A novel method for site-specific labeling of DNA with [hydrogen-3] and [carbon-14] which maintains plasmid structure and function.

摘要

Recent advances have brought gene therapy to the forefront of biomedical research. However, critical concerns about its clinical safety and efficacy still need to be addressed. The knowledge of in vivo disposition of therapeutic genes is important in this regard as it provides a basis for their therapeutic efficacy or lack thereof, and also helps in elucidation of toxicity linked to their biodistribution. Use of covalently radiolabeled DNA offers a convenient approach for studying the pharmacokinetics of plasmid DNA-based non-viral gene delivery systems. However, existing methods to covalently label plasmid DNA (pDNA) are known to affect its physical topology. This can be a serious disadvantage since plasmid topoforms are observed to differ in their biodistribution profiles. Thus, biodistribution profiles seen with labeled plasmid may not represent that of the original unlabeled plasmid. In addition, existing labeling methods lead to non-specific incorporation of label into pDNA, which can significantly inhibit the transcriptional capability of the transgene. Another limitation of existing labeling techniques is the inability to distinguish between intact, biologically active gene and its degraded fragments.; The overall goal of this dissertation project was to develop a novel radiolabeling method specifically for studying the tissue distribution of the plasmid DNA as well as for investigating its pharmacokinetic behavior and metabolic degradation. The labeling method utilizes the natural sequence-specificity of bacterial methylases to selectively incorporate radiolabel ([3H] and/or [14C]) into specific regions of supercoiled DNA. Moreover, the original plasmid topology and gene expression capability is not adversely affected by this labeling method. The ability of the labeling method to introduce two different radioisotopes into specific regions of the plasmid can also been applied to obtain double-labeled plasmids, which may be able to distinguish between intact, biologically active gene and its degraded fragments. We believe that this method will allow us to obtain a better correlation between gene delivery and gene expression, which will help in developing safer and more effective gene delivery vectors.