Technology of gene transfer: non-viral vectors

Haema 2016; 7(1): 72-80

by Aristidis Giannakopoulos, Eleana F. Stavrou, Aglaia Athanassiadou

Laboratory of General Biology, Medical School, University of Patras, Patras, Greece

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Non viral Vectors of gene transfer are usually plasmids, whose basic structure involves the transgene under the control of an efficient promoter, an origin of DNA replication and a gene for antibiotic resistance. Non viral vectors cannot transmit their DNA into cells, and therefore physical or chemical methods have been developed for this purpose. Their main advantage is that they do not intergrate into the endogenous genetic material and therefore they circumvent insertional mutatgenesis, an inherent problem of viral vectors. However, as a consequence of this, they tend to follow an erratic mitotic segregation that results in plasmid loss during culture. A major part of research on non viral vectors focuses on the development of structures that will give the plasmid the possibility of long term establishment in the cell nucleus, as an autonomously replicating unit. Replicating episomal vectors (REVs) and their derivatives are currently the main type of non viral vectors. REVs are maintained into the cell nucleus as autonomously replicating units and specific origins of replication, deriving from viral genomes (EBV and SV40), ensure their DNA replication. Most of the currently used REVs carry a Scaffold/Matrix Attachment Region – S/MAR that is rendering the plasmid capable for long term establishment in the cell nucleus. S/MARs are TA rich areas of DNA that belong to the ‘boundary elements’ of the genome, by defining chromatin loops and they are tethering chromatin to the nuclear matrix, for the maintenance of nuclear architecture and transcription of the transgene. These type of vectors have been shown to be functional in in vitro experiments with established cell lines and with primary cell cultures as well as in vivo experiments with whole organisms. Among other, it has been shown to have the capacity for stable transfection of the haematopoietic progenitor cells CD34+. Two types of derivative vectors that are devoid of prokaryotic DNA sequences, and therefore safer, are under development: (1) Vector pFAR (free of antibiotic resistance), which is constructed to be without a gene for antibiotic resistance. This vector needs a specially devised stain of E.Coli for culture. (2) Vector type minicirle, circular non viral vectors that are produced by in vivo recombination of the original plasmid, to contain only the eukaryotic transcription cassette. Finally, the transposon/transposase system SB1000X is a non viral vector, which, however, is integrated into the genome. This occurs quite randomly and no preference for the exonic or actively transcribed sequences is shown, and therefore the SB100X system is considered to be safer than the retroviral system. This system has been used in pre-clinical studies as well as in clinical trials.