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

基本信息

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
项目类别：
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

项目摘要

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，也称为马查多-约瑟夫病，是世界上最常见的显性遗传性共济失调。突变型 SCA3 疾病蛋白 ATXN3 通过显性毒性机制发挥作用，而缺乏 ATXN3 的小鼠表型正常，因此，抑制疾病基因 ATXN3 是一种有前途的治疗方法。减缓或阻断 SCA3 中的神经退行性级联反应。反义寡核苷酸 (ASO) 代表非病毒。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然而，这种方法存在三个主要局限性：i) 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- 该提案的目标是使用双特异性抗体解决这些限制。与 ASO (bAb-ASO) 缀合，以实现： i) 静脉内给药 ii) 在完整组织中有效运输；血脑屏障（BBB）和深入且广泛的大脑渗透；以及iii）选择性靶向神经元；使用针对介导神经元特异性内化和细胞内的细胞表面蛋白的抗体我们的方法具有三个主要组成部分：首先，我们使用经过验证的特定于 ASO 的 IgG (M6)。神经元膜蛋白（神经元糖蛋白 M6a）在哺乳动物中高度保守且高度其次，我们将单链抗体与 IgG M6 的 C 末端进行基因融合。识别出一个尚未被研究的 BBB 目标，用于介导有效转运至脑实质，即第三，我们将 ASO 附加到 M6/CD98hc 双特异性抗体上，该抗体将在抗体后释放。当前提案的总体目标是建立内化和介导基因沉默。使用 bAb-ASO 沉默转基因小鼠疾病特异性神经元基因并抑制的可行性我们的中心假设是双特异性抗体将能够在整个疾病表型中传递 ASO。 BBB 并进入神经元，导致细胞特异性基因沉默并改善疾病严重程度。假设，我们将首先评估 bAb-ASO 缀合物和 ATXN3 沉默的 PK/PD（目标 1）。将我们经过验证的 M6/CD98hc 双特异性抗体与优化的 ATXN3 ASO 结合并在细胞培养中进行测试。对于体内分析，我们将使用 YAC SCA3 转基因小鼠系，该小鼠系含有完整的人类 SCA3 我们将进行 PK/PD 分析以了解疾病基因并概括 SCA3 的分子和行为特征。接下来，我们将评估 ATXN3 蛋白的沉默以及重复给药的效果持续时间。在 SCA3 小鼠模型中评估使用 bAb-ASO 沉默 ATXN3 的功效（目标 2）。比较外周递送 bAb-ASO 缀合物与脑室内注射 bAb-ASO 缀合物的功效老化 SCA3 小鼠中的裸 ASO 以确定外周递送的 bAb-ASO 缀合物是否可预防疾病相关表型的发展，如果是的话，疗效是否等于或超过裸 ASO。