A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability,Topology,and In Vitro/In Vivo Functional Analysis

Abstract

The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice–grade pCMVβ reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at −20°C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

Get full access to this article

View all access options for this article.

References

Al-Dosari

M.S.

, and Gao

(2009). Nonviral gene delivery: principle, limitations, and recent progress. AAPS J., 11, 671–681.

Anselmetti

, et al. (1994). Biological materials studied with dynamic force microscopy. J. Vac. Sci. Technol. B, 12, 1500–1503.

Edelstein

M.L.

, et al. (2007). Gene therapy clinical trials worldwide to 2007—an update. J. Gene Med., 9, 833–842.

Escoffre

J.M.

, et al. (2012). New insight in the electrotransfer process: evidence for the involvement of the plasmid DNA topology. Curr. Gene Ther., 12, 417–422.

Fichtner

, et al. (2004). Anticancer drug response and expression of molecular markers in early-passage xenotransplanted colon carcinomas. Eur. J. Cancer, 40, 298–307.

Gill

D.R.

, et al. (2009). Progress and prospects: the design and production of plasmid vectors. Gene Ther., 16, 165–171.

Ginn

S.L.

et al. (2013). Gene therapy clinical trials worldwide to 2012 - an update. J. Gene Med., 15, 66–77.

Haase

, et al. (2010). pEPito a significantly improved non-viral episomal expression vector for mammalian cells. BMC Biotechnol, 10, 20.

Kim

B.I.

, et al. (2011). Long term structural changes of plasmid DNA studied by atomic force microscopy. Scanning, 33, 405–412.

10.

Lara

A.R.

, and Ramirez

O.T.

(2012). Plasmid production for therapeutic applications. Methods Mol. Biol., 824, 271–303.

11.

Lyubchenko

, et al. (1993). Atomic force microscopy of long DNA: imaging in air and under water. Proc. Natl. Acad. Sci. USA, 90, 2137–2140.

12.

Remaut

, et al. (2006). Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes. J. Control. Release, 115, 335–343.

13.

Schleef

, and Blaesen

(2009). Production of plasmid DNA as a pharmaceutical. Methods Mol. Biol., 542, 471–495.

14.

Schleef

, and Schmidt

(2004). Animal free production of ccc-supercoiled plasmids for research and clinical applications. J. Gene Med., 6, 45–53.

15.

Schleef

, et al. (2010). Production of non viral DNA vectors. Curr. Gene Ther., 10, 487–507.

16.

Schmidt

, et al. (1999). Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis. Anal. Biochem., 274, 235–240.

17.

Tolmachov

(2009). Designing plasmid vectors. Methods Mol. Biol., 542, 117–129.

18.

Voss

(2007). Production of plasmid DNA for pharmaceutical use. Biotechnol. Ann. Rev., 13, 201–222.

19.

Voss

, et al. (2003). Production of supercoiled multimeric plasmid DNA for biopharmaceutical application. J. Biotechnol., 105, 205–213.

20.

Voss

, et al. (2005). From bulk to delivery: plasmid manufacturing and storage. In DNA Pharmaceuticals. Schleef

, ed. (Wiley-VCH, Weinheim, Germany), pp. 23–42.

21.

Walther

, et al. (2001). Non-viral in vivo gene delivery into tumors using a novel low volume jet-injection technology. Gene Ther., 8, 173–180.

22.

Walther

, et al. (2003). Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer. Anal. Biochem., 18, 230–235.

23.

Walther

, et al. (2008). Novel jet-injection technology for nonviral intratumoral gene transfer in patients with melanoma and breast cancer. Clin. Cancer Res., 14, 7545–7553.

24.

Zheng

, et al. (2011). Identifying stabilizers of plasmid DNA for pharmaceutical use. J. Pharm. Sci., 100, 904–914.