Gene targeting of human embryonic stem cell derived neural precursor cells with adeno associated viral vectors

The derivation of human embryonic stem (hES) cells and their somatic derivatives like neural stem cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of these cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. Up to now only very few genetically modified human stem cell lines are available. This is due to a lack of tools for efficient genetic modification.
In this study, a new adeno-associated viral (AAV) system for site-specific integration was used for targeting the adenosine kinase (ADK) gene in human embryonic stem cell-derived neural stem cells (hES-NSCs). This system uses homology arms amplified from the locus of the target cells to direct the site of recombination.
To that end, five different targeting vectors for two exons of the ADK were constructed and used for production of recombinant AAV particles. rAAVs were able to infect hESC-NSCs and to stably integrate in ~0.3% of the cells. Under chemoselection, clones were raised and screened for correct targeting via PCR. Of 15 clones analysed, two showed evidence of site specific integration at one of the two alleles. By applying a second round of infection with an additional selection marker, a biallelic targeting of the ADK gene will be accomplished in the future. Thus, AAV- mediated gene targeting in hES cell and their somatic derivatives may serve as a powerful alternative tool for gene alteration in the human system.