Enhancement of Gene Therapy Outcomes for Fabry Disease

描述(申请人提供)：法布里病是第二大最常见的疾病 人类的溶酶体储存障碍(LSD)。它是X链接的，泛种族的 大约有1：40,000的雄性。在法布里病中， 溶酶体酶α-半乳糖苷酶A(a-GalA)主要表现在 血管内皮细胞。这些细胞在终末积聚过多的脂质。 通向血管的半乳糖残留物(主要是神经酰胺三己糖苷；Cth) 闭塞。Fabry病中Cth的一个主要来源是造血系统 系统--特别是巨噬细胞对红细胞的破坏。病人屈从于 到中年患上肾脏、心血管或脑血管疾病。当前 对这种疾病的治疗只是治标不治本。与许多其他LSD不同，有限 在Fabry病中观察到初级神经系统受累，甚至5 酶活性正常的百分率可改善临床病程。一个A-Gal A 已培育出缺陷小鼠，为研究小白鼠提供了一种模型。 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 改进基因治疗。存在一种称为新陈代谢协同性的现象 其中经过基因修正的细胞分泌水解酶--可以被吸收 并被旁观者细胞利用。这允许对较少数量的 影响治疗的细胞。我们之前已经创造了逆转录病毒载体和 纠正了各种Fabry患者和a-GalA缺陷小鼠细胞。我们有 也证明了代谢协同作用发生在体内。具体的 这项申请的目的是为了在 以改善法布里病的预后--可能还包括其他LSD。 假设1：在a-GalA缺陷小鼠中预先选择逆转录病毒 共表达可选择标记和治疗基因的转导细胞 与未浓缩的细胞相比，将改善代谢的协同性，因为 通过升高的α-GalA活性水平和降低的Cth水平进行系统测量 级别。 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α-A基因，从而导致 与Fabry病相关的转导细胞的特异性扩增 在体外和体内添加选择性活性的辅助因子。 假设：丙皂苷基因的共表达，这是一种与蛋白质有关的辅助因子 α-半乳糖A酶，导致基因矫正的更高的催化活性 细胞，而不是单独过表达α-GalA。

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.