Advancing Gene Therapy for Late Infantile Neuronal Ceroid Lipofuscinosis

描述（由申请人提供）：晚期婴儿神经性Ceroid脂褐质病（LINCL）是一种儿童期发病的常染色体隐性神经退行性疾病。这种疾病是一种溶酶体贮积病（LSD），由编码溶酶体水解酶三肽基肽酶I （TPP1）的基因CLN2突变引起。TPP1缺乏会导致癫痫发作、视力损害、认知和运动功能障碍，并且在儿童晚期总是致命的。目前，没有治疗或治愈这种毁灭性疾病的方法。治疗的主要障碍是生物活性TPP1酶向中枢神经系统的传递，这一目标受到血脑屏障（BBB）选择性的阻碍。在最近的研究中，我们的实验室设计了一种脑归巢腺相关病毒（AAV）基因传递载体。该病毒的脑向性是由一种名为GMNAFRA的肽赋予的，我们通过在LINCL小鼠模式下的体内噬菌体展示筛选鉴定了该肽。GMN肽对脑血管内皮细胞的管腔表面有亲和力，这是血脑屏障的主要成分。当外周递送时，这种肽修饰的病毒（GMN-AAV）到达并转导脑内皮。值得注意的是，在LINCL小鼠中递送编码CLN2的GMN-AAV可恢复脑内TPP1的表达并纠正神经病理缺陷。因此，这种新的基因治疗方法绕过血脑屏障，通过工程内皮细胞将重组酶直接分泌到潜在的神经瘤中。为了将这种令人兴奋和有前景的基因治疗方法用于LINCL患者，需要阐明与GMN肽相互作用的脑内皮受体分子的身份。本课题的总体目标是通过实验发现这种介导GMN-AAV脑靶向和转导的分子。我们的假设是，受体是一种质膜相关蛋白或多糖片段，在TPP1缺陷的大脑中表达于脑血管内皮细胞表面。在目标一中，我们将使用亲和纯化和定量质谱方法分离和鉴定GMN-AAV受体。在目标二中，我们将使用质谱分析和由美国国立卫生研究院运作的功能性糖组学联盟开发的哺乳动物聚糖阵列来确定GMN-AAV是否与可能存在于脑内皮细胞表面的特定聚糖或聚糖类结合。这些目标的完成将使我们能够推进这种新型基因疗法对LINCL儿童的翻译。此外，该治疗策略可广泛应用于其他导致中枢神经系统功能障碍和神经变性的lsd。

DESCRIPTION (provided by applicant): Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) is a childhood-onset autosomal recessive neurodegenerative disease. This disorder, a lysosomal storage disease (LSD), is caused by mutations in CLN2, the gene that encodes the lysosomal hydrolase tripeptidyl peptidase I (TPP1). TPP1 deficiency causes epileptic seizures, vision impairment, cognitive and motor dysfunction, and is invariably fatal by late childhood. Currently, there is no treatment or cure for this devastating disease. The major barrier to treatment is the delivery of bioactive TPP1 enzyme to the central nervous system, a goal hindered by the selectivity of the blood-brain-barrier (BBB). In recent studies, our lab has engineered a brain-homing adeno-associated virus (AAV) gene delivery vector. Brain tropism of the virus is conferred by a peptide, GMNAFRA, which we identified by in vivo phage display panning in a mouse mode of LINCL. The GMN peptide has affinity for the luminal surface of brain vascular endothelial cells, which are the primary component of the BBB. When delivered peripherally, this peptide-modified virus (GMN-AAV) travels to, and transduces brain endothelia. Significantly, delivery of GMN-AAV encoding CLN2 in LINCL mice restored TPP1 expression in the brain and corrected neuropathological defects. Thus, this novel gene therapy approach bypasses the BBB by engineering endothelial cells to secrete recombinant enzyme directly into the underlying neuropil. In order to advance this exciting and promising gene therapy approach to LINCL patients, elucidation of the identity of the brain endothelial receptor molecule that interacts with the GMN peptide is required. The overall goal of this proposal is to perform experiments to discover this molecule, which mediates GMN-AAV brain targeting and transduction. Our hypothesis is that the receptor is a plasma membrane-associated protein or glycan moiety expressed on the surface of brain vascular endothelial cells in TPP1 deficient brain. In aim one, we will use affinity purification and quantitative mass spectrometry methods to isolate and identify the GMN-AAV receptor. In aim two, we will use mass spectrometry glycan profiling, and a mammalian glycan array developed by the NIH-operated Consortium for Functional Glycomics to determine whether GMN-AAV binds to specific glycans or classes of glycans that may be present on the surface of brain endothelial cells. Completion of these aims will enable us to advance the translation of this novel gene therapy to children with LINCL. In addition, this therapeutic strategy could be broadly applied to other LSDs that cause central nervous system dysfunction and neurodegeneration.