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Zinc-finger Functional HIV Cure - Zinc-finger endonuclease targeting PSIP-1 inhibits HIV-1 integration
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"Genome editing is an emerging strategy to study virus-host interactions and to combat and cure HIV-1 infection (7, 8). An ideal therapy for HIV, or other chronic viral infections that course with latent reservoirs, is believed to involve the generation of a source of long-lived, self-renewing and multilineage hematopoietic stem cells that would repopulate the host with genetically modified cells refractory to infection (45, 46). Since the unique and exceptional case of HIV-1 sterilizing cure of a patient due to bone marrow transplantation with a matched donor homozygote for the CCR5delta32 mutation (47, 48), alternative strategies have aimed to reproduce the CCR5delta32 phenotype using genome-editing tools. Indeed, ZFNs targeting the HIV coreceptors gene have been successfully developed to generate human CD4+ T cells and human embryonic cell precursors and induced pluripotent stem cells which were refractory HIV-1 infection in different mice models (9-13)."
"Here, we have evaluated the feasibility and efficacy of generating LEDGF/p75 knockout cell, a key factor for the integration of viral DNA into the host genome, using ZFN targeting at the C-terminal region of the LEDGF/p75 protein, outside the best described functional domains PWWP and IBD. LEDGF/p75 has already been validated as a candidate for gene therapy in a model where engraftment of lentiviral transducer CD4+ T cells overexpressing LEDGF325-530 induced a 3-log reduction in plasma viral load of HIV-1 infected mice (23). Overexpression of the deficient mutant LEDGF325-530 in primary CD4+ T cells impeded but did not completely block viral replication, due to minimal wild type LEDGF/p75 expression. Thus, the use of ZNFLEDGF might be advantageous in gene therapy settings as it confers a permanent disruption of the target gene avoiding the presence of residual levels of the wild-type LEDGF/p75 form that might be highjacked by the HIV-IN to successfully replicate."
"Taken together, these results suggest that a complete LEDGF/p75 protein, including the C-terminal domain, is necessary to successfully tether HIV preintegration complex into active transcriptional units. Consistent with previous reports (20) (19) (21), LEDGF-/-KO cells were able to support inefficient but detectable viral integration and produce new viral particles confirming the presence of alternative pathways for HIV-1 replication in the absence of LEDGF/p75."
"In summary, we describe the generation of LEDGF/p75 knockout cells using a ZFN that successfully recognises and disrupts the sequence of the PSIP1 gene coding for the C-terminal end of the LEDGF/p75 protein. The truncation of the C-terminal end of the LEDGF/p75 results in a reduced protein stability that lead to the generation of KO cells with an impaired HIV-1 replication independent of genetic modification concerning the N-terminal functional domains or the IBD of LEGDF protein. Further studies must be carried out to elucidate the functional role of genetic variants in the coding regions of the PSIP1 gene in vivo. Our results confirm previous data indicating that other pathways rather than LEDGF/p75 might allow HIV integration. Finally, the ZFNLEDGF provides a new cellular model to study host factors involved in the HIV-1 integration process."
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Zinc-finger endonuclease targeting PSIP-1 inhibits HIV-1 integration
Roger Badia, Eduardo Pauls, Eva Riveira-Munoz, Bonaventura Clotet, Jose A. Este*
4 and Ester Ballana
1. IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autonoma de Barcelona, 08916 Badalona, Spain.
2.
ABSTRACT
Genome editing using zinc-finger nucleases (ZFN) has been successfully applied to disrupt CCR5 or CXCR4 host factors, inhibiting viral entry and infection. Gene therapy using ZFN to modify PSIP1 gene, encoding for LEDGF protein, might restrain an [underln]early[/underln] step of viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFNLEDGF) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of LEDGF/p75 protein. ZFNLEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frame shift of the coding region and resulted in the abolishment of LEDGF expression at mRNA and protein level. Functional assays revealed that infection with HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knock-out cells regardless of entry tropism, due to a blockade in HIV-1 provirus integration into host genome. However, residual infection was detected in LEDGF knock-out cells. Indeed, LEDGF knock-out restriction was overcome at high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than LEDGF/p75. Observed residual integration was, however, sensitive to the integrase inhibitor Raltegravir. These results demonstrate that the described ZFNLEDGF effectively targets PSIP1 gene which is involved at early steps of viral replication cycle and thus ZFNLEDGF may become a potential antiviral agent to restrict HIV-1 integration. Moreover, LEDGF knock-out cells represent a potent tool to elucidate the role of HIV integration cofactors in virus replication.
