Krabbe disease: Combined therapies for the central and peripheral nervous systems

描述(由申请人提供)：这是一项继续我们治疗Krabbe病或球状细胞白质营养不良(GLD)小鼠模型的研究，GLD是一种常染色体隐性遗传病，由半乳糖脑苷酶(GALC)活性不足引起。GALC负责半乳糖神经酰胺和神经氨酸的溶酶体分解代谢。在GLD中，神经氨酸在CNS和PNS中的积聚导致髓鞘形成细胞的凋亡性死亡。其他机制，包括驻留的小胶质细胞的激活，血巨噬细胞的募集和炎症，也在发病机制中发挥重要作用。目前对一些患者可用的唯一治疗方法是造血干细胞移植(HSCT)。虽然这种疗法有助于保持认知能力，但运动功能仍然是一个问题。在狗和小鼠模型上进行的病毒载体和造血干细胞移植治疗试验并未导致完全的临床、生化和病理纠正。很明显，要获得完全成功的治疗，需要综合治疗。与GALC早期病理改变、GALC活动时间和剂量有关的问题仍有待回答。在目标1中，我们将产生在四环素(TET)响应启动子控制下具有GALC表达的转基因小鼠。最初，我们将培育出在没有TET类似物的情况下具有高GALC表达的Tet-Off小鼠(TRG)。在对这些小鼠进行详细的鉴定后，它们将与杂合的GALC缺陷小鼠交配，产生除受tet类似物调控的GALC活性以外没有GALC活性的小鼠。TRG小鼠将被用来回答有关治疗时机和纠正所需的GALC活性剂量的重要问题，并作为移植干细胞的来源。在目标2中，我们将通过制备和测试体内和体外治疗试验的病毒载体来改善骨髓移植的结果。我们制备了一种高效价慢病毒载体，既可以提高骨髓细胞的GALC活性，也可以用于移植和脑内直接注射。此外，我们还将构建包含小鼠GALC基因和tet反应元件的慢病毒载体。该载体将被用于转导具有GALC高活性的小鼠BM细胞，高于正常供体小鼠未转导细胞的低水平，此外，还将在TET类似物的控制下表达GALC。我们将确定GALC-AAV2/1注射到后肢肌肉中获得高GALC表达的能力，以纠正接受骨髓移植的小鼠的残余运动缺陷。此外，脑内、脑室和小脑内注射mGALC-AAV2/1将与骨髓移植和批准的可以减少炎症和防止细胞凋亡的药物相结合。最初，这些药物将包括米诺环素和吲哚美辛。这些药物已经成功地减少了脱髓鞘，并在其他神经退行性疾病中提供了神经保护。使用GLD的狗模型的研究已经从这一应用中删除。Krabbe病是一种遗传性疾病，主要影响婴儿。目前治疗选择有限，但在动物模型中提出的研究可能会为改善人类患者的治疗方法铺平道路。可能需要结合治疗来阻止病理事件并提供长期纠正。

DESCRIPTION (provided by applicant): This is a proposal to continue our studies on the treatment of the mouse models of Krabbe disease or globoid cell leukodystrophy (GLD), an autosomal recessive disorder caused by the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide and psychosine. In GLD, the accumulation of psychosine in the CNS and PNS results in the apoptotic death of myelin-forming cells. Other mechanisms, including activation of resident microglial cells, recruitment of blood macrophages and inflammation, also play important roles in the pathogenesis. The only treatment currently available for some patients is hematopoietic stem cell transplantation (HSCT). While this treatment helps to maintain cognitive abilities, motor function remains a problem. Therapy trials with viral vectors and HSCT in the dog and mouse models have not lead to complete clinical, biochemical and pathological correction. It is clear that combined therapy will be required for entirely successful treatment. Questions related to early pathological changes and timing and dosage of GALC activity remain to be answered. In Aim 1, we will generate transgenic mice that have GALC expression under control of a tetracycline (tet) responsive promoter. Initially, we will produce Tet-Off mice (trG) that will have high GALC expression in the absence of tet analogues. After detailed characterization of these mice, they will be mated with heterozygous GALC-deficient mice to produce mice with no GALC activity except that regulated by tet analogues. The trG mice will be used to answer important questions regarding the timing of treatment and dosage of GALC activity needed for correction, and as a source of stem cells for transplantation. In Aim 2, we will improve upon the outcome of BMT by preparing and testing viral vectors for in vivo and ex vivo therapy trials. We have prepared a high titer lentiviral vector both for raising GALC activity in bone marrow cells for transplantation and for direct intracerebral injection. In addition we will construct a lentiviral vector containing both the mouse GALC gene and a tet responsive element. This vector will be used to transduce mouse BM cells that will have GALC high activity, above the low levels present in untransduced cells from normal donor mice and, in addition, will have the expression of GALC under control of tet analogues. We will determine the ability of GALC-AAV2/1 injected into the hind limb muscles to attain high GALC expression in order to correct the residual motor deficits in mice receiving BMT. Also, intracerebral, intraventricular and intracerebellar injections of mGALC-AAV2/1 will be combined with BMT and approved drugs that could reduce inflammation and prevent apoptosis. Initially these drugs will include minocycline and Indomethacin. These drugs have been successful in reducing demyelination and in providing neuroprotection in other neurodegenerative disorders. Studies using the dog model of GLD have been dropped from this application. Krabbe disease a genetic disease affecting mostly infants. Treatment options are limited at this time, but the studies proposed in animal models could lead the way to improved therapies for human patients. A combination of therapies may be needed to both stop the pathological events and provide long-term correction.