Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA p...

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Bibliographic Details
Published in:Analytical biochemistry 2003-07, Vol.318 (2), p.230-235
Main Authors: Walther, Wolfgang, Stein, Ulrike, Voss, Carsten, Schmidt, Torsten, Schleef, Martin, Schlag, Peter M
Format: Article
Language:English
Subjects:
Animals
CGE analysis
DNA - chemistry
Electrophoresis, Capillary
Gene therapy
Gene Transfer Techniques
Genetic Therapy - methods
Mice
Mice, Nude
Naked DNA
Nonviral gene transfer
Plasmid stability
Plasmids - chemistry
Specimen Handling
Time Factors
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Summary:The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVβ plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at −80 °C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 °C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at −80 °C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.
ISSN:0003-2697
1096-0309
DOI:10.1016/S0003-2697(03)00244-6