200. Bioengineering Scar for Tissue Regeneration After Traumatic Brain Injury

PURPOSE: Scarring and fibrosis often impede functional recovery following injury or disease, highlighting the need for innovative therapeutic strategies. Effective tissue repair requires the repopulation of damaged areas with functional cells. In traumatic brain injury (TBI), externally inflicted brain trauma leads to the activation, proliferation, and hypertrophy of glial cells, including astrocytes, which form a dense border around the injury site. Over time, a cystic cavity develops at the core, surrounded by a glial scar, resulting in prolonged neurological deficits and permanent disability. This study aims to develop and assess a novel bioengineering strategy that utilizes nanoparticle-mediated cellular transfection with transcription factors, including Sox2. This facilitates reprogramming of glial scar cells into functional progenitor neurons in vivo, promoting recovery following TBI. METHODS: We synthesized a library of poly(β-amino esters) (PBAEs) as gene delivery carriers. Various transfection conditions were screened using PBAEs (536, 546, and 446) at polymer/DNA ratios [10, 20, 30, and 60 weight-weight (w/w)], containing plasmids encoding Sox2-green fluorescent protein (GFP) (N=3). The specificity of transfection to astrocytes was evaluated by transfecting co-cultures of primary human astrocytes and human umbilical vein endothelial cells (HUVECs). Cell viability was assessed 24 hours after transfection using the AlamarBlue assay, while transfection efficiency was evaluated after 48 hours using flow cytometry and immunostaining. RESULTS: We identified a transfection condition (PBAE 446 with 10 w/w) with high cell viability in both types of cells (>98%), but with greater transfection efficiency in astrocytes. Sox2-GFP expression was observed in primary human astrocytes 48 hours after transfection. CONCLUSION: PBAEs/DNA nanoparticles can effectively deliver the SOX2 gene into primary human astrocytes, resulting in reprogramming of the cell into progenitor cells facilitating regeneration. Ongoing studies are focused on neuronal differentiation, electrophysiology, as well as in vivo efficacy and safety. This bioengineering strategy shows promise for repopulating lesion sites with functional neurons following TBI. This technique shows great promise for translation into general tissue regeneration, and could be applied to regenerate various damaged tissues, such as skin, adipose tissue, and peripheral nerves, while minimizing fibrosis.