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 这种病理在整个大脑中都存在，因为大多数脑细胞都存在代谢缺陷。 因此，基因或基因产物的全球分布是必要的。几种基因治疗策略正在进行中 因治疗全球脑部病变而接受调查。然而，所有的方法都有重要的 缺点，这在人类疾病的大型动物模型中变得明显。开发更多 这些模型的有效治疗将有助于将其转化为临床应用。 本项目将研究AAV载体介导的基因通过输注到脑内的方式进入大脑 脑脊液(CSF)，这可能导致基因在大型动物大脑中的播散性传递。这个 待评估的疾病模型是猫的α-甘露糖苷症(AMD)，一种溶酶体储存障碍(LSD) 由溶酶体酶(LE)结构基因α-甘露糖苷酶(MANB)突变引起。战略 治疗是基于交叉矫正，在这种治疗中，将MANB基因的正常拷贝转移到AMD 细胞导致这些基因转导细胞的代谢校正。此外，经过基因矫正的 细胞释放正常的MANB酶，该酶被周围的细胞吸收，并在代谢过程中纠正为 井。这一完善的交叉校正机制是大多数LSD治疗的基础。 临床将在活体动物身上监测疾病的进展和治疗后的改善 神经学评估、寿命延长、血清和脑脊液分析，以及非侵入性脑成像 磁共振波谱(MRS)和扩散张量成像(DTI)。在死后的动物中， 疾病矫正将定量分析媒介转导的分布，MANB的增加 酶活性，减少含甘露糖低聚糖底物积累，组织病理学 变化，以及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，以实现更多 完全矫正大脑：在目标1中，我们将调查不同的脑脊液输送途径的效果； 在目标2中，我们将研究剂量增加对病理消退程度的影响；在目标3中，我们将研究剂量增加对病理分辨率的影响。 我们将确定在疾病逐渐严重的阶段开始治疗的有效性 评估改善晚期疾病的可能性。

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.