Network modulation to improve gene therapy in CLN3 disease

项目摘要 三分之二的溶酶体贮积症（LSD）影响大脑，但大多数LSD治疗没有改善 中枢神经系统（CNS）症状。几项脑定向基因治疗的试验都未能证明 尽管恢复了CNS中的蛋白质表达，但仍具有临床益处。结果在以下受试者中尤其差： 出现神经功能缺损，这表明单独拯救表达可能不足以纠正 一旦患病的神经元回路建立， 在CLN 3疾病中，一种代表性的LSD和儿童痴呆症的最常见原因，患者发展为 失明癫痫和痴呆已经开发了几种CLN 3疾病小鼠模型。虽然所有 概括在患者中观察到的储存积累，行为表型是微妙和可变的。到 克服了这一局限性，我们在网络水平的电生理学上确定了稳健的、可重复的表型。 在两个CLN 3模型中的研究，敲除和人类突变模型。与组织病理学不同， 测量直接反映功能，因此可能是治疗开发的更好读数。 我们的工作表明，CLN 3疾病，传统上被认为是一种退行性疾病， 神经发育，特别是在海马中，CLN 3疾病的脆弱区域。开启体外电压 敏感染料成像（VSDI）和体内脑电图（EEG）记录，Cln 3-/-小鼠的脑电活动减少， 海马齿状回（DG）的兴奋性，更快的EEG背景活动，以及海马齿状回（DG）的丢失。 尖锐的波浪波纹，编码新记忆的振荡。此外，DG神经发生上调，可能是因为 一种代偿机制，在疾病早期（2个月），但不晚于（6个月）。类似的网络变化出现在其他 包括阿尔茨海默病（AD）在内的神经变性模型。内嗅神经深部脑刺激术 皮质已经显示出改善AD小鼠模型的结果。 以前，我们发现，在p0的非常早期的Cln 3基因替换纠正了Cln 3中的网络动力学。 敲除小鼠。我们的中心假设是，异常的神经元回路发育将限制神经元的活动。 时间窗口，即“治疗窗口”，当基因替换将改善网络生理学时， CLN 3疾病此外，我们预测，改变一个关键回路的活性可以改变治疗效果。 基因治疗的窗口期和有效性。我们的具体目标是：1）定义异常齿状回发育 在CLN 3疾病小鼠中，2）通过以下方法确定用于校正海马回路动力学的治疗窗： 基因替换，以及3）测试修改内嗅皮层活动是否改变电路缺陷和对基因的响应 疗法这样我们将以CLN 3疾病作为代表性的LSD，来探讨 网络活动和对基因治疗的反应。我们的长期目标是开发网络导向疗法 当与基因替代相结合时，可以改善LSD的结果。

PROJECT SUMMARY Two-thirds of lysosomal storage disorders (LSD) affect the brain, yet most LSD treatments do not improve central nervous system (CNS) symptoms. Several trials of brain-directed gene therapy have failed to show clinical benefit despite restoring protein expression in the CNS. Outcomes are especially poor in subjects who have developed neurologic deficits, suggesting rescue of expression alone may be insufficient to correct function once diseased neuronal circuits are established. In CLN3 Disease, a representative LSD and the most common cause of pediatric dementia, patients develop blindness, seizures, and dementia. Several CLN3 disease mouse models have been developed. While all recapitulate the storage accumulation seen in patients, behavioral phenotypes are subtle and variable. To overcome this limitation, we identified robust, reproducible phenotypes on network-level electrophysiology studies in two CLN3 models, a knockout and a human mutation model. Unlike histopathology, physiologic measures directly reflect function and, therefore, may be a better readout for therapy development. Our work suggests CLN3 disease, traditionally considered a degenerative disorder, disrupts early neurodevelopment, especially in the hippocampus, a vulnerable region in CLN3 disease. On in vitro voltage sensitive dye imaging (VSDI) and in vivo electroencephalogram (EEG) recordings, Cln3-/- mice have decreased excitability of the hippocampal dentate gyrus (DG), faster EEG background activity, and loss of hippocampal sharp wave ripples, oscillations that encode new memories. Also, DG neurogenesis is upregulated, perhaps as a compensatory mechanism, early (2mo) but not later (6mo) in disease. Similar network changes arise in other models of neurodegeneration including Alzheimer’s disease (AD). Deep brain stimulation of the entorhinal cortex has been shown to improve outcomes in mouse models of AD. Previously, we found that very early Cln3 gene replacement at p0 corrects network dynamics in a Cln3 knockout mouse. Our central hypothesis is that abnormal neuronal circuit development will limit the window of time, i.e. ‘therapeutic window’, when gene replacement will improve network physiology in CLN3 disease. Furthermore, we predict that altering activity in a key circuit could modify the therapeutic window and efficacy of gene therapy. Our Specific Aims are to: 1) define abnormal dentate gyrus development in CLN3 disease mice, 2) determine the therapeutic window for correction of hippocampal circuit dynamics by gene replacement, and 3) test if modifying entorhinal cortex activity alters circuit defects and response to gene therapy. In this way we will use CLN3 Disease as a representative LSD, to explore the relationship between network activity and response to gene therapy. Our long-term goal is to develop network-directed therapies that, when combined with gene replacement, improve outcomes in LSDs.