Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection.
BACKGROUND
Stability, integrity and retention of the DNA within the targeted tissue is decisive for efficient gene transfer using naked DNA. Pre-clinical and clinical studies require reproducible transfection rates by preventing rapid degradation of naked DNA in the transduced tissue. Tumor tissues contain nuclease activity, which can affect DNA stability if naked DNA is used. Therefore, inhibition of nuclease-mediated DNA degradation by the nuclease inhibitor aurintricarboxylic acid (ATA) might lead to improved gene transfer efficiency in tumor tissues.


METHODS
For both, DNA-degradation analysis and in vivo gene transfer experiments, the beta-galactosidase (LacZ)-expressing pCMVbeta and the cytosine deaminase (CD)-expressing pCMV-CD plasmid were used. Influence of the nuclease inhibitor ATA was determined in tumors, in which naked pCMVbeta or pCMV-CD DNA and ATA was co-administered by jet-injection. The nuclease activity and inhibition by ATA was analyzed using the DNase Alert detection system. The influence of ATA on LacZ expression was determined by specific ELISA and its effect on the therapeutic efficacy of CD gene transfer on tumor growth was determined in vivo.


RESULTS
The screening of different human mammary and colon carcinoma models revealed strong nuclease activity rapidly degrading naked plasmid DNA. Co-administration of ATA with pCMVbeta or pCMV-CD for in vivo jet-injection of tumors prevented DNA from nuclease degradation associated with either increased LacZ gene expression or improved reduction in tumor growth.


CONCLUSIONS
Tumor-associated nuclease activity is a notable hurdle in gene transfer of naked DNA and therefore inhibition of nucleolytic degradation of plasmid DNA facilitates intratumoral gene expression.
Walther
Wolfgang
W
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany. wowalt@mdc-berlin.de
Stein
Ulrike
U
Siegel
Robert
R
Fichtner
Iduna
I
Schlag
Peter M
PM
D001312:Aurintricarboxylic Acid / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D006801:Humans; D015552:Injections, Intralesional; D001616:beta-Galactosidase / Q000235:genetics
D004791:Enzyme Inhibitors; D001312:Aurintricarboxylic Acid; D001616:beta-Galactosidase