Diseño y desarrollo funcional de modelos vectoriales derivados de lentivirus deficientes en integración para la modificación génica específica de sitio

Transgenesis procedures in which the position and dosage of the inserted transgene are uncontrolled can lead to unpredictable effects due to disruption of host gene function, compromising data interpretation and placing severe limits on the applicability of this technology. Current methodologies for transgenesis suffer from a strong experimenter effect due to the uncontrolled position and number of inserted transgenes. Therefore, the availability of efficient procedures to generate isogenic cells aimed to obtain comparable results (because of the use of identically modified organisms) could allow to gain more reliable insights into gene function and ultimately, closer to those operating in physiological, pathological, and therapeutic conditions. We have addressed in this Ph.D. thesis project the development and functional assessment of novel designs aimed at providing solutions to the aforementioned problems. The designs are based on integration-deficient lentivirus (IDLV) and have followed three approaches: 1. Recombinase? mediated cassette exchange (RMCE) platform is based on the integration in the cell genome of an acceptor cassette (genomic landing pad Kas, standing for KO/KI acceptor site) to generate a receptor-modified cell. The second step is the delivery of a DONOR cassette driving the interchange (transfer Trina, standing for Transfer into acceptor) delivered by an IDLV. The IDLV encodes in an auto-limited manner the expression of cre recombinase that promotes the RMCE reaction on Kas. A promoter trapping strategy serves to monitor the authentic recombination switching the pattern of expression reporter genes. We have obtained reliable results in both human cell line (HEK293) and primary human cells (BJ fibroblasts) with high efficiency of RMCE (100% of the selected clones). The genomic analysis revealed proper insertion of the donor cassette as well as the absence of integrated copies of the lentivirus, assessed by high sensitivity qPCR. Finally we have demonstrated the versatility and applicability of the platform on episomal plasmids, random integrated copies and, importantly on singe copy insertion of Kas in the safe AAVS1 locus. 2. eGFP-driven fluorescence; excision of floxed genes by incoming cre; and sitespecific integration on AAVS1 mediated by rep78. 3. episomes. We have studied systematically the effect on episomal maintenance of several eukaryotic replication origins (ori) and S/MAR sequences on one-to-one combinations. The total generated bearing those sequences and reporter neoR and eGFP genes have been demonstrated to last the expression of the reporters in the absence of any selection for more than 3 months. The analysis of integration of the constructions by Southern blot on genomic and Hirt? s extracts, conventional PCR, and qPCR support the idea of non-integration at the end of the period. Finally, conclusive results were obtained by FISH analysis demonstrating than several combinations allow maintaining the phenotype with no-integrated copies in highly dividing cells (HEK293). Then we have developed novel and efficient episomes supporting partition, i.e. replication in the absence of viral proteins and segregation during karyokinesis. In summary, we have expanded the current use of vectors exploiting this capabilities to generate a collection of biological tools aimed at directing site specific integration in the genome with minima interference. We can anticipate a wide use in future transgenesis experiments both in research and, hopefully in therapies.