Microglia-specific promoter activities of humanHexBgene

Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains challenging. 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 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 had no activities in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene was also highly active in microglia but not in astrocytes. Considering that HexB is specifically expressed in microglia, not in monocytes/macrophages or other neuronal cells, these data suggest that the newly characterized 134 bp microglia-specific HexB promoter can be an ideal candidate for microglia-targeting AAV gene therapy, which could be developed for HIV eradication in the brain wherein microglia harbor the main HIV reservoirs in the CNS. Summary It is hard to overstate the importance of gene therapy that can remove viral genes from human cells. A cure for HIV would mean a lifetime free of treatment for patients who now must maintain a strict regimen of ART indefinitely. In order to develop a cure using AAV delivery, payload DNA must meet the AAV vector size limitations, and the payload genes must be expressed appropriately. Previous studies have identified microglia-specific HexB gene that shows stable expression during neural homeostasis and pathogenesis. Our study identified the essential HexB gene promoter (134 bp) as a strong candidate for AAV gene therapy to specifically target the brain microglia, the main cellular reservoirs of HIV in the central nervous system. Our studies continue to move us closer to identifying target-specific gene therapy for NeuroAIDS.