Translational studies on cerebrospinal fluid (CSF)-directed gene therapy for global neurometabolic brain disease

摘要 在大多数遗传性疾病中有效治疗中枢神经系统（CNS）的主要障碍是 这种病理学存在于整个大脑中，因为代谢缺陷存在于大多数脑细胞中。 因此，需要基因或基因产物的全球分布。几种基因治疗策略正在 研究用于治疗全脑病变。然而，所有这些方法都具有显著的 这些缺点在人类疾病的大型动物模型中变得明显。开发更 这些模型中的有效治疗将有助于转化为临床应用。 本项目将研究AAV载体介导的基因递送到脑中，通过输注到脑中， 脑脊液（CSF），其可导致基因在大型动物脑中的播散性递送。的 待评价的疾病模型是猫中的α-甘露糖苷沉积症（AMD），一种溶酶体贮积症（LSD）， 由溶酶体酶（LE）结构基因α-甘露糖苷酶（MANB）突变引起。战略 治疗是基于交叉校正，其中将MANB cDNA的正常拷贝转移到AMD中， 细胞导致那些基因转导细胞的代谢校正。此外，基因校正 细胞释放正常的MANB酶，被周围的细胞吸收，并在代谢上纠正它们， 好.这种完善的交叉校正机制是大多数LSD治疗的基础。 将通过临床试验在活体动物中监测疾病进展和治疗后的改善。 神经系统评估，寿命延长，血清和CSF分析，以及通过 磁共振波谱（MRS）和扩散张量成像（DTI）。在动物死后， 将定量分析疾病校正的载体转导分布、MANB增加 酶活性、含甘露糖寡糖底物积累的减少、组织病理学 变化和潜在的不利宿主反应。 我们将通过研究策略来解决影响翻译的重要问题， 大脑的完全矫正：在目标1中，我们将研究CSF递送的替代途径的效果; 在目标2中，我们将研究剂量递增对病理学消退程度的影响;在目标3中， 我们将确定治疗的有效性时，开始在逐步更严重的阶段的疾病， 评估改善晚期疾病的潜力。

ABSTRACT A major barrier to effective treatment of the central nervous system (CNS) in most inherited diseases is that pathology is present throughout the brain because the metabolic defect is present in most brain cells. Thus, global distribution of the gene or gene product is required. Several gene therapy strategies are being investigated for treatment of global brain lesions. However, all of the approaches have significant shortcomings, which become apparent in large animal models of human diseases. Development of more effective treatments in these models will facilitate translation into clinical usage. This project will investigate AAV vector mediated gene delivery into the brain by infusion into the cerebrospinal fluid (CSF), which can result in disseminated delivery of a gene in a large animal brain. The disease model to be evaluated is alpha-mannosidosis (AMD) in the cat, a lysosomal storage disorder (LSD) caused by mutations in the lysosomal enzyme (LE) structural gene, alpha-mannosidase (MANB). The strategy for treatment is based on cross-correction, in which transfer of a normal copy of the MANB cDNA into AMD cells results in the metabolic correction of those gene-transduced cells. Furthermore, the genetically corrected cells release normal MANB enzyme, which is taken up by surrounding cells and corrects them metabolically as well. This well-established cross-correction mechanism is the basis for treatments of most LSDs. The disease progression and improvement from treatment will be monitored in living animals by clinical neurological assessment, life-span increases, serum and CSF analyses, and non-invasive brain imaging by magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI). In post-mortem animals, the disease correction will be analyzed quantitatively for distribution of vector transduction, increases in MANB enzymatic activity, reduction of mannose-containing oligosaccharide substrate accumulation, histopathological changes, and potential adverse host responses. We will address important issues affecting translation by investigating strategies to attain more complete correction of the brain: in Aim 1 we will investigate the effects of alternative routes of CSF delivery; in Aim 2 we will investigate the effects of dose escalation on the extent of resolution of pathology; and in Aim 3 we will determine the effectiveness of therapy when initiated at progressively more severe stages of disease to evaluate the potential to ameliorate advanced disease.