Niibori Yosuke, Duba-Kiss Robert, Bruder Joseph T, Smith Jared B, Hampson David R
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.
Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 3M2, Canada.
Mol Ther Methods Clin Dev. 2023 Feb 4;28:330-343. doi: 10.1016/j.omtm.2023.01.007. eCollection 2023 Mar 9.
Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.
γ-氨基丁酸（GABA）能抑制性神经元功能受损与癫痫以及其他神经和精神疾病有关。基于重组腺相关病毒（rAAV）的针对GABA能神经元的基因治疗是治疗GABA相关疾病的一种有前景的方法。然而，需要开发能够在全脑的GABA能神经元中选择性驱动表达的与rAAV兼容的基因调控元件。在此，我们设计了几种新型的GABA能基因启动子。进行了包括进化保守DNA序列比对和GABA能神经元基因间转录因子结合位点搜索在内的分析，以揭示用作与rAAV兼容启动子的新序列。将rAAV（9型）注入新生小鼠的脑脊液和成年小鼠的脑实质中，以评估启动子特异性。在新生期注射的小鼠中，在多个脑区检测到转基因表达，具有非常高的神经元特异性和中度至高度的GABA能神经元选择性。这些GABA启动子的表达水平差异很大，并且在某些脑区，显示出明显不同的GABA能神经元转导模式。本研究首次报道了使用来自多个GABA能基因的分析设计的启动子在多个脑区具有功能的rAAV载体。这些新型的靶向GABA的载体可能是推进GABA相关疾病基因治疗的有用工具。
