Advancing Gene Therapy for Late Infantile Neuronal Ceroid Lipofuscinosis

描述(申请人提供)：晚期婴儿神经性干酪样脂褐质沉着症(LINCL)是一种儿童起病的常染色体隐性遗传性神经退行性疾病。这种疾病是一种溶酶体储存疾病(LSD)，由CLN2基因突变引起，CLN2基因编码溶酶体水解酶三肽基肽酶I(TPP1)。TPP1缺乏导致癫痫发作、视力障碍、认知和运动功能障碍，并总是在儿童后期致命。目前，还没有治疗或治愈这种毁灭性疾病的方法。治疗的主要障碍是将具有生物活性的TPP1酶输送到中枢神经系统，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的腺相关病毒(AAV)基因递送载体。该病毒的脑嗜性是由一种多肽GMNAFRA授予的，我们通过在LINCL小鼠模式中的体内噬菌体展示平移来鉴定它。GMN肽与脑血管内皮细胞的管腔表面具有亲和力，而脑血管内皮细胞是血脑屏障的主要成分。当这种多肽修饰的病毒(GMN-AAV)外周传递时，它会传播到大脑内皮细胞，并进行传导。值得注意的是，在LINCL小鼠体内交付编码CLN2的GMN-AAV恢复了脑内TPP1的表达，并纠正了神经病理缺陷。因此，这种新的基因治疗方法通过改造内皮细胞将重组酶直接分泌到潜在的神经纤维中，从而绕过了血脑屏障。为了推动这一令人兴奋和有希望的基因治疗方法应用于LINCL患者，需要阐明与GMN肽相互作用的脑内皮细胞受体分子的身份。这项提议的总体目标是进行实验，以发现这种分子，它介导GMN-AAV脑靶向和转导。我们的假设是，该受体是一种质膜相关蛋白或糖链，表达于TPP1缺陷脑组织中脑血管内皮细胞表面。在第一个目标中，我们将使用亲和纯化和定量质谱学的方法分离和鉴定GMN-AAV受体。在第二个目标中，我们将使用由美国国立卫生研究院运营的功能性糖链联合会开发的哺乳动物糖链阵列来确定GMN-AAV是否与可能存在于脑内皮细胞表面的特定糖链或类型的糖链结合。这些目标的实现将使我们能够推进本文件的翻译 儿童LINCL的新基因治疗。此外，这一治疗策略也可以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。 公共卫生相关性：拟议的研究将确定通过AAV基因递送载体介导脑血管内皮细胞结合和转导的细胞表面受体。该载体可以恢复TPP1酶的表达，并防止晚期婴儿神经元Ceroid Lipofuscinsis(LINCL)啮齿动物模型的神经病理，LINCL是一种目前无法治疗的致命神经退行性疾病。这些研究的结果有望推动这种极有希望的基因疗法应用于LINCL患者的治疗。

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 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. PUBLIC HEALTH RELEVANCE: The proposed research will identify the cell-surface receptor that mediates binding and transduction of brain vascular endothelia by an AAV gene delivery vector. This vector can restore TPP1 enzyme expression and prevent neuropathology in a rodent model of Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL), a currently untreatable fatal neurodegenerative disorder. Results of these studies are anticipated to advance this highly promising gene therapy to treatment of LINCL patients.