Reversible, covalent DNA condensation approach via chemical linkers for enhanced gene delivery

Introduction:: Viral approaches have been widely applied for gene delivery, but this approach suffers from limited gene packing size, host immune response, and high manufacturing costs. Non-viral gene delivery has been explored as an alternative approach but generally suffers from lower efficiency. In particular, efficient nucleus delivery of DNA has been the Achilles' heel of non-viral approach. Current non-viral DNA condensation systems are mainly based on electrostatic interaction between cationic polymer/liposomes and anionic DNA plasmids. However, the electrostatic interaction is too weak to keep DNA condensed to diffuse through the cytoplasm and transport into the nucleus for gene transcription initiation. Here, we report a two-stage condensation approach to achieve efficient cytoplasm diffusion and nuclear transport. Firstly, we utilize chemical linkers to crosslink DNA plasmids via a reversible covalent bond to form smaller-sized bundled DNA (b-DNA). Then, we package the b-DNA into cationic vectors to further condense b-DNA and enable efficient gene delivery to the nucleus. We demonstrate clear improvements in gene transfection efficiency with b-DNA, including using DNA plasmid with size up to 11.6 kbp. Furthermore, the improved gene transfection efficiency with b-DNA was also observed in primary cultured neurons. Finally, we have observed a clear improvement in lung-selective gene transfection efficiency in vivo by this two-stage condensation approach following intravenous administration. This reversible covalent assembly strategy demonstrates substantial value of non-viral gene delivery for clinical therapeutic applications.


Materials and Methods:: A covalent self-assembly method is developed to condense the DNA plasmid to a smaller size. Briefly, DNA and chemical crosslinkers were added to the PBS and reacted for 24h at room temperature. The chemical crosslinkers include disulfides(ethane-2,1-diyl) bis(4-nitrophenyl) bis(carbonate) (NPC), and the PEGylation of crosslinkers with different chain lengths (e.g. PEG 1k, PEG 2k, PEG 3.4k, PEG 5k) and topological structure (e.g. 4-arm PEG). Then, the mixture was collected and washed with PBS at least 5 times using Centrifugal Filters to remove the crosslinkers. The final volume of condensed DNA solution was adjusted to 0.5 mL with PBS (pH 7.4). The UV-Vis spectrum and dynamic light scattering (DLS) were used to measure the crosslinking density and size of DNA before and after condensation. The Polyethylenimine (PEI) and liposome-based nanoparticles were prepared by mixing DNA plamid with PEI/liposome, and incubated with kidney cell line 293T (HEK-293T) cells for 6 hours before characterization via fluorescence microscope and flow cytometry. For DIV3 primary embryonic cortical neurons, wells were treated with DNA nanocomposites consisting of 0.4 ug of pCMV-GFP DNA and 0.4 uL of jetOPTIMUS® diluted to 50 uL with Opti-MEM. 8-10 weeks old (20-25g). For in vivo gene delivery, Female C57BL/6 mice (n=5 pergroup) received i.v. tail-vein injections of in vivo-JetPEI nanocomposites formulated with 2 versions of b-DNA (PEG 2K and PEG 5K) and original DNA encoding for firefly luciferase (Luciferase-pcDNA3) at a dose of 1.5 mg/kg. In vivo bioluminescence imaging were done at 6, 12 and 24 hours post injection.


Results, Conclusions, and Discussions:: To test our strategy, we first chose 6.2 Kbp DNA as a model system to react with crosslinkers. The crosslinking density increased as the reaction progressed and became almost saturated after reaction 24 h, while the DNA hydration size reduced with increased crosslinking density (Figure 2a). After reaction for 24 h, the DNA hydration size was condensed from 600 nm to below 300 nm for all of the chemical linkers, with the smallest of 50 nm with 2000 molecular weight PEGylation crosslinker (Figure 2b). We then applied our condensed DNAs for gene delivery in HEK cells and demonstrated that the positive cell percentage increased from 60% in PEI/original DNA to up to 80% in PEI/b-DNA complexes with statistical significance shown in Figure 3a. In addition, we examined our DNA condensation strategy for delivery of very large 11.6 Kbp DNA plasmids and demonstrated that the percentage of the transfected cells increased from ~2% for PEI/original DNA to ~15% PEI/b-DNA (Figure 3b).Furthermore, the b-DNA complexes exhibited enhanced gene transfection efficiency compared to polymers/original-DNA complexes in primary neurons (Figure 4a). More specifically, the 4-arm PEG and PEG2k b-DNA/polymers complexes showed approximately a 2-fold increase in the percentage of neurons successfully expressing GFP (Figure 4b).Lastly, we investigated the in vivo gene delivery efficiency at targeted organs after intravenous administration. PEG 2k and PEG 5K condensed b-DNA encoding for firefly luciferase were complexed with the commercial cationic polymer, in vivo-JetPEI, which is known to enrich DNA delivery in lungs. After a 6-hour period following tail vein injection of beta-DNA complexes, enhanced luciferase protein expression was observed in mice treated with RECAST compared to the original DNA treated group, with the luciferase signal increasing approximately four-fold. (Figure 5a, b). After that, the luciferase signal decreased with the treatment time (Figure 5c) due to the clearance by the immune system.

In conclusion, we developed a strategy to achieve enhanced DNA nucleus import and improved gene delivery efficiency. Both in vitro and in vivo tests determined that it was an effective strategy to achieve improved gene transfection efficiency.








Acknowledgements (Optional): : We thank Dr. J. Stachowiak, Dr. L. Suggs, Dr. J. Tunnell, Dr. S. Parekh, Dr. A. Brock and Dr. R. Lu for allowing us to use their equipment and sharing with HEK cells with our lab. Dr. H. Wang acknowledges funding support from Robert A. Welsh Foundation Grant (No. F-2084-20210327), NIH Mentored Research Scientist Career Development Award (National Institute of Mental Health 1K01MH117490-01), NIH Maximizing Investigators’ Research Award (National Institute of General Medical Sciences 1R35GM147408), Craig Neilsen Foundation and the University of Texas and UT Austin Startup Fund.


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