GM2 Gangliosidosis Therapy Using Neurotropic Enzyme

使用神经营养酶治疗 GM2 神经节苷脂沉积症

• 批准号: 7897875
• 负责人: KOSTANTIN DOBRENIS
• 金额: $ 22.71万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897875
• 关键词: Affect; Affinity; Age; Amino Acids; Avidity; Axonal Transport; Behavioral; Binding; Binding Sites; Biochemical; Birth; Blood; Blood - brain barrier anatomy; Bone Marrow Transplantation; Brain; C-terminal; Cell membrane; Cells; Cessation of life; Child; Childhood; Chimera organism; Chimeric Proteins; Clinical Trials; Code; Coupling; Disease; Effectiveness; Endocytosis; Engineering; Enzyme Tests; Enzymes; Extracellular Space; G(M2) Ganglioside; Gangliosidoses GM2; Gene Proteins; Genes; Genetic; Hematopoietic; Hematopoietic stem cells; Hexosaminidases; Human; Immunologics; Injection of therapeutic agent; Intramuscular; Intramuscular Injections; Intraperitoneal Injections; Intravenous; Investigation; Knockout Mice; Lead; Lentivirus Vector; Lysosomal Storage Diseases; Lysosomes; Manuscripts; Mediating; Membrane; Mental Retardation; Methods; Microglia; Modality; Modification; Motor Neurons; Mus; Neuraxis; Neurons; Open Reading Frames; Pathology; Patients; Peptides; Perikaryon; Peripheral; Property; Proteins; Recombinant Proteins; Recombinants; Regimen; Replacement Therapy; Route; Sandhoff Disease; Solutions; Stem cells; Synapses; System; Tay-Sachs Disease; Testing; Tetanus Toxin; Therapeutic; Therapeutic Agents; Tongue; Translating; Transplantation; base; beta-n-acetylhexosaminidase; cellular transduction; enzyme activity; enzyme replacement therapy; fusion gene; gene replacement; gene replacement therapy; gene therapy; improved; in vivo; mouse model; neuronal cell body; neurotropic; public health relevance; receptor; relating to nervous system; retrograde transport; success; theories; therapeutic effectiveness; uptake

DESCRIPTION (provided by applicant): Neuronal storage disorders result from genetic deficiency in lysosomal enzymes, or accessory proteins, leading to substrate accumulation throughout the central nervous system (CNS), and remain incurable. Enzyme replacement (ERT), hematopoietic stem cell replacement (HSCRT) and gene replacement therapies variably rely on delivery of exogenous or secreted enzyme to lysosomes of host deficient neurons through endocytic uptake. Effectiveness of CNS therapy has been limited by the blood brain barrier, the need for global CNS delivery, and low neuronal endocytic rates. The atoxic C-fragment (Hc) of tetanus toxin is a potential solution to all these problems. Hc binds universally to neuronal membrane, is efficient endocytosed, can undergo axonal retrograde transport permitting CNS entry via peripheral terminals of motoneurons, and is retrogradely transsynaptically transferred to higher order neurons. We previously demonstrated that efficient neuronal uptake and depletion of lysosomal storage could be attained by chemically coupling beta- hexosaminidase (Hex), the deficient enzyme in GM2 gangliosidosis, to Hc. In recent studies on a 34 residue peptide (Hc1282) derived from Hc, we have found Hc1282 binds to neurons, enhances macromolecular uptake, can further enhance neuronal uptake when in multivalent form, and displays more efficient lysosomal delivery than Hc, We hypothesize that a fusion gene of the Hex beta-subunit (hexb) coding region and Hc or an Hc subsequence can yield a functional chimeric protein that will lead to an efficacious treatment for GM2 gangliosidosis. Hexb fusion genes will be constructed with Hc, the Hc C-terminal half, the Hc1282 peptide, and a multicopy version of Hc1282. Recombinant proteins will be screened for enzyme activity and enhanced lysosomal delivery using neuronal cultures from a mouse model of GM2 gangliosidosis, and for retrograde transport from the periphery using intramuscular injections. The best candidate construct(s) will then be tested on disease mice using: ERT initiated at birth; HSCRT, at 5 weeks of age, with ex vivo gene replacement where stem cell-derived microglia will secrete the high-uptake recombinant enzyme within the CNS; and potentially in a combined ERT/HSCRT regimen. The Hc-based strategy is applicable to numerous neuronal storage diseases that make up a significant portion of pediatric pathology and could be translated to human therapy. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop a therapeutic strategy relevant to lysosomal storage diseases that affect 1 in 8000 children. About half of these disorders, like Tay Sachs disease, also affect the brain, frequently result in early death and remain without a cure. This investigation will explore a gene modification strategy of the enzyme deficient in Tay Sachs disease in the context of enzyme replacement therapy and in hematopoietic stem cell replacement therapy which are currently used in human patients. If successful this modification will allow entry of enzyme into brain from the blood, and promote both widespread and efficient delivery of active enzyme to lysosomes of neurons. In this manner it may make these therapeutic approaches highly effective for treating diseased brain in Tay Sachs and related diseases, and could be relatively quickly translated to human clinical trials.

