Kathy Y. Wei, Jamie Bergen, Dr. Suzie Pun Department of Bioengineering, UW

1. Abstract DNA Complexation Ability of PEI-VDP HeLa Transfections with PEI-VDP The transfection efficiencies of polyplexes formed from PEI and PEI-VDP at various N/P ratios were measured using HeLa as a model cell line. Gene therapy promises to treat neurological disorders, such as Alzheimer's disease, Huntington’s disease, and Parkinson’s disease, that currently have limited or no available treatment. Non-viral gene delivery vehicles offer several advantages over viral-based vectors because they are potentially safer and more customizable. The specific non-viral vehicles used in this experiment are polyplexes, which are polymer/DNA complexes. The major challenge faced by these materials is the inefficiency of polyplexes at overcoming barriers to gene delivery, especially in non-dividing cell types such as neurons. Intracellular barriers to nuclear delivery of foreign DNA include targeting (getting to the cell), uptake (getting into the cell), endosomal escape (getting out of the endosome), retrograde transport (getting to the nucleus), and nuclear localization (getting into the nucleus). This project focuses on attaching peptide ligands that target specific barriers to the surface of polyplexes to increase DNA delivery efficiency. The DNA incorporation efficiency, polyplex size, and polyplex charge were measured for various formulations of a virally-derived peptide conjugated to polyethylenimine, a cationic polymer that is widely used to deliver DNA. This peptide, which may have endosomal escape as well as retrograde transport capabilities, increased gene delivery in a model cell line by up to a factor of 26-fold. Also, the synergistic effect of combining the peptide and Tet1, a neuron targeting peptide based on tetanus toxin, is being studied in cultured neuron-like cells. Success in increasing gene delivery with safe, customizable polyplexes will bring us closer to treating not only neurological diseases, but also diseases like cancer. The presence of a virally-derived peptide on the polymer slightly increases the ability of the polymer to complex DNA. Size and Zeta Potential of PEI-VDP-based Polyplexes The incorporation of a virally-derived peptide into polyplexes does not significantly alter their size of surface charge. Overcoming the Barriers In order to effectively deliver exogenous DNA into the nucleus of cells, delivery vehicles must overcome several intracellular barriers. The delivery vehicles can be viral or non-viral and, while viral vectors are more efficient, non-viral vehicles like polyplexes are potentially safer. The goal of the project is to improve the efficiency of non-viral vectors in neurons. Combining PEI-VDP and PEI-Tet1 5. Nuclear Localization 4. Retrograde Transport Axon Neuron-like PC-12 cells were transfected with polyplexes incorporating various combinations of PEI-VDP and PEI-Tet1. 1. Targeting Nucleus 2. Uptake 3. Endosomal Escape Soma = polyplex Conclusions + = Compared to polyplexes formulated with PEI, polyplexes formulated with PEI-VDP increase reporter gene expression in HeLa cells up to 26-fold. The increase in transfection is not due to any changes in polyplex size or zeta potential that may have been introduced by attaching the peptide to PEI. Further, PEI-VDP does not appear to be more toxic than PEI at concentrations of interest. Combining both peptides increases the ability of polyplexes to transfect PC-12 cells as much as the individual peptides do. polymer polyplex DNA Cellular Toxicity of PEI-VDP The cellular toxicities of PEI and PEI-VDP do no differ significantly at concentrations relevant for gene delivery. Future Work The next step for the work on the VDP peptide is to test controls. Specifically, peptide sequences of VDP that are mutated to remove the retrograde transport ability without affecting endosomal escape and vice versa. This will determine whether the increases in the transfections were due to non-specific effects and which, if any, of the components of the VDP peptide is contributing to the increase in transfection. The next step for the work on combining the VDP peptide and Tet1 peptide is to determine why there is no synergistic effect when combining the peptides. Tet1 VDP Acknowledgements Amgen Scholars Program: Amgen Foundation, Janice DeCosmo, Jennifer Harris, Jessica Salvador, and Tracy Nyerges. Pun Lab: Dr. Suzie Pun, Jamie Bergen, Teresa Peterson, and everyone in the lab. The Molecular Swiss Army Knife:Designing multifunctional non-viral vehicles for gene delivery to neurons Kathy Y. Wei, Jamie Bergen, Dr. Suzie PunDepartment of Bioengineering, UW PEI, N/P 1.5 PEI-VDP, N/P 1.5 PEI, N/P 1.7 PEI-VDP, N/P 1.7 PEI, N/P 1.8 PEI-VDP, N/P 1.8 PEI, N/P 1.9 PEI-VDP, N/P 1.9 PEI-VDP, N/P 1.6 PEI, N/P 1.6 * Figure 4: Gel retardation assay comparing the abilities of PEI and PEI-VDP to condense DNA and form polyplexes. PEI complexes DNA at N/P=1.8 and PEI-VDP complexes DNA at N/P=1.7. PEI-VDP is slightly better at complexing DNA. complexed DNA ** migration of uncomplexed DNA Figure 8: Luciferase reporter gene expression in HeLa cells. There were significant increases in transfection for N/P = 3 and 5. At N/P=3 PEI-VDP was 26 times more effective than PEI (**p<0.01)and at N/P=5 it was 14 times more effective (*p<0.05). Figure 5: Particle size of polyplexes formed with PEI or PEI-VDP was measured by dynamic light scattering. The two types of polyplexes have similar sizes of about 100 nm in diameter, except at N/P=2, where sizes of the PEI polyplexes are large probably because the polyplexes are aggregating. Figure 9: PC-12 transfection with PEI-VDP and PEI-Tet1 at N/P = 3. Both PEI-VDP and PEI-Tet1 appear to increase transfection compared to PEI alone. The 50/50 combination of PEI-VDP and PEI-Tet1 show similar levels to either peptide alone. Figure 6: Zeta potential, or surface charge, of polyplexes formed with PEI or PEI-VDP was measured by dynamic light scattering. The zeta potentials of the two types of polyplexes are similar at all measured N/P ratios. Figure 1: Barriers to delivering exogenous DNA into the nucleus include: 1) Targeting: deliver only to specific cells types 2) Uptake: DNA must be internalized by the cell 3) Endosomal escape: DNA must exit the endosome before it becomes degraded by the low pH levels inside 4) Retrograde transport: DNA must travel to the nucleus. This is especially applicable in neurons if polyplexes are delivered to axon terminals, which can be up to a meter from the neuronal nucleus 5) Nuclear localization: DNA must pass through the nuclear envelope. Figure 2: Polyplexes are non-viral gene delivery vehicles that consist of a DNA core wrapped in a positively charged polymer coat. The N/P ratio, or amine to phosphate ratio, is a measure of the amount of polymer to DNA used for a particular polyplex formulation. Figure adapted from: Pack et al. (2005) Nat Rev Drug Discovery 4:581-593. Figure 7: A cell viability assay (MTS) was used to measure toxicity of free polymer to HeLa cells. The IC50 is 0.0048 mg/ml forfree PEI and 0.0038 mg/ml for PEI-VDP. In the polymer concentration range of interest, less than 0.001 mg/ml, PEI-VDP is not more toxic than PEI. Figure 3: One approach to improving the efficiency of polyplexes is by attaching peptide ligands that target specific barriers. For example, Tet1, a peptide based on tetanus toxin, can be included for targeting and uptake and VDP, a virally-derived peptide, can be incorporated for endosomal escape and retrograde transport. The resulting multifunctional vehicle functions like a molecular Swiss Army knife.