Gene therapy for Alzheimer's disease using virally delivered Aβ variants

摘要 持续的抗抗体Aducanumab的传奇已经产生了第一个阳性的3期结果 自2003年美金刚被批准以来，阿尔茨海默病(AD)就一直在治疗中。这一前景看好，尽管有争议的结果 为降低抗体的治疗潜力注入了新的活力，并使正在进行的 探索其他方法，以长期和安全地减轻抗体。过去的研究表明，小肽 可以很容易地定制抑制剂来防止抗体聚集，但体内递送基于多肽的药物是 受半衰期短和脑渗透力差的限制。我们已经鉴定出两个符合以下条件的抗体序列变体 潜在治疗用途的标准，因为它们1)防止WT抗体在体外聚集，2)促进分解 3)减轻抗体低聚物的毒性，更重要的是，4)不自聚集。交付 在体内，我们开发了一种新的微型基因来在血浆中表达我们的变异肽 通过g-分泌酶的裂解，它们被释放到细胞外空间的膜。通过将这个打包 将迷你基因导入AAV载体，并将其注射到APP/PS1小鼠体内，我们的实验数据表明 变异型抗体可降低抗体负荷并延缓斑块的形成。目前的提案将以这些成果为基础 通过以下具体目标。首先，我们将破译相互作用的生物物理机制 变异型和野生型Aβ多肽。我们将使用CD光谱分析方法，SEC层析， EM和抗体图谱来定义我们的变体防止/逆转的结构机制 野生型抗体的聚集性。第二，我们将确定变体Aβ的剂量、时机和路线 给药影响体内疗效。我们将使用病毒策略来比较介入治疗后 淀粉样变性发病，从出生开始预防性治疗，确定变异：野生型的最低有效率 Ab型需要改变斑块的形成和认知功能，并测试是否通过脑脊液传递 可以达到神经元转导的效果。第三，我们将询问变种的神经免疫反应。 Aβ作为减少斑块的帮凶。我们将使用组织学和转录图谱来评估 对变异肽或其AAV载体的神经免疫反应是否对斑块起作用 体内预防，以及神经免疫反应是否随年龄变化。最后，我们将测试潜在的 变体用于减慢AD聚合播种的β。我们预计变种Ab会减缓AD Ab萃取物的播种速度， 但更重要的是，将测试变异型抗体是否可以缓解带有淀粉样蛋白的小鼠体内tau的交叉播种。如果 成功地，这种自我抑制的策略也可能适用于其他蛋白质错误折叠疾病 由于技术上的原因，多肽治疗一直被回避，现在可以通过 表达工程和病毒技术。

SUMMARY The continuing saga of anti-Ab antibody aducanumab has produced the first positive phase 3 outcome for Alzheimer's disease (AD) since memantine was approved in 2003. This promising, albeit controversial, result has breathed new life into the therapeutic potential for Ab-lowering strategies and legitimized the ongoing exploration of other means to chronically and safely mitigate Ab. Past work had shown that small peptide inhibitors can be readily tailored to prevent Ab aggregation, but in vivo delivery of peptide-based drugs was limited by short half-life and poor brain penetration. We have identified two Ab sequence variants that meet criteria for potential therapeutic use, as they 1) prevent aggregation of WT Ab in vitro, 2) promote disassembly of Ab existing fibrils, 3) mitigate toxicity of Ab oligomers, and importantly, 4) do not self-aggregate. To deliver these peptides in vivo, we have developed a novel mini-gene to express our variant peptides at the plasma membrane where they are released into the extracellular space by g-secretase cleavage. By packaging this minigene into an AAV vector that is injected into APP/PS1 mice, our pilot data show that viral expression of variant Ab lowers Ab load and delays plaque formation. The current proposal will build on these results through the following specific aims. First, we will decipher the biophysical mechanism of interactions between variant and wild type Aβ peptides. We will use analytical methods of CD spectroscopy, SEC chromatography, EM, and antibody profiling to define the structural mechanism by which our variants prevent/reverse aggregation of wild-type Ab. Second, we will determine how dosage, timing and route of variant Aβ administration influence efficacy in vivo. We will use viral strategies to compare interventional treatment after amyloid onset with preventative treatment starting at birth, determine the lowest effective ratio of variant:wild- type Ab needed to modify plaque formation and cognitive function, and test whether delivery through the CSF can match the effect of neuronal transduction. Third, we will interrogate the neuroimmune reaction to variant Aβ as an accomplice to plaque reduction. We will use histological and transcriptional profiling to assess whether a neuroimmune response to either the variant peptide or its AAV carrier contribute to plaque prevention in vivo, and if the neuroimmune response changes with age. Finally, we will test the potential for variant Aβ to slow AD aggregate seeding. We anticipate that variant Ab will slow seeding by AD Ab extracts, but will more importantly test whether variant Ab can assuage cross-seeding of tau in amyloid-bearing mice. If successful, this strategy for self-inhibition may also be applicable to other protein misfolding diseases where peptide treatments have been eschewed for technical reasons that can now be overcome through expression engineering and viral technology.