Sustained Transgene Expression Using Non-Viral Enzymatic Systems for Stable Chromosomal Integration

Abstract

Gene delivery technologies have been developed for various biotechnology applications. In gene therapy, they are promising for the treatment of several inherited and acquired human diseases. When therapies require the transfection of a transgene, the vector integration is one of the solutions that is used for maintaining and sustaining expression. On the basis of their origin, vectorisation technologies are currently divided in two fields, gathering on one hand viral vectors and, on the other hand, non-viral approaches. In the case of the non-viral therapies, three main sub-fields are in progress to integrate transgenes. The first uses oligonucleotides to stimulate targeted gene repair by homologous recombination. The second is based on site-specific endonucleases for which the cleavage activity is used to stimulate the host recombination mechanisms in the presence of a DNA vector. The third one is developed from phage and transposon enzymatic systems. The two lasts sub-fields use non-viral enzymes and are the scope of this review. Here, our objective was to overview the main non-viral enzymatic systems able to integrate DNA cassettes. Their molecular and functional characteristics are summarized, and their properties and limits in the current state of the art highlighted. An overview of the safety and quality issues is also presented and discussed, taking into account the solutions that might circumvent problems, intellectual property and economic status for each system. As a conclusion, we propose projections of the future technological developments in the context of the different interests for public and private bodies.

Transposon tools: worldwide landscape of intellectual property and technological developments

“…There are four different statutes: on the one hand (1) granted patents, and on the other hand the applications which are not issued: (2) publications that have been published (3), those currently undergoing examination, and (4) those deemed to have been withdrawn. These findings confirm those of a previous study of the families of published patents concerning transposases and transposons (Palazzoli et al 2008 Two other co-authoring applicants in the top 10 ( Fig. 4b) are the Transgenrx Company and the Louisiana State University and Agricultural and Mechanical College.…”

“…At any moment, the patent can be withdrawn, and then the invention falls into the public domain, and becomes available for free commercial exploitation Genetica (2010) 138:285-299 287 some space for letters and articles that discuss patent landscapes, including critical and relevant analyses of the commercial R&D and academic research, open-science alternatives, and the strategy of the technology's owners (Vrtovec and Vrtovec 2007;Bergman and Graff 2007;Denker 2008;Vrtovec and Scott 2008;Chandrasekharan et al 2009). In a few cases, most of the articles in purely scientific reviews have in fact included a section about IP (Palazzoli et al 2008). Patent contents and portfolios are of interest at two main levels (Fig.…”

Modified Transposases for Site-Directed Insertion of Transgenes

“…The integrated gene must be correctly regulated and expressed long-term, and integration must not cause any deleterious effects for the cells (Palazzoli et al 2008 ) . One strategy that might comply with these constraints, and help to overcome the dif fi culties associated with integration into a single copy genomic site, would be to use a non-heterochromatic repeated sequence.…”

Site-directed integration of transgenes: transposons revisited using DNA-binding-domain technologies

“…There are several constraints: correct regulation and long-term expression of the transgene, and integration without any deleterious effects for the cells (Palazzoli et al 2008). Several repeated sequences comply with these conditions.…”

“…Using such vectors could have negative consequences, leading to insertional mutagenesis or position effects due to random gene integration. Alternatively, they might be very inefficient, leading to the insertion of transgenes into unexpressed genomic regions (Palazzoli et al 2008).…”