Enhancement of Gene Therapy Outcomes for Fabry Disease

提高法布里病的基因治疗效果

• 批准号: 6797751
• 负责人: JEFFREY A MEDIN
• 金额: $ 20万
• 依托单位: ONTARIO CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-29 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6797751
• 关键词: Fabry' s disease; alpha galactosidase; carbohydrate receptor; cell line; cofactor; enzyme mechanism; gene therapy; hematopoietic tissue transplantation; laboratory mouse; nonhuman therapy evaluation; receptor mediated endocytosis; recombinant proteins; transfection /expression vector

DESCRIPTION (provided by applicant): Fabry disease is the 2nd-most prevalent lysosomal storage disorder (LSD) in humans. It is X-linked and pan-ethnic with a frequency of about1:40,000 males. In Fabry disease the deficiency of the lysosomal enzyme alpha-galactosidase A (a-gal A) are mainly manifested in the vascular endothelium. These cells build up excessive lipids with terminal galactose residues (mainly ceramide trihexoside; CTH) leading to vessel occlusions. A major source of CTH in Fabry disease is from the hematopoietic system-specifically from the breakdown of RBCs by macrophages. Patients succumb to renal, cardiovascular, or cerebrovascular disease in mid life. Current treatment for the disorder is only palliative. Unlike many other LSDs, limited primary nervous system involvement is observed in Fabry disease and even 5 percent of normal enzyme activity may improve the clinical course. An a-gal A deficient mouse has been created that offers a model for studies on the pathophysiology and the development of therapeutic strategies. Fabry disease and this model offer compelling systems to develop and test methods for the improvement of gene therapy. A phenomenon called metabolic cooperativity exists wherein genetically corrected cells secrete the hydrolase-which can be taken up and used by bystander cells. This allows for correction of a lower number of cells to impact therapy. We have previously created retroviral vectors and corrected a variety of Fabry patient and a-gal A-deficient mouse cells. We have also demonstrated that metabolic cooperativity occurs in vivo. The Specific Aims of this application are designed to further this therapeutic approach in order to improve outcomes for Fabry disease-and possibly for other LSDs. Hypothesis 1: In the a-gal A-deficient mouse pre-selection of retrovirally transduced cells co-expressing a selectable marker and the therapeutic gene will improve metabolic cooperativity over that seen with non-enriched cells, as measured systemically by increased levels of a-gal A activity and decreased CTH levels. Hypothesis 2: Retroviral vectors (including novel recombinant lentiviruses) can be generated that encode marking or the therapeutic a-gal A gene that lead to the specific amplification of transduced cells relevant to Fabry disease upon the addition of selectively active co-factors both in vitro and in vivo. Hypothesis: Co-overexpression of the prosaposin gene, a protein co-factor for the a-gal A enzyme, leads to higher catalytic activity in genetically corrected cells than overexpression of a-gal A alone.

描述（由申请人提供）：法布里病是第二常见的疾病 人类溶酶体贮积症（LSD）。它是 X 连锁的、泛种族的 频率约为 1:40,000 男性。在法布里病中，缺乏 溶酶体酶α-半乳糖苷酶A（a-gal A）主要表现在 血管内皮。这些细胞会积累过多的脂质，并最终导致 通向血管的半乳糖残基（主要是神经酰胺三己糖苷；CTH） 遮挡。法布里病中 CTH 的主要来源是造血系统 系统特异性地来自巨噬细胞对红细胞的分解。患者屈服 中年时患肾脏、心血管或脑血管疾病。当前的 这种疾病的治疗只是姑息治疗。与许多其他 LSD 不同，它的作用有限 在法布里病中观察到原发性神经系统受累，甚至 5 正常酶活性的百分比可以改善临床病程。一个 a-gal A 缺陷小鼠的诞生为研究提供了一个模型 病理生理学和治疗策略的发展。法布里病 该模型提供了令人信服的系统来开发和测试方法 基因治疗的改进。存在一种称为代谢协同性的现象 其中经过基因修正的细胞会分泌水解酶——可以被吸收 并被旁观者细胞使用。这允许校正较少数量的 细胞影响治疗。我们之前已经创建了逆转录病毒载体 纠正了多种 Fabry 患者和 a-gal A 缺陷小鼠细胞。我们有 还证明了体内发生代谢协同性。具体 本申请的目的旨在进一步推动这种治疗方法 以改善法布里病以及其他 LSD 的治疗结果。 假设1：在a-gal A缺陷小鼠中逆转录病毒预选 共表达选择标记和治疗基因的转导细胞 与非富集细胞相比，将提高代谢协同性，如 通过 a-gal A 活性水平升高和 CTH 降低进行系统测量 水平。 假设2：逆转录病毒载体（包括新型重组慢病毒）可以 产生编码标记或治疗性 a-gal A 基因，从而导致 与法布里病相关的转导细胞的特异性扩增 添加体外和体内选择性活性辅助因子。 假设：prosaposin 基因（一种蛋白质辅因子）的共同过度表达 a-gal A 酶，在基因校正中具有更高的催化活性 细胞比单独过度表达 a-gal A 更有效。