Zinc Finger Endonuclease Targeting PSIP1 Inhibits HIV-1 Integration

Abstract

Genome editing using zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and infection. Gene therapy using ZFNs to modify the PSIP1 gene, which encodes the lens epithelium-derived growth factor (LEDGF) protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFN LEDGF ) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of the LEDGF/p75 protein. ZFN LEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of LEDGF expression at the mRNA and protein levels. Functional assays revealed that infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual infection was detected in the LEDGF knockout cells. Indeed, LEDGF knockout restriction was overcome at a high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through LEDGF/p75. However, the observed residual integration was sensitive to the integrase inhibitor raltegravir. These results demonstrate that the described ZFN LEDGF effectively targets the PSIP1 gene, which is involved in the early steps of the viral replication cycle; thus, ZFN LEDGF may become a potential antiviral agent for restricting HIV-1 integration. Moreover, LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.

Cyclin D3-dependent control of the dNTP pool and HIV-1 replication in human macrophages

“…For quantification of proviral DNA, a primer and probe set that is able to amplify both unintegrated and integrated viral DNA was used as described before. 40 Infections were stopped at 16 h to measure only early events of viral infection (reverse transcription). DNA was extracted using a DNA extraction kit (Qiagen) and proviral DNA quantifications were performed.…”

Potential mechanisms for cell-based gene therapy to treat HIV/AIDS

“…The DNA will be repaired, thereby introducing deletions or insertions at the site of cleavage through the error-prone non-homologous end joining pathway [69]. Most studies have focused on the HIV-1 co-receptor CCR5 and CXCR4 [9, [70][71][72], but the integrated HIV-1 DNA genome can also be targeted [73,74]. CCR5-and CXCR4-specific ZFNs have been studied in in vitro and in vivo models and showed resistance to HIV-1 infection [71,72].…”

“…Trans-dominant protein [37][38][39][40] Repeat sequence LTR ZNF [73] Crisp-Cas9 [84] PSIP-1 gene Zinc finger nuclease [74] TALEN [81] Transcription Tat Trans-dominant protein [31,169] Intrabody [45,47] TAR decoy [93,94,96] Ribozyme tat/vpr [15,102,103] TAR antisense [108][109][110] siRNA [170] shRNA [130] Translation Rev Trans-dominant protein [29,30] Rev-responsive element decoy [95,99] Ribozyme rev/tat [104] siRNA [170] …”

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

“…Wild-type iPSCs were seamlessly modified by NHEJ-mediated deletion of the CCR5 gene, at an average rate of 14% with TALENs and 33% with CRISPR [98]. To increase resistance to HIV infection, other genes were targeted in addition to CCR5 , including C-X-C chemokine receptor type 4 ( CXCR4 ), which encodes a coreceptor, and PC4 and SFRS1 interacting protein 1 ( PSIP1 ), which encodes the lens epithelium-derived growth factor (LEDGF)/p75 protein required for HIV integration [99,116,117]. …”