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

Abstract

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 are less than 0.03 endotoxin units/micrograms plasmid DNA and residual protein is undetectable. This process was used to produce 100 mg of VCL-1005 for use in an active clinical protocol.

Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine

“…46 Plasmid DNA was prepared by bacterial fermentation and purification, and quality assurance analysis was performed as previously described. 43,47 Plasmid DNA was resuspended in phosphate buffered saline vehicle (PBS, 10 mm NaH 2 PO 4 , pH 7.2, 0.9% NaCl) at the desired concentration and stored at −20°C until use.…”

Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: Inhibition of primary tumor growth and metastatic spread

“…19 Plasmid DNA was isolated and purified from contaminating endotoxin using Sephacryl columns 24 ( Sephacryl S-1000 Superfine, 1.6Â100 cm; Pharmacia Biotech, Freiburg, Germany ). Endotoxin levels in purified DNA (0.03 EU / g of DNA ) were determined using a kit containing Limulus Amebocyte Lysate ( Biowhittaker, Rockland, ME ) as specified.…”

Sodium phosphate enhances plasmid DNA expression in vivo

“…Plasmid DNA purification Plasmid DNA was transformed into Escherichia coli DH10B competent cells and highly purified covalently closed circular plasmid DNA was isolated by a modified lysis procedure 55 followed by standard double CsCl-ethidium bromide gradient ultracentrifugation and dialysis. 56 SEAP and preproinsulin encoding plasmid DNAs were purified using Giga columns from Qiagen (Valencia, CA, USA) according to the kit instructions.…”