Gene targeting by homologous replacement makes it possible to precisely manipulate genomic DNA and maintains genetic integrity by retaining the relationship between the protein coding sequences and the gene-regulatory elements (5
). This aspect of homologous replacement overcomes any potential for inappropriate gene expression either in the amount of protein produced or in the type of cell expressing the gene (33
). A recent study suggests that preclinical experimental treatments involving transgenes should include long-term follow-up before they enter clinical trials (34
). Authors reports a long latency period before lymphomas develop in mice transplanted with cells that have been transduced with LV-IL2RG. This observation further highlights the need to develop vectors capable of regulated therapeutic gene expression.
Oligonucleotide-mediated modification has been applied by a number of different groups both in vitro
and in vivo
to modify both plasmid and genomic DNA targets (35
). Among the various oligonucleotide-based gene targeting approaches, SFHR has been shown to correct specific mutations at a target locus (5
). In a recent study SFHR was shown to restore the SMN
full length protein in human SMA cells obtained from chorionic villi, demonstrating the feasibility of using this approach to stably correct human fetal cells (8
). Another study described genotypic and functional correction of a point mutation in the gene encoding the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (21
). In addition, a number of studies have shown specific modification of the CFTR gene by SFHR (2
Based on these studies, the potential of SFHR-mediated modification for “in vivo
” or “ex vivo
” gene therapy of monogenic disorders is significant when compared to the cDNA-based “gene complementation” approaches (5
This study showed that it was possible to insert a 3-bp (ΔF508) deletion into the genomic DNA Cftr gene of mouse embryonic stem cells by SFHR following electroporation (nucleofection) of a 783-bp ΔF508 fragment containing the unique KpnI restriction site. As a result, the SDF-derived ΔF508 mutant mRNA was expressed.
Furthermore potential PCR artefacts that could result from the presence of free SDF within the cell (20
) was not detected. To minimize the potential for artifact, the PCR primers used were outside the region of homology defined by the SDF. In addition, the SDF copy number at the time of analysis was about 3200 molecules per cell, assuming that there was no degradation or loss of the transfected SDF. This number is less than that required to give rise to any PCR artifacts as already reported in DNA mixing reconstitution analyses (20
). Moreover, the treatment of the isolated RNA with DNase eliminates any contaminating SDF that might be present in the crude RNA isolate. Consistently with the molecular analysis, CFTR channel activity was significantly reducted in transfected ES cells. Using spectrofluorimetric measurements of the entire population of the cells, we specifically found that CFTR-dependent chloride secretion was 58% lower in ES electroporated cells with respect to controls. Video-imaging measurements performed on single ES clone, demonstrated in the same time that each clone is composed by homogeneous cells but not all clones underwent to SFHR-mediated modification. In fact different regions within the same clone exhibited the same CFTR-dependent chloride efflux, but only 8 of 12 examined clones showed a complete inhibition of CFTR-dependent chloride efflux.
As far as we know, the present study applies for the first time a functional test for evaluating the specific SFHR-induced modification in ES cells, avoiding any artefacts due to the presence of the free SDF, not integrated within genomic DNA, as recently reported (45
In addition to its role as a tool for developing an in vitro
means for understanding the pathophysiology of monogenic disorders, SFHR can be applied to ES cells for therapeutically correcting genetic mutations and repairing disease dependent tissue damage (5
). SFHR has already been used for modifying hematopoietic stem cells (5
) that have been shown to have the capacity to differentiate into human airway epithelial cells (52
). Mouse ES cells have also been shown to generate a fully differentiate and functional tracheobronchial airway epithelium (53-55) and could also potentially be applied to repair damaged CF airways.
Moreover, mutating genes in ES cells by homologous recombination has been a powerful research tool for developing animal models of human disease. The approach described here could potentially augment these classical homologous recombination strategies in mice to develop a range of animal models through nuclear transfer (5
In conclusion, the present study represents the basis for developing innovative cell and gene-based therapeutic strategies for CF or other monogenic disease. While it has not yet been possible to effectively carry out somatic cell nuclear transfer in human oocytes, the potential of generating patient derived stem cells with corrected mutant genes could conceivably translate into a significant improvement and possible cures for many inherited diseases.