Acute Hepatic Porphyrias: Pathogenesis & Treatment

该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，这将导致翻译研究的独立学术生涯。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，黑人家庭干细胞研究所所长，将分别担任导师和共同导师。申请人将95%的精力投入到职业发展计划中，其中包括：1）进行拟议的实验室研究，2）干细胞生物学和iPS技术培训，3）参与遗传学和干细胞生物学“正在进行的工作”会议，期刊俱乐部和研讨会，并参加国家科学会议，以及4）与导师的双周会议。拟议研究的具体目标是：1）探讨急性间歇性卟啉病（AIP）中危及生命的急性神经系统发作的发病机制，AIP是最常见的肝性卟啉病[由于羟甲基胆烷合酶活性不足（HMB-合酶）]，和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（iPS）细胞的概念验证-AIP和其他复发性急性肝卟啉病患者的基础治疗。为了实现这一目标，Aim 1将在先前产生的具有“诱导型”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（R167 Q +/+）中研究疾病发病机制。目的2探讨原代肝细胞移植和骨髓移植对R167 Q +/+小鼠血浆和尿卟啉前体、4-氨基酮戊酸（ALA）和胆色素原（PBG）的清除作用。这些研究将确定清除循环卟啉前体所需的HMB合酶感受态细胞的数量，以及清除是否也下调肝5 '-氨基乙酰丙酸合酶1（ALAS 1）活性。目的3将评估移植过表达HMB合酶的肝细胞和骨髓（在移植前用慢病毒载体离体转导）是否可以有效地减少清除R167 Q +/+小鼠中ALA和PBG积累所需的移植细胞的数量。这是特别重要的，因为对于缺乏肝细胞损伤的疾病如AIP，实现高水平的供体细胞植入和增殖是一个主要挑战，因此不为供体细胞提供增殖优势。基于目标2和3的结果，目标4将应用最新的iPS技术开发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以预防AIP急性发作的基本原理，这些研究表明，腹膜内注射过表达HMB-合酶的AAV 2/8载体有效且连续地预防了AIP的T1/T2小鼠模型中急性发作的生化诱导（参见初步结果，第C.3节）。这个综合的研究和教育计划，加上西奈山优秀的研究环境，应该有利于申请人过渡到学术生涯作为一个独立的翻译研究人员。 公共卫生相关性：拟议的研究调查肝细胞和骨髓细胞的移植是否可以治疗急性间歇性卟啉症（AIP），这是一种由合成血红素的酶缺乏引起的肝脏疾病。开发安全有效的治疗方法应保护患者免受AIP危及生命的急性神经系统发作，并改善其生活质量。

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.