Developing an inducible mouse model for gene replacement therapy in Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD)

项目摘要/摘要(30行) 琥珀酸半醛脱氢酶缺乏症(SSADHD)是一种罕见的先天性代谢疾病，由 Aldh5a1突变。Aldh5a1编码SSADH，它是抑制物分解代谢所必需的 神经递质γ-氨基丁酸。在SSADHD中，GABA及其受体的病理性积聚 代谢产物羟丁酸(γ-羟基丁酸酯，GHb)可导致广谱脑病。矛盾的是，尽管环境 GABA升高，SSADHD患者易癫痫发作和猝死 (SUDEP)，强调了代偿性GABA受体减少相对于病理性GABA构建的意义。 向上。SSADHD尚未得到满足的一个主要医疗需求是直接治疗潜在的酶 缺乏如酶替代疗法(ERT)和基因疗法。概念验证系统性ERT和 肝脏导向的aldh5a1过表达增加了aldh5a1-/-小鼠的存活率。然而，酶或病毒载体 在aldh5a1-/-小鼠体内注射导致难以控制的、非特异性的SSADH修复难以评估 治疗效果和量效关系。目前尚不清楚何时、以何种速度或以何种方式恢复SSADH 细胞类型足以进行表型逆转。因此，我们建议开发一种新的小鼠模型，该模型 允许在独立的Cre或强力环素调节下有条件地重新激活aldh5a1。在这只新奇的老鼠里 Aldh5a1基因活性在基础水平上被破坏，但在cre介导的重组后被重组。 或受多西环素水平可逆调节。我们在这项为期两年的探索性拨款中的具体目标是 目的：1)建立aldh5a1lox-RTTA-Stop小鼠模型，并对其基线表型轨迹进行表征。 发展。2)当SSADH修复是安全的时，测试与年龄相关的治疗窗口 通过在不同的发育时间点将AAV-Cre注射到这只小鼠体内，是有效的。3)测试是否 SSADH的突然恢复会导致癫痫发作，而逐渐的SSADH恢复是否对癫痫发作有影响 早期单次注射AAV-Cre与多次低剂量注射AAV-Cre的治疗优势 后天发育。4)测试抑制性细胞定向部分SSADH修复是否可能 通过用GAD2-IRES-Cre小鼠培育aldh5a1lox-RTTA-Stop小鼠，足以进行表型逆转。这部小说 小鼠模型能够测试SSADH恢复策略的临床前准备情况，如基因治疗和 以受控的、可量化的和细胞特有的方式进行ERT。这个项目的长期目标有两个： 1)提供对SSADH病理生理学的机械性见解，以及SSADH修复如何 抢救症状。所提出的小鼠模型允许有条件的SSADH耗尽和恢复，因此 SSADHD的病理机制和SSADH修复的影响可以进行更详细的研究。 2)治疗性SSADH修复体关键参数的确定。建议的模型提供了 对SSADH修复的剂量、速度和细胞特异性的必要见解，推进未来 翻译和临床研究，包括SSADHD患者的ERT和基因治疗。

Project summary/abstract (30 lines) Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is a rare inborn metabolic disorder caused by aldh5a1 mutations. Aldh5a1 encodes SSADH which is essential for the catabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA and its metabolite γ-hydroxybutyrate (GHB) leads to broad spectrum encephalopathy. Paradoxically, despite ambient GABA is heightened, SSADHD patients are susceptible to seizures and sudden unexpected death in epilepsy (SUDEP), highlighting the significance of compensatory GABA receptor reduction over pathologic GABA build- up. A major unmet medical need for SSADHD is treatment directly addressing the underlying enzyme deficiency such as enzyme replacement therapy (ERT) and gene therapy. Proof-of-concept systemic ERT and liver-directed aldh5a1 over-expression increased aldh5a1-/- mice survival. However, enzyme or viral vector injections in aldh5a1-/- mice lead to uncontrollable, non-specific SSADH restoration difficult to evaluate therapeutic efficacy and dose-response relationship. It is unclear restoring SSADH at what time, rate or in what cell types would suffice for phenotype reversal. We thus propose to develop a novel mouse model which allows conditional aldh5a1 reactivation under independent Cre or doxycycline regulation. In this novel mouse strain, aldh5a1 gene activity is disrupted at basal level, but is reconstituted upon Cre-mediated recombination or is reversibly regulated by the level of doxycycline. Our specific aims in this two-year exploratory grant are to: 1) Develop the aldh5a1lox-rtTA-STOP mouse model and characterize its baseline phenotype trajectory across development. 2) Test for an age-dependent therapeutic window when SSADH restoration is safe and effective, by injecting AAV-Cre into this mouse at contrasting developmental time points. 3) Test whether abrupt SSADH restoration leads to epileptic seizures, and whether gradual SSADH restoration has a therapeutic advantage, by injecting single dose versus multiple lower doses of AAV-Cre across days in early postnatal development. 4) Test whether inhibitory cell-directed partial SSADH restoration might be sufficient for phenotype reversal, by breeding aldh5a1lox-rtTA-STOP mice with Gad2-IRES-Cre mice. This novel mouse model enables testing of preclinical readiness of SSADH-restoring strategies such as gene therapy and ERT in a controlled, quantifiable and cell-specific manner. This project's long-term objectives are two-fold: 1) Provision of mechanistic insights into SSADH pathophysiology and how SSADH restoration might rescue symptoms. The proposed mouse model allows conditional SSADH depletion and restoration, so that pathological mechanisms of SSADHD and the impacts of SSADH restoration can be studied in great details. 2) Establishment of key parameters for therapeutic SSADH restoration. The proposed model provides necessary insights into the dose, pace and cell-specificity of SSADH restoration, advancing future translational and clinical studies including ERT and gene therapy for SSADHD patients.