权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

SCA 3，也称为Machado-Joseph病，是世界上最常见的显性遗传性共济失调。突变SCA 3疾病蛋白ATXN 3通过显性毒性机制起作用，缺乏ATXN 3的小鼠都是正常的因此，抑制疾病基因ATXN 3代表了一种有希望的方法，减缓或阻断SCA 3中的神经变性级联反应。反义寡核苷酸（ASO）代表了一种非病毒的基因抑制方法已经成为治疗SCA 3和其他疾病的令人信服的治疗策略。神经退行性疾病然而，这种方法受到三个主要限制：（鞘内或脑室内）给药途径; ii）脑深部渗透性差，和iii）缺乏细胞- 具体目标。本提案的目标是使用双特异性抗体解决这些限制中的每一个 - 缀合至ASO（bAb-ASO）以使得能够：i）静脉内施用; ii）有效转运穿过完整的细胞; 血脑屏障（BBB）和深而广泛的脑渗透;和iii）选择性靶向神经元使用靶向介导神经元特异性内化和细胞内释放ASO。我们的方法有三个主要组成部分。首先，我们使用经验证的特异性IgG（M6），神经元膜蛋白（神经元糖蛋白M6 a），在哺乳动物中高度保守，在大脑中表达。其次，我们将单链抗体基因融合到IgG M6的C末端，认识到一个未充分研究的BBB靶点，用于介导有效转运到脑实质中，即 CD98hc。第三，我们将ASO连接至M6/CD 98 hc双特异性抗体，其将在抗体产生后释放。内化和介导基因沉默。本提案的总体目标是建立使用bAb-ASO沉默转基因小鼠中疾病特异性神经元基因并抑制疾病表型我们的中心假设是，双特异性抗体将使得能够跨膜递送阿索。 BBB和进入神经元，导致细胞特异性基因沉默和疾病严重程度的改善。为了验证这一假设，我们将首先评估bAb-ASO缀合物的PK/PD和ATXN 3的沉默（目的1）。我们将将我们验证的M6/CD 98 hc双特异性抗体与优化的ATXN 3阿索偶联并在细胞培养物中测试。对于体内分析，我们将使用YAC SCA 3转基因小鼠系，其携带完整的人SCA 3基因。疾病基因，并概括了SCA 3的分子和行为特征。我们将进行PK/PD分析，评估ATXN 3蛋白的沉默和重复给药的这种作用的持续时间。接下来我们就评估在SCA 3的小鼠模型中使用bAb-ASO沉默ATXN 3的功效（目的2）。我们会直接比较外周递送的bAb-ASO缀合物与脑室内注射的bAb-ASO缀合物的功效。在老化的SCA 3小鼠中的裸阿索，以确定外周递送的bAb-ASO缀合物是否防止了疾病相关表型的发展，如果是，疗效是否等于或超过裸体的律师