University of Texas at Austin Austin, Texas, United States
Purpose: Recombinant DNA or plasmid (pDNA) is a crucial research tool and platform for gene therapy and vaccine applications. Disruption and degradation of plasmid conformations compromise the accuracy of PCR assays1, bacteria transformation and cell transfection2. The objective of this study was to adapt a novel thin film matrix to stabilize pDNA conformation and maintain transfection and PCR amplification capabilities3. Successful stabilization of pDNA within thin films reduce the reliance on cold chain for transportation and storage and the overall cost of plasmid based vaccines and biomedicines. Methods: A model plasmid (pAAV LacZ) was used for these studies. Film base was mixed and casted under aseptic conditions. Electrophoresis was utilized to identify different pDNA conformations on agarose gels. Linear and nicked pDNA standards were produced by digestion with BsaI and Nb.BsrD I accordingly. Transfection efficiency was assessed via transgene expression in HEK293 cells detected by β-galactosidase colorimetric assays. Percent recovery was calculated by comparison of transfection efficiency of pDNA in buffer stored at -20oC to that of formulated pDNA. qPCR with primers and probe targeting AAV ITR regions was used to evaluate amplification efficiency. Results: The screening of more than 40 excipients for thin film formulation yielded the selection of a base polymer, amino acid and sugars that support pDNA stability in the dry form. Films made from a combination of these compoents showed comparable supercoiled form, transfection (96.7 ± 1.6%) and amplification efficiency (101.7 ± 18.9%) to frozen pDNA stock. Plasmids stored within this optimized matrix for 12 weeks at 25oC/ RH 60% retained at least 80% transfection efficiency. Films remained in an amorphous state as determined by XRD. Conclusion: An optimized film matrix protected pDNA from degradation under ambient temperature for 3 months. Film base polymer plays the most influential role in preserving transfection efficiency of pDNA during the drying process while amino acids and sugars contributed to long-term stability. Additional studies are underway to characterize beneficial physical and chemical interactions between excipients and pDNA within the film matrix. References: 1. Lin, C. H., et al. PLoS One 6, (2011) e29101. 2. Chancham, P. & Hughes, J. A. J. Liposome Res. 11, 139–152 (2001). 3. Bajrovic, I., et al. Sci. Adv. 6, eaau4819 (2020).
Acknowledgements: This project was supported by a research grant from Asklepios BioPharmaceutical (MAC), College of Pharmacy Endowed Fellowships donated by Duane A. Boyle, Dr. Feng Zhang & James McGinity (TD).
