(M1330-04-23) Reversible, Covalent DNA Condensation Approach via Chemical Linkers for Enhanced Gene Delivery

Purpose: Viral approaches have been widely applied for gene delivery, but this approach suffers from limited gene packing size, host immune response and high manufacture cost. Non-viral gene delivery has been explored as an alternative approach but it generally suffers from lower efficiency. Specifically, the current non-viral DNA condensation systems are mainly based on electronic interaction between cationic polymer/liposomes and anionic DNA plasmids. However, the electronic interaction is too weak to keep DNA condensed to diffuse through the cytoplasm and transport into the nucleus to initiate gene transcription. Here, we developed a two-stage condensation approach to achieve efficient nucleus transport. Firstly, we utilized chemical linkers to crosslink DNA plasmids via a reversible covalent bond to form bundled DNA (b-DNA) with smaller sizes. Then, we package the b-DNA into the cationic vectors to further condense b-DNA and enable efficient delivery to the nucleus. We demonstrated clear improvements in gene delivery efficiency with b-DNA with different sized b-DNA plasmids using our strategy.

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 24 h at room temperature. The chemical crosslinkers include disulfides(ethane-2,1-diyl) bis(4-nitrophenyl) bis(carbonate) (NPC), and the PEGylation of NPC crosslinkers with different chain lengths (e.g. PEG 1k, PEG 2k, PEG 3.4k and PEG 5k) and topological structure (e.g. 4-arm PEG). Then, the mixture was collected and washed with PBS at least 5 times using Amicon Ultra 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.

Results: 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 1A). 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 1B). We then applied our condensed DNAs for gene delivery in HEK cells and demonstrated that PEI/b-DNA exhibited greater transfection abilities than PEI/original DNA, shown in Figures 2A and 2B. 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 2C, without affecting cells toxicity (Figure 2D). Finally, we examined our DNA condensation strategy for delivery of very large 11.6 Kbp DNA plasmids. The flow cytometry data demonstrated that the percentage of the transfected cells increased from ~2% for PEI/original DNA to ~15% PEI/b-DNA and the percentage of the transfected cells increased from ~5% for Lipofectamine 2000/original DNA to >20% for Lipofectamine 2000/ b-DNA (Figure 3A and 3B).

Conclusion: In conclusion, we developed a covalent DNA condensation approach via chemical linkers to achieve enhanced DNA nucleus import and improved gene delivery efficiency. The DNA plasmids were firstly bundled to smaller size via reversible covalent crosslinking method, and then packaged into the cationic polymers or liposomes for cell uptake. We demonstrated high gene delivery efficiency even at a low concentration of cationic vectors with 6.2 Kbp DNA and a significant improvement in gene transfection efficiency even when DNA plasmid size increased to 11.6 Kbp.

Acknowledgements: We would like to acknowledge 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), and the University of Texas at Austin Startup Fund. We declare that a patent application relating to this work is currently being filed


Figure 1. DNA condensation with chemical linkers. (A) The measurements of DNA crosslinking density and DNA hydration size changes with the reaction time, where crosslinker PEG2000 is used. (B) The final DNA hydration size after reacting with various linkers for 24 h (n=3 per group, one-way ANOVA).


Figure 2. Enhanced green fluorescence protein (GFP) expression of 6.2 Kbp b-DNA through RECAST in HEK cells. (A) GFP expression images of HEK cells by various PEI/DNA complexes. (i) original DNA, and condensed DNA via (ii) NPC, (iii) 4-arm PEG linker, (iv) PEG1000 linker, (v) PEG2000 linker, (vi) PEG3400 linker, (vii) PEG5000 linker, scale bar is 100 μm; (B) the corresponding fluorescence intensity quantification analysis of (A) (n > 4 per group, one-way ANOVA); (C) the flow cytometry quantification analysis of various PEI/ DNA complexes (n = 5 per group, one-way ANOVA); (D) the cell viability tests of various PEI/ DNA complexes at N/P of 10 (n > 3 per group, one-way ANOVA). All plots show mean ± SEM unless otherwise mentioned. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.


Figure 3. Enhanced green fluorescence protein (GFP) expression of 11.6 Kbp b-DNA through RECAST in HEK cells. (A) the flow cytometry quantification analysis of PEI/DNA complexes with different chemical linkers; (B) Lipofectamine 2000/DNA complexes with different chemical linkers (n = 3 per group, one-way ANOVA). All plots show mean ± SEM unless otherwise mentioned. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.