Abstract
Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains a challenge. Overcoming this hurdle is essential for gene editing in the central nervous system (CNS). Here, we characterized the minimal/native promoter of the HEXB gene, which is known to be specifically and stably expressed in the microglia during homeostatic and pathological conditions. Dual reporter and serial deletion assays identified the critical role of the natural 5’ untranslated region (−97 bp related to the first ATG) in driving transcriptional activity of the mouse Hexb gene. The native promoter region of mouse, human, and monkey HEXB are located at −135, −134, and −170 bp to the first ATG, respectively. These promoters were highly active and specific in microglia with strong cross-species transcriptional activities, but did not exhibit activity in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene that was highly active in microglia but not in astrocytes. Considering that HEXB is specifically expressed in microglia, these data suggest that the newly characterized microglia-specific HEXB minimal/native promoter can be an ideal candidate for microglia-targeting AAV gene therapy in the CNS.
Highlights
Adeno-associated virus (AAV) delivery of gene editors is one of the foremost technologies in development for gene therapy of central nervous diseases (CNS), including human immunodeficiency virus (HIV) infection
Microglia-specific gene promoters have been studied in the mouse microglia cell models using mouse myeloid cellspecific promoter including the CD68 promoter (Kettenmann et al, 2011; Rosario et al, 2016); it is not clear whether these promoters are effective in human primary
Based on previous reports on the promoter activity of the mouse Hexb gene (Norflus et al, 1996; Urbanelli et al, 2014), we initially evaluated the activity of Hexb promoter region −330 bp upstream of ATG (Figure 1A)
Summary
Adeno-associated virus (AAV) delivery of gene editors is one of the foremost technologies in development for gene therapy of central nervous diseases (CNS), including human immunodeficiency virus (HIV) infection. Finding an effective gene delivery system is essential. One way is to find an effective promoter and express the selected gene editor in the brain microglia. Microglia-specific gene promoters have been studied in the mouse microglia cell models using mouse myeloid cellspecific promoter including the CD68 promoter (Kettenmann et al, 2011; Rosario et al, 2016); it is not clear whether these promoters are effective in human primary. Since CD68 is expressed in both microglia and macrophages, it may not be specific to microglia in the human brain
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