Walther Wolfgang, Stein Ulrike, Siegel Robert, Fichtner Iduna, Schlag Peter M
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
J Gene Med. 2005 Apr;7(4):477-85. doi: 10.1002/jgm.690.
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.
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.
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.
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.
在使用裸DNA进行高效基因转移时，靶向组织内DNA的稳定性、完整性和保留情况起着决定性作用。临床前和临床研究需要通过防止裸DNA在转导组织中快速降解来实现可重复的转染率。肿瘤组织含有核酸酶活性，如果使用裸DNA，这种活性会影响DNA的稳定性。因此，核酸酶抑制剂金精三羧酸（ATA）抑制核酸酶介导的DNA降解可能会提高肿瘤组织中的基因转移效率。
在DNA降解分析和体内基因转移实验中，均使用了表达β-半乳糖苷酶（LacZ）的pCMVβ质粒和表达胞嘧啶脱氨酶（CD）的pCMV-CD质粒。通过喷射注射将裸pCMVβ或pCMV-CD DNA与ATA共同施用于肿瘤，以确定核酸酶抑制剂ATA的影响。使用DNase Alert检测系统分析核酸酶活性以及ATA的抑制作用。通过特异性ELISA确定ATA对LacZ表达的影响，并在体内确定其对CD基因转移治疗肿瘤生长疗效的影响。
对不同的人乳腺癌和结肠癌模型进行筛选发现，存在很强的核酸酶活性，可迅速降解裸质粒DNA。将ATA与pCMVβ或pCMV-CD共同用于肿瘤的体内喷射注射，可防止DNA被核酸酶降解，这与LacZ基因表达增加或肿瘤生长抑制改善相关。
肿瘤相关的核酸酶活性是裸DNA基因转移中的一个显著障碍，因此抑制质粒DNA的核酸降解有助于肿瘤内基因表达。
