Cancer Gene Therapy Using Plasmid DNA: Purification of DNA for Human Clinical Trials

作者：Nancy A. Horn, Jennifer Meek, Gregg Budahazi, Magda Marquet · 发表于：Human Gene Therapy · 年份：1995 · DOI：10.1089/hum.1995.6.5-565 · 被引用次数：244 · 研究领域：RNA Interference and Gene Delivery、Immunotherapy and Immune Responses、Bacteriophages and microbial interactions

A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals. Defined pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. The overall process yield of purified plasmid DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli DNA levels are reproducibly below 1% as measured by Southern analysis. Endotoxin levels a...