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

描述（由申请方提供）：这是一项继续研究克拉伯病或球样细胞脑白质营养不良（GLD）小鼠模型治疗的建议，GLD是一种由半乳糖苷酶（GALC）活性缺乏引起的常染色体隐性遗传疾病。GALC负责半乳糖神经酰胺和精神病的溶酶体催化。在GLD中，CNS和PNS中精神病碱的积累导致髓鞘形成细胞的凋亡性死亡。其他机制，包括激活驻留的小胶质细胞，募集血液巨噬细胞和炎症，也发挥重要作用的发病机制。目前对某些患者可用的唯一治疗方法是造血干细胞移植（HSCT）。虽然这种治疗有助于维持认知能力，但运动功能仍然是一个问题。在狗和小鼠模型中使用病毒载体和HSCT的治疗试验尚未导致完全的临床、生化和病理纠正。很明显，要完全成功地治疗，需要联合治疗。有关早期病理变化和GALC活性的时间和剂量的问题仍有待回答。在目标1中，我们将产生在四环素（泰特）响应性启动子控制下具有GALC表达的转基因小鼠。最初，我们将产生Tet-Off小鼠（trG），其在不存在泰特类似物的情况下具有高GALC表达。在对这些小鼠进行详细表征后，将其与杂合GALC缺陷小鼠交配，以产生除泰特类似物调节外无GALC活性的小鼠。trG小鼠将用于回答关于治疗时间和校正所需的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用于体内和离体治疗试验的病毒载体来改善BMT的结果。我们已经制备了一种高滴度的慢病毒载体，用于提高骨髓细胞中GALC的活性，用于移植和直接脑内注射。此外，我们将构建含有小鼠GALC基因和泰特应答元件的慢病毒载体。该载体将用于转染小鼠BM细胞，所述小鼠BM细胞将具有高于来自正常供体小鼠的未转导细胞中存在的低水平的GALC高活性，此外，将具有在泰特类似物控制下的GALC表达。我们将确定注射到后肢肌肉中的GALC-AAV 2/1获得高GALC表达以校正接受BMT的小鼠中的残余运动缺陷的能力。此外，mGALC-AAV 2/1的脑内、脑室内和小脑内注射将与BMT和可减少炎症和防止细胞凋亡的批准药物组合。最初这些药物将包括米诺环素和吲哚美辛。这些药物已成功地减少脱髓鞘，并在其他神经退行性疾病中提供神经保护。使用狗GLD模型的研究已从本申请中删除。 Krabbe病是一种遗传性疾病，主要影响婴儿。目前的治疗选择有限，但在动物模型中提出的研究可能会为人类患者提供更好的治疗方法。可能需要联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.