这些研究的成功完成将代表着向以肝细胞为基础的治疗大量以肝脏为基础的遗传性代谢疾病迈出了重要的一步，包括PH1。公共卫生相关性：这项研究项目旨在开发基于移植肝细胞广泛再生的原发性高草酸尿症-1的新疗法，并设计规避肝细胞同种异体移植排斥反应的策略。

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.