High-Yield Production of pBR322-Derived Plasmids Intended for Human Gene Therapy by Employing a Temperature-Controllable Point Mutation

Abstract

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.

Scalable Technology to Produce Pharmaceutical Grade Plasmid DNA for Gene Therapy

“…With this culture procedure larger amounts of plasmid pIDKE2 can be obtained in DH10B cells. Several researchers have reported the behaviour of plasmid volumetric yield (mg/L) for different fermentation strategy (Figure 1) (Diogo, 2000(Diogo, , 2001Durland, 1999;Lahijani, 1996;Shmidt, 2001Shmidt, , 2003Wang, 2001). As it is shown, plasmid production under non optimized laboratory conditions invariably leads to very low volumetric titters (5-70 mg/L) and DNA production processes that employ simple batch cultivation technology yield relatively low biomass and correlatively support low plasmid volumetric yields too (<100 mg/L).…”

Therapeutic angiogenesis following intramuscular gene transfer of vascular endothelialgrowth factor 121 in a dog model of hindlimb ischemia

“…agitation, and an air flow rate of 5 L/h. For increased copy number, the temperature was raised to 42ºC at mid-log phase (Lahijani et al 1996). After bacterial alkaline lysis, the mixture was neutralized and clarified by tangential flow filtration (TFF) using a nominal molecular weight cutoff TFF polyethersulfone membrane (1,000,000 Da).…”

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

“…In the past twenty years, many efficient strategies for recombinant-cell fermentation had been developed to attain high productivity of proteins in E. coli, B. subtillis, yeast, even in animal cell culture systems (SivaKesava et al, 1999;Ebisu et al, 1992;Cheng et al, 1997;Churgay et al, 1997). However, few papers were focused on the plasmid DNA production in recombinant E. coli fermentation process (Lahijani et al, 1996;O'Kennedy et al, 2000). It was reported that the media composition affected cell growth rate, and thus influenced plasmid copy number (Kleinman et al, 1986;Kim and Shuler, 1990).…”