COMPREHENSIVE GENE THERAPY FOR MUCOPOLYSACCHARIDOSIS VII

描述(申请人提供)：我们的长期目标是开发安全和有效的方法来综合治疗粘多糖病，粘多糖病的特征是溶酶体糖胺聚糖(GAG)沉积，这是由于遗传缺乏GAG逐步降解所需的酶所致。由于GAG沉积会影响多个系统，包括肝、脾、骨骼、大脑和眼睛，因此这些疾病的基因治疗面临着特殊的挑战。在这个项目中，我们将以粘多糖症-VTI(MPS-VII，β-葡萄糖苷酸酶缺乏症，Sly综合征)为目标，因为已经有了一个特征良好的小鼠模型。在初步研究中，我们已经证明，使用基于SV40的重组载体转移β-葡萄糖醛酸酶(GUSB)基因有可能改善大脑以及肝脏和脾中的GAG沉积。第一个具体目标是优化基于SV40的载体，以实现安全和高效的系统性基因转移。新的病毒基因缺失载体将被用来调节启动子，以便在各种组织中高效地长期表达转基因。绝缘体将被引入，以保护宿主基因组中的邻近基因不会被载体中的增强子意外激活。在具体目标2中，我们将确定最佳给药途径、基因转移的有效性、组织和血清GUSB水平以及减少含有GAG的溶酶体囊泡。除了评估肝和脾的表型纠正外，还将特别强调在静脉、脑室或脑内注射载体后，将基因转移到大脑不同区域的各种类型的脑细胞。我们还将确定将这些载体注射到新生或胚胎小鼠体内的潜力，以纠正发育早期GUSB缺陷的能力，从而防止MPS VII中常见的早期脑损伤。由于基于SV-40的载体无毒、无免疫原性，并且能够整合到宿主基因组中，该项目的成功完成将为与内脏、骨骼和中枢神经系统损伤相关的所有形式的溶酶体储存障碍的全面分子治疗提供安全有效的方法。

DESCRIPTION (provided by applicant): Our long-term objective is to develop safe and effective methods for comprehensive gene-based therapies for mucopolysaccharidoses, which are characterized by lysosomal glycosaminoglycan (GAG) deposition, resulting from inherited deficiency of enzymes needed for stepwise degradation of GAGs. Because GAG deposits affect multiple systems, including the liver, spleen, bones, brain and eyes, gene therapy for these disorders present a special challenge. In this project, we will target mucopolysaccharidosis-VTI (MPS-VII, beta-glucuronidase deficiency, Sly syndrome), since a well-characterized murine model is available. In preliminary studies, we have shown that transfer of the beta-glucuronidase (GUSB) gene using recombinant SV40-based vectors has the potential to ameliorate the GAG deposits in the brain as well as in the liver and spleen. The first specific aim is to optimize SV40- based vectors for safe and efficient systemic gene transfer. Novel viral gene-deleted vectors will be generated to accommodate promoters for efficient long-term transgene expression in various tissues. Insulators will be introduced to protect neighboring genes in the host genome from being accidentally activated by enhancers within the vector. In Specific Aim 2, we will determine optimal routes of administration, efficacy of gene transfer, tissue and serum GUSB levels and reduction of GAG-containing lysosomal vesicles. In addition to evaluating phenotypic correction of the liver and spleen, special emphasis will be placed on gene transfer to various types of brain cells in different regions of the brain after intravenous, intracerebro-ventricular or intracerebral injection of the vectors. We will also determine the potential of injecting these vectors into newborn or fetal mice on the ability to correct GUSB deficiency early in development, thereby preventing early brain damage, which is common in MPS VII. Since SV-40-based vectors are non-toxic, non-immunogenic and capable of integrating into the host genome, successful completion of this project should provide a safe and effective method for comprehensive molecular therapy of all forms of lysosomal storage disorders that are associated with visceral, skeletal and central nervous system injury.