Acute Hepatic Porphyrias: Pathogenesis & Treatment

描述(由申请者提供)：NIDDK导师研究科学家发展奖的总体目标是为申请者提供一个综合的研究和教育计划，使其在翻译研究领域拥有独立的学术生涯。遗传学和基因组科学教授兼副院长兼基因组研究副院长、医学博士罗伯特·J·德斯尼克和基因与细胞疗法教授、黑人家庭干细胞研究所主任伊霍尔·莱米施卡博士将分别担任导师和共同导师。这位申请者将把95%的精力投入到职业发展计划中，包括：1)承担拟议的实验室研究，2)干细胞生物学和iPS技术培训，3)参加遗传学和干细胞生物学“进行中的工作”会议，杂志俱乐部和研讨会，以及出席国家科学会议，以及4)与导师的双周会议。这项研究的具体目的是：1)探讨急性间歇性门静脉综合征(AIP)--一种最常见的[由于羟甲基胆烷合成酶(HMB-Synthase)活性缺乏]引起的急性神经功能损害的发病机制，以及2)评估肝细胞和骨髓移植治疗AIP的有效性，作为未来诱导多能干细胞(IPS)细胞为基础的治疗AIP和其他反复发作的急性肝门脉疾病患者的概念验证。为了实现这一目标，Aim 1将研究先前产生的具有“可诱导”生化表型的T1/T2 AIP敲除小鼠和具有严重生化和临床表型的新产生的AIP敲入小鼠(R167Q+/+)的疾病发病机制。目的2观察原代肝细胞移植和骨髓移植对严重感染R167Q+/+小鼠血、尿中积累的卟啉前体、4-氨基酮戊酸(ALA)和胆红素原(PBG)的清除作用。这些研究将确定清除循环中的卟啉前体所需的HMB合酶活性细胞的数量，以及清除是否也下调肝脏5‘-氨基酮丙酸合酶1(ALAS1)的活性。目的3评估移植高表达HMB合酶的肝细胞和骨髓(移植前用慢病毒载体体外转导)是否能有效减少R167Q+/+小鼠体内清除ALA和PBG所需的移植细胞数量。这一点特别重要，因为对于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个重大挑战，因此不能为供体细胞提供增殖优势。根据AIMS 2和AIMS 3的结果，AIM 4将应用最新的iPS技术来开发AIP小鼠的治疗方法，作为未来在患者中应用的模型。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的T1/T2小鼠模型中，腹膜内注射过表达HMB合酶的AAV2/8载体有效并持续地防止急性发作的生化诱导(见初步结果，C.3节)。这一整合的研究和教育项目，加上西奈山出色的研究环境，应该有助于申请者过渡到作为独立翻译研究员的学术生涯。 公共卫生相关性：这项拟议的研究调查了肝细胞和骨髓细胞移植是否可以治疗急性间歇性卟啉病(AIP)，这是一种由于缺乏合成血红素的酶而引起的肝脏疾病。开发一种安全有效的治疗方法应该保护患者免受AIP危及生命的急性神经发作的影响，并提高他们的生活质量。

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.