Capotondo A, Cesani M, Pepe S, Fasano S, Gregori S, Tononi L, Venneri M A, Brambilla R, Quattrini A, Ballabio A, Cosma M P, Naldini L, Biffi A
San Raffaele Telethon Institute for Gene Therapy, 20132 Milan, Italy.
Hum Gene Ther. 2007 Sep;18(9):821-36. doi: 10.1089/hum.2007.048.
Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.
基于造血干/祖细胞（HSPCs）或直接进行中枢神经系统（CNS）基因转移的异染性脑白质营养不良（MLD）成功基因治疗方法，突出了需要高水平的芳基硫酸酯酶A（ARSA）表达才能在小鼠模型中实现疾病表现的纠正。因此，全面评估ARSA表达高于生理水平的安全性是这些方法临床转化的先决条件。在这里，我们使用慢病毒载体（LVs）生成了两个相关模型，用于严格评估转导细胞中ARSA过表达的后果。我们首先证明，在克隆形成试验和新生血液嵌合小鼠模型中，人HSPCs中ARSA过表达不会影响其克隆形成和多谱系分化能力。此外，我们通过生成转基因小鼠来研究全身组织中ARSA的过表达，这些转基因小鼠通过LV使ARSA酶的过表达比正常范围高15倍，并在其基因组中携带多个LV拷贝。对这些小鼠的表征证明了在两个主要基因治疗靶点HSPCs和神经元中ARSA过表达的安全性，在超生理酶水平存在的情况下，造血和神经系统的复杂功能得以维持。在ARSA过表达的HSPCs和转基因小鼠中测试了依赖于相同共同激活剂硫酸酯酶修饰因子-1（SUMF1）的其他硫酸酯酶的活性，排除了饱和现象的发生。总体而言，这些数据表明，从临床转化的角度来看，可以安全地实现ARSA过表达的治疗水平。此外，它们展示了一个用于新基因治疗方法安全性临床前评估的实验平台。
