Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integrat Show more
Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91(-/-) cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91(phox). Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91(phox) expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e77; doi:10.1038/mtna.2013.4; published online 5 March 2013. Show less
Rik Gijsbers, Keshet Ronen, Sofie Vets+5 more · 2010 · Molecular therapy : the journal of the American Society of Gene Therapy · Nature · added 2026-04-24
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 integrat Show more
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. Show less