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 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 pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees 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 degrees 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 degrees 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.
Walther
Wolfgang
W
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany. wowalt@mdc-berlin.de
Stein
Ulrike
U
Voss
Carsten
C
Schmidt
Torsten
T
Schleef
Martin
M
Schlag
Peter M
PM
D000818:Animals; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000737:chemistry*; D013048:Specimen Handling; D013997:Time Factors
D004247:DNA