Minicircle Biopharmaceuticals–An Overview of Purification Strategies

Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are plasmid-derived circular DNA molecules that are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates a minicircle containing the eukaryotic therapeutic cassette of interest and a miniplasmid containing the prokaryotic backbone. The production process results thus in a complex mixture, which hinders the isolation of minicircle molecules from other DNA molecules. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles in compliance with the regulatory guidelines for therapeutic use. In minicircle purification, the characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. This review summarizes the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.

Brief report: Mutations in SIV Nef that disrupt and restore tetherin downregulation

The H196 residue in SIVmac239 Nef is conserved across nearly all HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) interacting domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317). Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196, which is associated with loss of multiple AP-2 dependent functions in SIVmac239. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this mutation. Variants were engineered into the SIVmac239 parental plasmid and mutant viruses were used to test tetherin downregulatory capacity in primary CD4 T cells using flow cytometry. SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.

Multiple Copies of qnrA1 on an IncA/C2 Plasmid Explain Enhanced Quinolone Resistance in an Escherichia coli Mutant

ABSTRACT In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1. Strain J53 Cipr 8-2 showed an increase in the copy number and transcription level of qnrA1. We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related StreptomycesPlasmid pSB24.2

ABSTRACT A database search revealed extensive sequence similarity betweenStreptomyces lividans plasmid pIJ101 andStreptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, thecis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korBfunctions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.

Genes Leave Their Characteristic Marks on the Melting Profile of Plasmid DNAs

High resolution melting profiles of four linearised plasmids (pUC9, pGV403, pHP2, and pBR322) were recorded by means of UV absorbance vs. teperature scaning. The set of transitions obtained for each plasmid are compared to each another and to the transitions obtained for their excised particular antibiotica resistance gene. It can be shown that each gene leaves a charactersitic mark on the melting profile of its parental plasmid