GENE THERAPY FOR ERYTHROPOIETIC PROTOPORPHYRIA

红细胞生成性原卟啉症（EPP）是一种遗传性疾病，其中铁螯合酶（将铁插入原卟啉的酶）缺陷。在EPP中，原卟啉积聚在红细胞中，渗漏到约5%的患者体内。由于已经证明血浆、皮肤和肝脏中发现的绝大多数原卟啉来自红细胞，我们建议针对骨髓的基因治疗可以治愈EPP。这对于严重光敏或对药物治疗有耐药性的早期肝病患者尤其有益。利用小鼠EPP（纯合隐性突变）模型，我们最近证明了将正常同基因Balb/C供者的骨髓移植到辐照的EPP受体中可以完全治愈这种疾病的表型。我们还表明，逆转录病毒介导的人铁螯合酶cDNA转移到人EPP患者外周血BFU-E中，纠正了特异性原卟啉介导的荧光。此外，我们正在通过慢病毒载体和体内选择协议开发改进的造血干细胞基因转移方法，将在EPP基因治疗模型中进行验证和优化。基于这些初步结果，我们的具体目的如下：具体目的1：证明将人铁螯合酶cDNA逆转录病毒转移到EPP供体小鼠的造血干细胞中，然后将其移植到EPP受体小鼠中，可以治愈或显著改善疾病表型。特异性目的2：比较基于小鼠白血病病毒-病毒HIV-1慢病毒载体转染小鼠造血干细胞并通过骨髓移植纠正EPP表型的效率。特异性目的3：评估EPP基因治疗方案的疗效，该方案结合骨髓移植前最小骨髓消融、多药耐药（MDR）或二氢叶酸还原酶（DHFR）逆转录病毒载体和相应的体内选择方案。

Erythropoietic protoporphyria (EPP) is a genetic disease in which ferrochelatase, the enzyme that inserts iron into protoporphyrin, is defective. In EPP, protoporphyrin accumulate in erythrocytes, leaks into about 5% of patients. Since it has been demonstrated that the vast majority of the protoporphyrin found in plasma, skin and liver derives from the erythrocytes, we propose that gene therapy directed at the bone marrow could cure EPP. This would be especially beneficial for patients with severe photosensitivity or incipient liver disease resistant to pharmacological treatment. Using a mouse model of EPP (homozygous recessive mutation), we have recently demonstrated that transplantation of bone marrow from normal syngeneic Balb/C donors into irradiated EPP recipients completely cures the disease phenotype. We have also shown that retrovirus-mediated transfer of human ferrochelatase cDNA into peripheral blood BFU-E from human EPP patients corrects the specific protoporphyrin-mediated fluorescence. In addition, we are developing improved gene transfer methods for hematopoietic stem cells by means of Lentiviral vectors and in vivo selection protocols, which will be validated and optimized in the EPP gene therapy model. Based on these preliminary results, our Specific Aims are as follows: Specific Aim 1: To demonstrate that retroviral transfer of human ferrochelatase cDNA into hematopoietic stem cells of EPP donor mice followed by their transplantation into EPP recipient mice will cure or significantly ameliorate the disease phenotype. Specific Aim 2: To compare the efficiencies of Murine Leukemia Virus-verus HIV-1 Lentivirus-based vectors to transduce murine hematopoietic stem cells and correct the EPP phenotype by bone marrow transplantation. Specific Aim 3: To evaluate the efficacy of gene therapy protocols for EPP that combine minimal myeloablation prior to bone marrow transplantation, Multi-drug Resistance (MDR) or Dihydrofolate Reductase (DHFR) retroviral vectors and corresponding in vivo selection regimens.