In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons

• Published in: Molecular therapy. Methods & clinical development 2023-03, Vol.28, p.330-343
• Main Authors: Niibori, Yosuke, Duba-Kiss, Robert, Bruder, Joseph T., Smith, Jared B., Hampson, David R.
• Format: Article
• Language: English
• Subjects: autism; DLX; epilepsy; GAD1; GAD2; gene therapy; Original; schizophrenia; SLC32A1; Vgat; γ-aminobutyric acid
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.01.007

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. In silico 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 in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders. [Display omitted] Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. We designed and tested several novel GABAergic gene promoters for use in gene therapy. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.