LEDGF Hybrids Efficiently Retarget Lentiviral Integration Into Heterochromatin

Abstract

Correction of genetic diseases requires integration of the therapeutic gene copy into the genome of patient cells. Retroviruses are commonly used as delivery vehicles because of their precise integration mechanism, but their use has led to adverse events in which vector integration activated proto-oncogenes and contributed to leukemogenesis. Here, we show that integration by lentiviral vectors can be targeted away from genes using an artificial tethering factor. During normal lentivirus infection, the host cell-encoded transcriptional coactivator lens epithelium-derived growth factor/p75 (LEDGF/p75) binds lentiviral integrase (IN), thereby targeting integration to active transcription units and increasing the efficiency of infection. We replaced the LEDGF/p75 chromatin interaction-binding domain with CBX1. CBX1 binds histone H3 di- or trimethylated on K9, which is associated with pericentric heterochromatin and intergenic regions. The chimeric protein supported efficient transduction of lentiviral vectors and directed the integration outside of genes, near bound CBX1. Despite integration in regions rich in epigenetic marks associated with gene silencing, lentiviral vector expression remained efficient. Thus, engineered LEDGF/p75 chimeras provide technology for controlling integration site selection by lentiviral vectors.

Role of the PWWP Domain of Lens Epithelium-derived Growth Factor (LEDGF)/p75 Cofactor in Lentiviral Integration Targeting

“…C_LEDGF BC-Ires-Bsd was described earlier (17). pLN-C_LEDGF BC D366A-Ires-Bsd was constructed by replacing the 5Ј end of the LEDGF/p75 cDNA after XhoI-StuI digest.…”

“…Cells depleted for LEDGF/p75 (13,14) or somatic knock-out cells (15,16) inhibit productive HIV infection. In addition, fusion proteins in which the LEDGF/p75 DNA binding portion is replaced with an alternative chromatin binding domain efficiently tether the PIC to the host cell chromatin and support lentiviral vector transduction (17)(18)(19)(20). We and others demonstrated that these fusion proteins retarget proviral integration to the regions bound by the specific chromatin binding domain (17,18).…”

“…In addition, fusion proteins in which the LEDGF/p75 DNA binding portion is replaced with an alternative chromatin binding domain efficiently tether the PIC to the host cell chromatin and support lentiviral vector transduction (17)(18)(19)(20). We and others demonstrated that these fusion proteins retarget proviral integration to the regions bound by the specific chromatin binding domain (17,18).Defects in lentiviral infection are only observed after potent knockdown of LEDGF/p75 because residual protein levels are sufficient to support integration (13,14,21). This initially blurred the interpretation causing controversy with two studies that failed to observe a reduction in HIV replication after partial * This work was supported, in whole or in part, by National Institutes of Health …”

“…In an effort to understand the role of the PWWP domain in LEDGF/p75-mediated tethering and targeting of the PIC, we employed potent LEDGF/p75 knockdown cell lines to permit unambiguous analysis in the absence of endogenous LEDGF/ p75 (17). Deletion of the PWWP domain disrupts association of LEDGF/p75 with condensed mitotic chromatin (37).…”

Single-Cell Imaging of HIV-1 Provirus (SCIP)

“…We next applied SCIP to cells where HIV-1 integration is retargeted toward heterochromatin and intergenic regions, thus altering the physiological lentiviral integration preference toward gene-rich units (36). This experimental system was obtained by expressing in target cells a chimera LEDGF/p75 engineered to contain an alternative chromatin-binding domain, CBX1, at its N terminus (CBX-LEDGF ).…”