Author Description

"Combination antiretroviral therapy (cART) can effectively inhibit replication of the human immunodeficiency virus (HIV) and prevent progression to AIDS. However none of the currently available drugs inactivate the HIV DNA that is integrated on one of the chromosomes of all infected cells. As a consequence, current cART does not lead to a cure of the virus infection and HIV-infected individuals need lifelong suppressive therapy to prevent AIDS. Novel strategies are being developed that aim at the inactivation of the integrated HIV DNA in infected cells, including the use of DNA cleaving CRISPR-Cas systems. In this thesis, I describe how we investigated the possibilities and risks of such antiviral therapy by analyzing the intended and unintended CRISPR-Cas effects. We demonstrate that CRISPR-Cas treatment of HIV-infected cells can permanently inactivate the viral DNA by causing small insertions and deletions (indels) at the target sites, or by causing excision or inversion of the DNA fragment between two target sites. However, we demonstrate that such treatment also causes much larger deletions, which can include flanking cellular DNA sequences. Using a novel quantitative assay we demonstrate that CRISPR-Cas treatment of cells causes large deletions at a surprisingly high frequency, with on average one large deletion for every two indels. As large chromosomal deletions may trigger oncogenic transformation and turn the cell into a cancer cell, this high frequency poses a serious safety risk for clinical application of the CRISPR-Cas systems. Therefore, it is crucial to closely monitor for such unintended effects when applying CRISPR-Cas systems in humans, not only in antiviral approaches but also in other gene therapy applications."--