INTRODUCTION
Human immunodeficiency virus (HIV) requires the host cellular machinery to successfully replicate (1). Developing of genome editing tools, such as zinc finger endonucleases (ZFNs) (2), transcription activator-like [TAL] effector nuclease (TALEN) (3) or clustered regulatory interspaced short palindromic repeat (CRISPR) (4-6) become a promising alternative to modify essential host factors along the replication cycle of HIV (7, 8). ZFNs have demonstrated their applicability to reproduce in vitro the CCR5∼32 phenotype by successfully cleaving the CCR5 gene, generating human CD4+ T cells refractory to HIV-1 infection (9-12). Similarly, ZFNs approach successfully cleaved the alternative HIV-1 coreceptor CXCR4 in CD4+ T cells from humanized mice model resulting in impaired HIV-1 infection (13). Genome editing as anti-HIV therapy is currently under study in at least 2-3 clinical trials using ZFNs targeting CCR5. However, similar strategies targeting host cellular factors affecting later steps of the virus replication cycle have not been evaluated. A crucial step of the viral replication cycle is exerted by the lens epithelium derived growth factor (LEDGF/p75), a member of the hepatoma-derived growth factor (HDGF) related protein (HRP) family. HRPs are characterized by a conserved N terminal PWWP domain, an ,90- to 135-amino acid module found in a variety of nuclear proteins (14). Six human HRP family members have been described: HDGF, HRP1, HRP2, HRP3, LEDGF/p75, and LEDGF/p52. Two of them, LEDGF/p75 and HRP2, possess affinity for HIV-1 integrase (IN), given by a second evolutionary conserved domain within their C-termini that mediates the interaction with HIV-1 IN, hence the term ''IN-binding domain (IBD)''(15). Initially identified as IN associated protein (16), LEDGF/p75 was revealed as a lentivirus-specific cellular cofactor required for HIV integration into the host genome (see references (17, 18) and (19) for review). LEDGF/p75 directly interacts with viral HIV-IN, tethering viral reintegration complex into active transcription units of the cellular chromatin. The role of LEDGF/p75 in HIV-1 replication was studied using RNA interference (RNAi) targeting LEDGF/p75 and LEDGF KO murine embryonic fibroblasts (MEF). Although both strategies potently downregulate or completely abolished LEDGF/p75 expression, residual replication was observed. Thus, all studies point to a key but not essential role for LEDGF/p75 in lentiviral replication and suggested that the existence of alternative cellular cofactors, such as HRP2, were responsible for the residual replication observed in the absence of LEDGF/p75 (20, 21).
Nevertheless, the LEDGF/p75 interaction with HIV-IN has been suggested as valid target for antiviral therapy (22-24). In that sense, recently developed allosteric LEDGF/p75-IN interaction inhibitors (LEDGINs and ALLINIS) have been proved to target the LEDGF/p75 binding pocket of HIV-IN and to inhibit the catalytic activity of the IN. Moreover, LEDGINs and ALLINIS also exert antiviral activity by promoting IN multimerisation. Aberrant IN complexes lead to the formation of defective regular cores during the maturation process, resulting in an impaired the infectivity of the new viral particles (25-27). On the other hand, a series of PSIP1 single nucleotide polymorphisms (SNP) were associated to HIV-1 disease progression in cohorts of African and Caucasian HIV-1 positive individuals (28) (29). In addition, two missense mutations were identified in two samples belonging to a LTNP cohort (30). All missense mutations identified are located in the helix-turn-helix (HTH) motifs at the C-terminal region of the protein, after the IBD domain. Although none of the mutations restricted HIV replication in vitro (29, 31, 32), these findings suggested that genetic variation in PSIP1 may influence susceptibility to HIV-1 infection and disease progression. Here, we describe a novel genome editing ZFN that specifically disrupt the PSIP1 gene encoding LEDGF/p75 in its C-terminus, after all relevant functional domains and nearby the missesense mutations described in patients (ZFNLEDGF). ZFNLEDGF was able to generate LEDGF/p75 cells expressing a truncated protein that become refractory to HIV-1 integration. Generated LEDGF/p75 knock-out cells represent a potent tool to further investigate the function of LEDGF/p75 protein and may help to elucidate the role of HIV integration cofactors in virus replication. Moreover, the ZFNLEDGF may become a potential antiviral strategy to restrict HIV-1 integration and virus replication in vivo.
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