GENE THERAPY FOR ERYTHROPOIETIC PROTOPORPHYRIA

红细胞生成性原卟啉(EPP)是一种遗传性疾病，在这种疾病中，将铁插入原卟啉的铁络合酶存在缺陷。在EPP中，原卟啉在红细胞中积聚，渗入约5%的患者体内。由于已证实血浆、皮肤和肝脏中的原卟啉绝大部分来自红细胞，我们认为针对骨髓的基因治疗可以治愈EPP。这对严重光敏或对药物治疗有抵抗力的早期肝病患者尤其有益。利用EPP(纯合子隐性突变)小鼠模型，我们最近证明了将正常同基因Balb/C供者的骨髓移植到辐射后的EPP受体中完全治愈了疾病表型。我们还表明，逆转录病毒介导的人铁络合酶基因从人EPP患者的外周血BFU-E中转移到BFU-E可以纠正原卟啉介导的特异性荧光。此外，我们正在开发利用慢病毒载体和体内选择方案进行造血干细胞基因转移的改进方法，这些方法将在EPP基因治疗模型中得到验证和优化。基于这些初步结果，我们的具体目标如下：具体目标1：证明逆转录病毒转导人铁络合酶基因到EPP供体小鼠的造血干细胞，然后将其移植到EPP受体小鼠体内，可以治愈或显著改善疾病的表型。特异性目的2：比较小鼠白血病病毒-真性HIV-1慢病毒载体转导小鼠造血干细胞的效率，并通过骨髓移植纠正EPP表型。具体目的3：评价联合骨髓移植前微量清髓、多药耐药(MDR)或二氢叶酸还原酶(DHFR)逆转录病毒载体及相应的体内选择方案治疗EPP的疗效。

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.