Neuronal Silencing of ATXN3 Using Peripherally Administered Antibody/ASO Conjugates That Penetrate the Blood-Brain Barrier

使用可穿透血脑屏障的外周给药抗体/ASO 缀合物对 ATXN3 进行神经元沉默

• 批准号: 10646563
• 负责人: Peter M Tessier
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646563
• 关键词: Acceleration; Address; Aging; Amino Acid Transport System L; Antibodies; Antisense Oligonucleotides; Ataxia; Behavioral; Behavioral Assay; Binding; Biochemical; Biological Assay; Bispecific Antibodies; Blood - brain barrier anatomy; Brain; Cell Culture Techniques; Cell Surface Proteins; Cell surface; Cells; Cessation of life; Conjugating Agent; Data; Development; Disease; Dose; Drug Kinetics; GPM6A gene; Gene Silencing; Genes; Goals; Human; Immunoglobulin G; In Vitro; Inherited; Injections; Label; Laboratories; MJD1 protein; Machado-Joseph Disease; Maleimides; Mammals; Measures; Mediating; Membrane Proteins; Methods; Molecular; Motor; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Penetration; Peptides; Peripheral; Pharmacodynamics; Phenotype; Proteins; Research; Route; Severity of illness; Sulfhydryl Compounds; Technology; Testing; Therapeutic; Transgenic Mice; antibody conjugate; blood-brain barrier penetration; brain parenchyma; comparative efficacy; disease phenotype; efficacy evaluation; fluorophore; high reward; high risk; improved; in vivo; interest; intravenous administration; motor control; mouse model; mutant; novel therapeutics; prevent; radiotracer; therapeutic development

SCA3, also known as Machado–Joseph disease, is the most common dominantly inherited ataxia in the world. The mutant SCA3 disease protein, ATXN3, acts through a dominant toxic mechanism, and mice lacking ATXN3 are phenotypically normal. Thus, suppression of the disease gene, ATXN3, represents a promising approach to slow or block the neurodegenerative cascade in SCA3. Anti-sense oligonucleotides (ASOs) represent a nonviral gene suppression approach that has emerged as a compelling therapeutic strategy for treating SCA3 and other neurodegenerative disorders. However, this approach suffers from three main limitations: i) highly invasive (intrathecal or intracerebroventricular) route of administration; ii) poor deep brain penetration, and iii) lack of cell- specific targeting. The goal of this proposal is to address each of these limitations using bispecific antibodies conjugated to ASOs (bAb-ASOs) to enable: i) intravenous administration; ii) efficient transport across the intact blood-brain barrier (BBB) and deep and widespread brain penetration; and iii) selective targeting of neurons using antibodies that target cell-surface proteins that mediate neuron-specific internalization and intracellular release of ASOs. Our approach has three main components. First, we use a validated IgG (M6) specific for a neuronal membrane protein (Neuronal Glycoprotein M6a) that is highly conserved within mammals and highly expressed in the brain. Second, we genetically fuse a single-chain antibody to the C-terminus of the IgG M6 that recognizes an understudied BBB target for mediating efficient transport into the brain parenchyma, namely CD98hc. Third, we attach ASOs to the M6/CD98hc bispecific antibody that will be released after antibody internalization and mediate gene silencing. The overall objective of the current proposal is to establish the feasibility of using bAb-ASOs to silence disease-specific neuronal genes in transgenic mice and suppress disease phenotypes. Our central hypothesis is that the bispecific antibody will enable delivery of ASO across the BBB and into neurons, resulting in cell-specific gene silencing and improvement in disease severity. To test this hypothesis, we will first evaluate PK/PD of bAb-ASO conjugates and silencing of ATXN3 (Aim 1). We will conjugate our validated M6/CD98hc bispecific antibodies to an optimized ATXN3 ASO and test in cell culture. For in vivo analysis, we will use the YAC SCA3 transgenic mouse line, which harbors the full human SCA3 disease gene and recapitulates molecular and behavioral features of SCA3. We will perform PK/PD analysis to evaluate the silencing of ATXN3 protein and the duration of this effect with repeated dosing. Next, we will evaluate the efficacy of silencing ATXN3 using bAb-ASOs in mouse models of SCA3 (Aim 2). We will directly compare the efficacy of peripherally delivered bAb-ASO conjugate to that of intracerebroventricular injection of the naked ASO in aging SCA3 mice to determine whether peripherally delivered bAb-ASO conjugate prevents the development of disease-associated phenotypes and, if so, whether the efficacy equals or surpasses that of naked ASOs.

SCA3，又称Machado-Joseph病，是世界上最常见的遗传性共济失调。 突变的SCA3疾病蛋白ATXN3通过显性毒性机制发挥作用，而缺乏ATXN3的小鼠 都是表型正常的。因此，抑制疾病基因ATXN3代表了一种很有希望的方法 延缓或阻断SCA3的神经退行性级联反应。反义寡核苷酸(ASO)代表一种非病毒 基因抑制方法已成为治疗SCA3和其他疾病的引人注目的治疗策略 神经退行性疾病。然而，这种方法有三个主要限制：i)高度侵入性 (鞘内或脑室内)给药途径；ii)脑深部渗透性差，以及iii)缺乏细胞- 特定的目标。这项提议的目标是使用双特异性抗体来解决这些局限性。 结合ASOS(BAB-ASOS)以实现：i)静脉给药；ii)有效地跨完整的 血脑屏障(BBB)和深度和广泛的脑渗透；以及iii)选择性靶向神经元 使用针对细胞表面蛋白的抗体，该抗体介导神经元特异性内化和细胞内 释放阿索斯。我们的方法有三个主要组成部分。首先，我们使用一种有效的免疫球蛋白(M6)来识别 神经细胞膜蛋白(神经糖蛋白m6A)，在哺乳动物中高度保守，在 在大脑中表达。其次，我们在基因上将一种单链抗体融合到免疫球蛋白M6的C末端， 识别一个未被充分研究的血脑屏障靶向脑实质的有效转运，即 CD98hc。第三，我们将ASO结合到M6/CD98hc双特异性抗体上，该抗体将在抗体后释放 内化和介导基因沉默。目前建议的总体目标是建立 Bab-ASOS沉默转基因小鼠疾病特异性神经元基因的可行性 疾病表型。我们的中心假设是，双特异性抗体将使ASO能够通过 BBB并进入神经元，导致细胞特异性基因沉默和疾病严重程度的改善。为了测试这一点 假设，我们将首先评估Bab-ASO偶联物的PK/PD和ATXN3的沉默(目标1)。我们会 将我们验证的M6/CD98hc双特异性抗体与优化的ATXN3 ASO偶联，并在细胞培养中进行测试。 对于活体分析，我们将使用YAC SCA3转基因小鼠系，它携带完整的人类SCA3 疾病基因，并概括了SCA3的分子和行为特征。我们将进行PK/PD分析以 评估ATXN3蛋白的沉默作用以及重复给药的持续时间。接下来，我们将 在SCA3小鼠模型中评价Bab-ASOS沉默ATXN3的效果(目标2)。我们会直接 Bab-ASO结合物外周给药与脑室注射的疗效比较 在衰老的SCA3小鼠中进行裸鼠ASO研究，以确定外周递送的Bab-ASO结合物是否可以防止 疾病相关表型的发展，如果是这样，其疗效是否等于或超过 赤身裸体的阿索斯。