Hepatocyte - Based Therapies of Primary Hyperoxaluria 1

描述（由申请方提供）：原发性高尿酸I型（PH 1）是一种常染色体隐性遗传疾病，由过氧化物酶体丙氨酸-乙醛酸氨基转移酶（AGT）缺乏导致肝细胞产生过量草酸盐引起，导致乙醛酸转化为草酸盐增加。尿草酸排泄增加引起肾钙质沉着症和尿石症，导致肾衰竭和随后的组织草酸盐沉着症，并伴有危及生命的并发症。肝肾联合移植是PH 1的决定性治疗方法，但由于体内草酸盐储存量大，因此并不总是成功的。肝细胞移植，这是侵入性小得多，可能被用作一个潜在的先发制人的治疗。由于肝细胞在PH 1中过量产生草酸盐，突变宿主肝细胞的显著部分必须被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，同种异体肝细胞的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)我们已经产生了一个新的Agxt-1基因缺失的PH 1小鼠模型（Agxt-/-）和一个新的转基因小鼠表达突变的人AGXT，这是最常见的PH 1患者，在Agxt无效的背景。第二，我们已经设计了策略，通过提供增殖优势移植肝细胞，使用制备性肝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，大规模地重新填充肝脏。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是确定最小有效HIR剂量和使用Agxt-/-受体和同源LacZ-转基因供体小鼠的HIR、HGF表达和肝细胞移植之间的最佳时间关系。我们将确定在口服乙二醇或单侧肾切除术后，治疗性显著改善高尿症和预防肾钙质沉着症和尿石症所需的肝再生水平。我们将确定细胞周期调节蛋白，所需的肝细胞再增殖反应为基础。在具体目标2中，我们将评估两种消除同种异体移植物排斥的方法：（a）从Agxt- /-小鼠切除的肝叶分离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-/-受体中之前用AdE 3基因或针对Fas的shRNA离体转导。这些研究的成功完成将代表以肝细胞为基础治愈大量以肝脏为基础的遗传性代谢疾病（包括PH 1）的重要一步。公共卫生相关性：该研究项目旨在开发基于移植肝细胞的肝脏广泛再增殖的原发性高尿症-1的新疗法，并设计规避肝细胞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.