Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection

Gene therapy has long held promise to correct a variety of human diseases. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas) and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgenic animals and plants, and transcriptional modulation. 

One potential application for CRISPR/Cas9 currently being explored is cell re-engineering to combat HIV/AIDS.

To fulfill a productive infection cycle the human immunodeficiency virus (HIV) relies on host-cell factors. Interference with these co-factors has demonstrated to be effective in protecting cells against HIV infection, as exemplified by the natural occurring CCR5 del mutations and the ablation of HIV co-receptor CCR5 using zinc finger nucleases.

An alternative target being explored in this thesis is LEDGF/p75. LEDGF/p75, encoded by the PSIP1 gene, is used by the lentiviral integrase (IN) protein in the pre-integration complex of HIV to bind host-cell chromatin and thus, facilitating proviral integration. LEDGF/p75 depletion results in defective HIV replication. However, as its cellular function, LEDGF/p75 tethers cellular proteins and their respective complexes to the host-cell genome. We assessed CRISPR KO and, in addition, used site-specific editing of the PSIP1 locus using CRISPR/Cas to target the aspartic acid residue in position 366 and mutated it to asparagine (D366N) to disrupt the interaction with HIV IN but retain LEDGF/p75 cellular function. The resulting cell lines demonstrated successful disruption of the LEDGF/p75 HIV-IN interaction without affecting binding with cellular binding partners. In line with LEDGF/p75 depleted cells, D366N cells did not support HIV replication, in part due to the limited integration efficiency. In addition, we have confirmed that the provirus that had managed to integrate showed only negligible transcriptional activity thus, in effect, remained transcriptionally silent.

Taken together, these results support the potential of site-directed CRISPR/Cas9 mediated knock-in to render cells more resistant to HIV infection and provides an additional strategy to protect patient-derived T-cells against HIV-1 infection as part of cell-based therapy.