Di Domenico C, Di Napoli D, Gonzalez Y Reyero E, Lombardo A, Naldini L, Di Natale P
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, and Center for Animal Experimentation, Cardarelli Hospital Naples, 80128 Naples, Italy.
Hum Gene Ther. 2006 Nov;17(11):1112-21. doi: 10.1089/hum.2006.17.1112.
Mucopolysaccharidosis type I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in the accumulation of glycosaminoglycans (GAGs) in many of the cells of affected patients. Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We have previously shown in a murine model the therapeutic potential of lentiviral IDUA vector-mediated gene therapy, in which human IDUA cDNA was driven by the cytomegalovirus promoter. However, the major limitation of this approach was the induction of an immune response against the therapeutic protein, which limited the efficacy and long-term duration of treatment. In this study, we evaluate the potential of liver-directed gene therapy, that is, programming of murine hepatocytes to secrete the enzyme with mannose 6-phosphate (M6P), which can be taken up by distant cells. Eight- to 10-week-old mice were injected via the tail vein with a lentiviral vector expressing human IDUA cDNA driven by the albumin gene promoter selectively expressed in hepatocytes. One month after treatment, IDUA activity was present in the liver and spleen of treated mice; an expression level of 1% normal IDUA activity was sufficient to reduce the GAG level in liver, spleen, kidney, heart, and lung. Interestingly, 6 months after a single injection of this vector, IDUA activity was detectable in several murine tissues; the level of enzyme activity was low but sufficient to maintain the decrease in GAG levels in liver, spleen, kidney, heart, and lung. Also, the level of enzyme-specific antibodies reached at 6 months postinjection was nearly null, and real-time polymerase chain reaction analysis showed high levels of vector DNA content in liver and spleen. Thus, these results show that the use of LV with the albumin gene promoter selectively expressed in hepatocytes limited the immune response to the transgene and allowed stable and prolonged expression of the IDUA enzyme and a partial correction of the pathology.
由于α-L-艾杜糖醛酸酶（IDUA）活性缺乏导致的I型黏多糖贮积症（MPS I），会使受影响患者的许多细胞中糖胺聚糖（GAGs）蓄积。通过体内给予慢病毒载体（LVs）进行稳定的基因替代，对代谢紊乱和其他全身性疾病具有治疗潜力。我们之前在小鼠模型中展示了慢病毒IDUA载体介导的基因治疗的潜力，其中人IDUA cDNA由巨细胞病毒启动子驱动。然而，这种方法的主要局限性是诱导了针对治疗性蛋白的免疫反应，这限制了治疗效果和治疗的长期持续性。在本研究中，我们评估了肝脏定向基因治疗的潜力，即对小鼠肝细胞进行编程，使其分泌带有6-磷酸甘露糖（M6P）的酶，该酶可被远处的细胞摄取。通过尾静脉给8至10周龄的小鼠注射一种慢病毒载体，该载体表达由在肝细胞中选择性表达的白蛋白基因启动子驱动的人IDUA cDNA。治疗一个月后，在治疗小鼠的肝脏和脾脏中存在IDUA活性；正常IDUA活性1%的表达水平足以降低肝脏、脾脏、肾脏、心脏和肺中的GAG水平。有趣的是，单次注射该载体6个月后，在多个小鼠组织中可检测到IDUA活性；酶活性水平较低，但足以维持肝脏、脾脏、肾脏心脏和肺中GAG水平的降低。此外，注射后6个月时酶特异性抗体的水平几乎为零，实时聚合酶链反应分析显示肝脏和脾脏中载体DNA含量很高。因此，这些结果表明，使用在肝细胞中选择性表达白蛋白基因启动子的LVs，限制了对转基因的免疫反应，并允许IDUA酶稳定且长期表达，以及对病理状况的部分纠正。
