iBB

Despite very good safety records, clinical trials using plasmid DNA have failed due to low transfection efficiency and brief transgene expression. One of the reasons for this failure is poor plasmid design. In a paper published in Biotechnology and Genetic Engineering Reviews, researchers from iBB-BERG led by Gabriel Monteiro review critical aspects of plasmid design including promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences. The impact of detrimental elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats is also examined. Click on title to learn more.

Photo details: CHO cells transfected with plasmid vector coding for GFP.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, a team led by Gabriel Monteiro and Miguel Prazeres from iBB-BERG and Kristala Prather from MIT, optimized the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA) using a predictive thermodynamic model. The optimized parA gene was then inserted in the chromosome of an E. coli strain with improved arabinose uptake. overall the results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.The research was developed under the framework of the MIT-Portugal program and is a direct result of Michaela Simcikova's Ph D. thesis in Bioengineering. Click on title to read the publication in Applied Microbiology and Biotechnology.