iBB

The determination of the recombination efficiency and the monitoring of parental plasmids (PP), minicircles (MC) and miniplasmid (MP) species during processing and in the final product are critical aspects of minicircle manufacturing. A paper published in Analytical Biochemistry by BERG-iBB researchers Cláudia Alves, Miguel Prazeres and Gabriel Monteiro describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The results presented indicate that the method can be applied for the determination of recombination efficiency in pure DNA samples.

Description: plate for screening CRISPR modifications, featured photo by Cláudia Alves, Copyright BERG-iBB and University of Florida 2019.

Context: BIOTecnico PhD student Cláudia Alves, under the supervision of BERG-iBB researchers Gabriel Monteiro and Miguel Prazeres and in collaboration with Christopher Reisch from the University of Florida, is using CRISPR/Cas9 to create mutations in E. coli that improve minicircle production.

Peripheral artery disease (PAD) is a debilitating condition characterized by the blockage of arteries, which leads to limb amputation in more severe cases. Researchers from SCERG- and BERG-iBB propose the use of human bone marrow (BM) MSC transiently transfected with minicircles encoding for VEGF as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The data shows that VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays.These results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach to improve angiogenesis in the context of PAD. The work was published in Human Gene Therapy.

The ability to purify supercoiled minicircles (sc MC) from related miniplasmid (MP) and parental plasmid (PP) impurities is critical for a wider application of MC vectors in gene therapy research. BERG-iBB researchers developed a process that relies on multimodal chromatography with a 2-step NaCl elution to separate sc MC from open circular impurities. A detailed study of the DNA-ligand interactions underlying the separation shows that the order of elution reflects the increasing degree of base exposure of the species. Data further suggests that nucleic acid bases most likely engage in cation-π, π-π stacking and H-bonding with ligand. The work was published in Separation and Purification Technology.