Acute Hepatic Porphyrias: Pathogenesis & Treatment

描述（由申请人提供）：NIDDK指导研究科学家发展奖的总体目标是为申请人提供一个综合的研究和教育计划，这将导致申请人在转化研究方面的独立学术生涯。Robert J. Desnick博士，医学博士，遗传学和基因组科学教授兼主席，基因组研究副院长，Ihor Lemischka博士，基因和细胞治疗教授兼黑人家族干细胞研究所所长，将分别担任导师和联合导师。申请人将把95%的精力投入到职业发展计划中，包括：1)进行拟议的实验室研究，2)干细胞生物学和iPS技术的培训，3)参加遗传学和干细胞生物学“工作进展”会议，期刊俱乐部和研讨会，参加国家科学会议，4)每两周与导师举行会议。拟议研究的具体目的是：1)研究急性间歇性卟啉症（AIP）（最常见的肝性卟啉症[由于缺乏羟甲基双烷合成酶（hhb -synthase）活性]中危及生命的急性神经系统发作的发病机制；2）评估肝细胞和骨髓移植治疗AIP的有效性，为未来以诱导多能干细胞（iPS）为基础的治疗AIP和其他复发性急性肝性卟啉症患者提供概念证明。为了实现这一目标，Aim 1将研究先前产生的具有“诱导”生化表型的T1/T2 AIP敲入小鼠和新产生的具有严重生化和临床表型的AIP敲入小鼠（R167Q+/+）的疾病发病机制。目的2将评估原代肝细胞移植和骨髓移植在清除严重感染的R167Q+/+小鼠血浆和尿液中积累的卟啉前体、4-氨基乙酰丙酸（ALA）和卟啉胆汁素原（PBG）的有效性。这些研究将确定清除循环卟啉前体所需的hmb合成酶胜任细胞的数量，以及清除是否也下调肝脏5'-氨基乙酰丙酸合成酶1 （ALAS1）活性。目的3将评估移植过表达hmb合成酶的肝细胞和骨髓（移植前用慢病毒载体进行体外转导）是否能有效减少R167Q+/+小鼠清除ALA和PBG积累所需的移植细胞数量。这是特别重要的，因为对于诸如AIP等缺乏肝细胞损伤的疾病，实现高水平的供体细胞植入和增殖是一项重大挑战，因此不能为供体细胞提供增殖优势。基于Aims 2和3的结果，Aim 4将应用最新的iPS技术开发针对AIP小鼠的治疗方法，作为未来应用于患者的模型。我们实验室最近的研究支持了细胞移植预防AIP急性发作的基本原理，该研究表明，在AIP T1/T2小鼠模型中，腹腔注射过表达hmb合酶的AAV2/8载体有效且持续地阻止了AIP急性发作的生化诱导（见初步结果，第C.3节）。这个综合的研究和教育项目，加上西奈山优秀的研究环境，应该有助于申请人作为一名独立的转化研究人员过渡到学术生涯。

DESCRIPTION (provided by applicant): The overall goal of this NIDDK Mentored Research Scientist Development Award is to provide an integrated research and educational program for the applicant that will lead to an independent academic career in translational research. Robert J. Desnick, PhD, MD, Professor and Chair of Genetics and Genomic Sciences and Associate Dean for Genome- Based Research, and Ihor Lemischka, PhD, Professor of Gene and Cell Therapy and Director of the Black Family Stem Cell Institute, will serve as Mentor and Co-Mentor, respectively. This applicant will devote 95% of her effort to the career development program which includes: 1) undertaking the proposed laboratory research, 2) training in stem cell biology and iPS techniques, 3) participation in Genetics and Stem Cell Biology "Work in Progress" sessions, journal clubs and seminars, and attendance at national scientific meetings, and 4) bi-weekly meetings with Mentors. The specific aims of the proposed research are: 1) to investigate the pathogenesis of the life-threatening acute neurological attacks in Acute Intermittent Porphyria (AIP), the most common hepatic porphyria [due to deficient activity of hydroxymethylbilane synthase (HMB-synthase)], and 2) to evaluate the effectiveness of hepatocyte and bone marrow transplantation for AIP as proof-of-concept for future induced pluripotent stem (iPS) cell-based therapies for patients with AIP and the other acute hepatic porphyrias who have recurrent attacks. Towards this goal, Aim 1 will investigate disease pathogenesis in the previously generated T1/T2 AIP knock-down mice that have an "inducible" biochemical phenotype and in the newly generated AIP knock-in mice (R167Q+/+) that have a severe biochemical and clinical phenotype. Aim 2 will evaluate the effectiveness of primary hepatocyte transplantation and bone marrow transplantation in clearing the accumulated plasma and urinary porphyrin precursors, 4-aminolevulenic acid (ALA) and porphobilinogen (PBG) in the severely affected R167Q+/+ mice. These studies will determine the number of HMB-synthase competent cells necessary to clear the circulating porphyrin precursors and whether the clearance also down-regulates hepatic 5'- aminolevulinic acid synthase 1 (ALAS1) activity. Aim 3 will assess whether transplanting HMB-synthase overexpressing hepatocytes and bone marrow (transduced ex vivo with lentiviral vectors prior to transplantation) can effectively decrease the number of engrafted cells required to clear ALA and PBG accumulation in the R167Q+/+ mice. This is of particular importance, as it is a major challenge to achieve high levels of donor cell engraftment and proliferation for diseases such as AIP that lack hepatocellular damage and thus do not provide the donor cells a proliferative advantage. Based on the results of Aims 2 and 3, Aim 4 will apply the latest iPS technology to develop treatment for the AIP mice as a model for future applications in patients. The rationale for cell transplantation to prevent the acute attacks in AIP is supported by recent studies in our laboratory demonstrating that intraperitoneal injection of an AAV2/8 vector overexpressing HMB-synthase effectively and continuously prevented the biochemical induction of an acute attack in the T1/T2 mouse model of AIP (see Preliminary Results, Section C.3.). This integrated research and educational program, together with the outstanding research environment at Mount Sinai, should facilitate the applicant's transition to an academic career as an independent translational researcher. PUBLIC HEALTH RELEVANCE: The proposed research investigates whether transplantation of hepatocytes and bone marrow cells can treat Acute Intermittent Porphyria (AIP), a hepatic disorder resulting from the deficiency of an enzyme that synthesizes heme. Development of a safe and effective treatment should protect patients from the life-threatening acute neurological attacks of AIP and improve their quality of life.