Enhancement of Gene Therapy Outcomes for Fabry Disease

描述（由申请人提供）：法布里病是第二大流行病 溶酶体贮积症（LSD）。它是X连锁的，泛种族的， a frequency频率of about 1：40，000 males男性.在法布里病中， 溶酶体酶α-半乳糖苷酶A（α-gal A）主要表现在 血管内皮这些细胞积累过多的脂质， 半乳糖残基（主要是神经酰胺三己糖苷; CTH）导致血管 闭塞。法布里病中CTH的主要来源是造血干细胞。 巨噬细胞对红细胞的破坏是系统特异性的。患者死亡 肾脏、心血管或脑血管疾病。电流 对该病症的治疗仅是姑息性的。与许多其他LSD不同， 在法布里病中观察到原发性神经系统受累， 正常酶活性的百分比可以改善临床过程。一个a-gal A 已经建立了缺陷小鼠，为研究 病理生理学和治疗策略的发展。法布里病 这个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。存在一种叫做代谢协同性的现象 其中遗传校正的细胞分泌水解酶， 并被旁观者细胞利用。这允许校正较低数量的 细胞来影响治疗。我们以前已经创造了逆转录病毒载体， 校正了多种法布里病患者和α-gal A缺陷小鼠细胞。我们有 也证明了代谢协同性发生在体内。具体 本申请的目的是为了进一步促进这种治疗方法， 以改善法布里病的预后，并可能改善其他LSD的预后。 假设1：在α-gal A缺陷小鼠中，逆转录病毒的预选择 共表达选择标记和治疗基因的转导细胞 将改善与非富集细胞相比的代谢协同性， 通过增加的α-半乳糖醛酸A活性水平和降低的CTH来全身测量 程度. 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-gal A基因，其导致 与法布里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辅因子。 假设：鞘脂激活蛋白原基因的共过表达，鞘脂激活蛋白原基因是一种蛋白质辅因子， α-半乳糖醛酸A酶，导致更高的催化活性，在遗传校正 细胞比单独的α-gal A过表达。

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.