描述（由申请人提供）：神经元存储障碍是由溶酶体酶或辅助蛋白的遗传缺乏引起的，导致整个中枢神经系统（CNS）的底物积累，并保持无法治愈。酶替代（ERT），造血干细胞置换（HSCRT）和基因替代疗法各种各样的依赖于通过内吞摄取的外源性或分泌酶传递到宿主缺乏神经元的溶酶体的递送。中枢神经系统治疗的有效性受到血液脑屏障的限制，全球CNS递送的需求和较低的神经元内吞率。破伤风毒素的毒性c-fragment（HC）是解决所有这些问题的潜在解决方案。 HC与神经元膜的普遍结合，有效地内吞，可以通过运动神经元的外围末端进行轴突逆行转运，允许中枢神经系统进入，并且经过连续性地转移到高阶神经元中。我们先前证明，可以通过化学偶联β-己糖胺酶（Hex）来实现有效的神经元摄取和溶酶体储存的耗竭，GM2神经节病中的缺乏酶，可用于HC。在最近关于源自HC的34个残留肽（HC1282）的研究中，我们发现HC1282与神经元结合，增强了大分子分子的摄取，可以在多价形式中进一步增强神经元的吸收，并且比HC更有效地递送了Hex and fusion Gene and fusion（Hex）。 HC或HC子序列可以产生功能性嵌合蛋白，从而导致对GM2神经节病的有效治疗。 HEXB融合基因将由HC，HC C末端半，HC1282肽和HC1282的多拷贝版本构建。重组蛋白将使用来自GM2神经节病小鼠模型的神经元培养物以及使用肌肉内注射从周围的外周进行逆行转运，从而筛查重组蛋白的酶活性和增强的溶酶体递送。然后，最佳候选构建体将使用以下疾病小鼠进行：出生时启动的ERT； HSCRT在5周龄时，具有离体基因置换，其中干细胞衍生的小胶质细胞将分泌中枢神经系统内的高摄取重组酶；并有可能在联合ERT/HSCRT方案中进行。基于HC的策略适用于众多构成小儿病理学的大部分的神经元储存疾病，可以将其转化为人类治疗。公共卫生相关性：该提案旨在制定与影响8000名儿童中有1例的溶酶体储存疾病有关的治疗策略。这些疾病中约有一半（如Tay Sachs病）也会影响大脑，经常导致早期死亡，并且无法治愈。这项研究将探索在酶替代疗法和目前在人类患者中使用的造血干细胞替代疗法的酶的基因修饰策略。如果成功，这种修饰将允许将酶从血液中进入大脑，并促进活性酶的广泛和有效递送到神经元的溶酶体。通过这种方式，可以使这些治疗方法非常有效地治疗Tay Sachs和相关疾病中的患病大脑，并且可以相对较快地转化为人类临床试验。