Modification of Integration Site Preferences of an HIV-1-Based Vector by Expression of a Novel Synthetic Protein

Abstract

HIV-1-based lentiviral vectors are a promising tool for gene therapy. However, integration of a lentiviral vector into host cell genes may lead to the development of cancer. Therefore, control of integration site selection is critical to the successful outcome of gene therapy approaches that use these vectors. The discovery that integration site selection by HIV-1 and HIV-1-based vectors is controlled by the LEDGF=p75 protein has presented new opportunities to control integration site selection. In this study, we tested the hypothesis that a fusion protein containing the C-terminal HIV integrase-binding portion of LEDGF=p75, and the N-terminal chromodomain of heterochromatin protein-1a (HP1a), can target HIV-1 vector DNA outside of genes. We show that this fusion protein, termed TIHPLE, associates with the heterochromatin hallmark trimethylated Lys-9 of histone H3 (H3K9me3). Transient overexpression of TIHPLE alters integration site selection by an HIV-1-based vector and decreases the number of integration events that occur in genes. This change in integration site selection was achieved without a reduction in overall integration efficiency. Furthermore, we show that TIHPLE increases integration in the vicinity of H3K9me3 and in repetitive DNA sequences. These data provide a novel approach to address the problem of the tendency of retroviral vectors to integrate at undesirable sites of the human genome.

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

“…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).…”

Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

“…In cells depleted of LEDGF/p75, HIV-1 integration and replication were significantly affected, while the residual HIV-1 integration sites were less enriched in transcriptional units (30-32). Furthermore, it was possible to retarget HIV-1 integration by chimeric proteins containing the IN binding domain (IBD) of LEDGF/p75 and alternative chromatin binding domains (33)(34)(35).Several cellular proteins, including transcription factors and chromatin and RNA binding proteins, were recently identified as potential interaction partners for MLV IN (36). This diverse group of proteins included BRD2, a member of the bromodomain and extraterminal domain (BET) family of chromatin binding…”

“…In cells depleted of LEDGF/p75, HIV-1 integration and replication were significantly affected, while the residual HIV-1 integration sites were less enriched in transcriptional units (30-32). Furthermore, it was possible to retarget HIV-1 integration by chimeric proteins containing the IN binding domain (IBD) of LEDGF/p75 and alternative chromatin binding domains (33)(34)(35).…”

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

“…LEDGF/p75 has a C-terminally located IN-binding domain (IBD) (24,25) and N-terminal elements that bind chromatin (26,27). Novel fusion proteins comprising the LEDGF/p75 IBD and foreign chromatin binding domains redirect HIV-1 to integrate at positions where the heterologous binding partner preferentially binds, demonstrating a direct role for the host factor in targeting lentiviral integration (28)(29)(30). Neither the preference for the local DNA sequence at the site of HIV-1 integration (20)(21)(22) nor the preference for gene-dense regions of chromosomes (12,20) seems to be influenced by LEDGF/p75.…”