Polymeric Nanoparticle-Mediated GFAP-TIMP-1 Gene Delivery to Human Astrocytes

Purpose: The neuroprotective functions of astrocyte tissue inhibitor of metalloproteinases-1 (TIMP-1) during HIV-1-induced apoptosis are documented. However, astrocyte TIMP-1 levels decrease during chronic inflammation typical of HIV-associated neurocognitive disorders (HAND). We hypothesize that nanoparticle (NPs) mediated, astrocyte-targeted TIMP-1-gene delivery could restore TIMP-1 levels and serve as a HAND therapy. Optimally biocompatible polymeric NPs with validated in vivo reporter gene expression would serve as gene delivery vehicles. TIMP-1 overexpression may be restricted to astrocytes, using a glial fibrillary acidic protein (GFAP) promoter-driven gene expression, without adversely affecting the astrocyte structure and functions. Methods: We tested biocompatibility, dose-time kinetics, and in vivo gene delivery with arginine-modified polyethylenimine (PEI) analogs (AnPn) in vitro (primary human neural cells) and in vivo (mice) using a cytomegalovirus (CMV) promoter-driven luciferase plasmid (pLuc). Truncated and full-length GFAP promoter-driven plasmids encoding luciferase-reporter (gfa-Luc) and TIMP-1 (gfa-TIMP-1) were sub-cloned. Astrocytes transfected with AnPn-delivered gfa-Luc and gfa-TIMP-1 were evaluated for morphological changes, inflammatory biomarker profiles, and glutamate clearance to evaluate if critical astrocyte functions are affected. Results: Select PEI analogs (AnPn) led to robust and sustained reporter gene expression in astrocytes. Successful in vivo reporter gene delivery to the brain was confirmed by luminescence assay and immunohistochemistry. Truncated GFAP promoters led to a detectable gene expression in astrocytes compared to full-length GFAP promoters, which was regulated by inflammatory stimuli. Lastly, gfa-TIMP-1 plasmids were successfully delivered using AnPn to astrocytes in the presence and absence of HIV-relevant stimuli. Immunocytochemistry, and proinflammatory biomarker profiles of transfected astrocytes were analyzed to delineate NP-specific versus TIMP-1-specific changes. Conclusions: These studies confirmed successful polymeric NPs-mediated reporter gene delivery in vitro and in vivo. Subsequently, NPs-mediated GFAP promoter-driven reporter and TIMP-1 gene expression was demonstrated in vitro. These findings serve as proof-of-concept towards testing therapeutic TIMP-1 gene delivery and demand future investigations geared towards clinical translations for HAND treatment.