Riassunto analitico
The genome editing procedure aims to manipulate the genome in a selected region resulting in gene disruption, correction or addition. Recently the site-specific gene addition came out as a safer alternative to the random gene insertion mediated by the retroviral/lentiviral vectors. Site-specific gene insertion has been remarkably improved thanks to the development of custom made zinc-finger nucleases (ZFNs). This platform includes two zinc-finger DNA binding motifs, that bind predetermined DNA sequences, fused to the catalytic domain of the endonuclease FokI, which generates a double strand break (DSB). DSB occurring into the genome can be repaired by homologous recombination (HR) with the sister chromatid or with designed donor DNA cassette in which the gene of interest is flanked by sequences homologous to the target site. The donor templates are usually delivered by non-integrating viral vectors, such as integrase-defective lentiviral vector (IDLV) carrying a mutation in the viral pol gene (D64V), adenoviral vector (Ad) or adeno-associated vector (AAV). The aim of this work is to molecularly compare the AAV2, Ad and IDLV as vector scaffold to deliver a GFP expression cassette to the predetermined AAVS1 site on chromosome 19, which has been reported to be a “safe harbor” for hosting and robustly expressing an exogenous gene. Human keratinocyte cell line (HaCaT) were transduced with IDLV-, Ad- and AAV2-donor together with an Adenoviral vector carrying the ZFNs specific for the AAVS1 site. Under all conditions tested, I achieved between 30-80% transduction, depending on viral vector used as measured 3 days after infection by cytofluorimetric analysis (FACS) of GFP expression. Cells cultures were carried on for 14-20 days in order to serially dilute the un-integrated GFP cassette and to measure the percentage of cells harboring the GFP cassette stable integrated into the genome. The results showed that AAV2-donor provides an HR frequency lower than IDLV-donor, but higher that Ad-donor. To deeply investigate the HR process occurring in the AAV-donor transduced cells, the AAV2-donor/AdZFNs transduced bulk population was sorted out for GFP-expression and cloned by serial dilution. Molecular analysis (site-directed PCR and Southern blot) on single clones demonstrated the presence of single copy of the GFP cassette into the AAVS1 locus in the majority of the clones (14 out of 21). Interestingly none of the clones showed the presence of concatamer of the integration cassette previously described in clones stably transduced with an IDLV-donor .To deeply investigate the role of the lentiviral defective integrase used to package the IDLV-donor, I constructed 2 more IDLV donors carrying 3 (IDLV3x-donor) or 4 (IDLV4x-donor) point mutations in pol gene leading to single amino acid substitutions of 3 (K258, K264, K266) or 4 lysine (K258, K264, K266, K273) to arginines in the C-terminal of the integrase. These lysine sites have been reported to play a role in the process of integration of the HIV genome during infection. HaCaT cells were transduced with AdZFNs in combination with IDLV3x-, IDLV4x- and IDLV-donor as control in order to understand if the 3 or the 4 mutations have an impact on the HR process. The results showed that IDLV3x- and IDLV4x-donor vectors have a defect in the viral packaging as demonstrated by p24 titer compared to GFP transducing efficiency nevertheless I could not detect any difference in the HR efficiency, suggesting that the mutated lysine sites in the integrase domain are not crucial in the HR process after ZFN-mediated DSBs. Overall, these experiments demonstrate that AAV2 represent a promising vector to deliver an homologous integration cassette upon ZFN cleavage on the target site although the on-target integration efficiency is lower compared to the IDLV-donor. Further studies are required to deeply investigate the role of the pre-integration complex of the lentiviral vector in the HR repair process
|