Gene delivery offers therapeutic promise for the treatment of neurological diseases and spinal cord injury. Several studies have offered viral vectors as vehicles to deliver therapeutic agents, yet their toxicity and immunogenicity, along with the cost of their large-scale formulation, limits their clinical use. As such, non-viral vectors are attractive in that they offer improved safety profiles compared to viruses. Poly(ethylene imine) (PEI) is one of the most extensively studied non-viral vectors, but its clinical value is limited y its cytotoxicity. Recently, chitosan/DNA complex nanoparticles have een considered as a vector for gene delivery. Here, we demonstrate that DNA nanoparticles made of hyaluronic acid (HA) and chitosan have low cytotoxicity and induce high transgene expression in neural stem cells and organotypic spinal cord slice tissue. Chitosan-TPP/HA nanoparticles were significantly less cytotoxic than PEI at various concentrations. Additionally, chitosan-TPP/HA nanoparticles with pDNA induced higher transgene expression in vitro for a longer duration than PEI in neural stem cells. These results suggest chitosan-TPP/HA nanoparticles may have the potential to serve as an option for gene delivery to the spinal cord.