Poster X15 - Gene and drug delivery targeted to hyperexcited neuronal cells with glucose dendrimers

Introduction: Non-viral gene therapy has potential to overcome the limitations of conventional gene delivery approaches using viral vectors, such as broad tropism, limited DNA packaging capacity, immunogenicity, and scalability of vector production. One of the key challenges in gene and drug therapy for neuropathies is targeting and intracellular delivery into neuronal cells, selectively at the site of pathology. We recently developed glucose dendrimer that specifically targeted neurons at the site of pathology in CNS animal models, and produced superior efficacy compared to free drugs. This study seeks to understand the key mechanisms behind this selective neuronal targeting by glucose dendrimers (without any targeting ligands), through detailed studies in neuronal and retinal ganglion cell cultures.

Materials and

Methods: We successfully synthesized and characterized generation-two glucose-dendrimer (GD2) composed primarily of glucose and ethylene glycol building blocks. We compared human retinal ganglion cells (RGCs) and mouse hippocampal cells (HT-22) uptake of GD2 to hydroxyl-terminated, generation-four poly(amidoamine) dendrimer (HD) by using confocal microscopy (Treatment time=6h). Altered cellular uptake of GD2 was studied in vitro using glutamate-stimulated neuronal cells to mimic neuronal injury. We explored the cellular uptake mechanism and the role of Glut transporters by blocking relevant uptake pathways. Further, Cy5-labelled GD2 was analyzed with intracellular trafficking along with semiquantitative and morphological analysis. RGCs were provided by Dr. Donald Zack’s group (Dr. Cindy Berlinicke).

Results, Conclusions, and Discussions:

Results:
Glucose dendrimers appear to be rapidly taken up by neuronal cells (within minutes), with an injury-dependent extent of uptake. Interestingly, GD2 demonstrated >40-fold more uptake by RGCs and HT-22 cells respectively than HD which showed minimal uptake. GD2-Cy5 showed intracellular distribution (cytoplasm and nucleus) with relatively higher localization at the perinuclear region. Moreover, cellular uptake occurred in as little as 30 min with GD2 in both RGCs and HT-22 cells. Further blocking glucose transporters (GLUT) using cytochalasin B and glutor demonstrated reduced GD2 uptake by neurons. Additionally, in glutamate hyperexcited RGCs and HT-22 cells, GD2 demonstrated enhanced uptake in comparison to healthy cells. Our studies have shown close to cell uptake efficiency close to 100% by RGCs and HT-22 cells.

Conclusions:
Drug and gene delivery into neuronal cells has been a major challenge. Glucose dendrimers (without targeting ligands) have potential to selectively transfect hyperexcited neuronal cells, making them valuable for non-viral gene and drug delivery, in order to prevent or improve treatment of neuropathies.

Acknowledgements (Optional):