Title: Employing the HIV Integrase cellular co-factor, LEDGF/p75, for gene therapy
Other Titles: Het gebruik van de HIV Integrase cellulaire co-factor, LEDGF/p75, voor gentherapie
Authors: Vets, Sofie; S0106307
Issue Date: 19-Dec-2012
Abstract: This work employs the current insights and knowledge on lens epithelium-derived growth factor (LEDGF/p75), a pivotal cellular binding partner of HIV integrase (IN) essential for efficient HIV integration and replication. On the one hand we evaluated LEDGF/p75 as a target for HIV gene therapy, to protect primary cells from HIV infection. On the other hand, we generated artificial fusion proteins where we replaced the natural DNA-binding domain of LEDGF/p75 with alternative DNA binding domains to retarget viral vector integration in the genome of the host cell, providingproof-of-principle that integration can be targeted to specific regionswithin the genome and opening possibilities for the development of safer viral vectors for gene therapy. LEDGF/p75 was identified and characterized in our group as a cellular binding partner of HIV IN. The protein contains several chromatin binding motifs in its N-terminal domain and an integrase binding domain (IBD, aa347-429) in its C-terminal domain . LEDGF/p75 is hijacked by the viral integrase and orchestrates chromosomal tethering and integration of the provirus into thehost cell chromatin . Proviral integration is an attractive target for therapy because of its central role in the HIV replication cycle. Integration forever links the fate of the integrating virus with that of thetarget cell. Although RNAi and overexpression of truncation mutants in laboratory cell lines were employed to validate the pivotal role of LEDGF/p75 in HIV replication , the impact of LEDGF/p75 KD and/or LEDGF325-530 overexpression on HIV replication has not been studied in primary cells. LEDGF/p75 and HIV After more than thirty years of research exploiting different approaches to tackle HIV infection, still no cure exists. In 2011, more than 34 million people world-wide were living with HIV, 2.5 million people got newly infected and 1.7 million died because of the infection (UNAIDS). Where HAART(highly active antiretroviral therapy) is available, the diagnosis of AIDS becomes less frequent; HIV-1 infection is no longer considered as a lethal disease but rather a chronic, manageable infection. However, recent studies show that HAART does not restore life-expectancy completely . Furthermore, as those living with HIV-1 grow older, age-related toxicities emerge as well as other ART co-morbidities such as increased risk of cardiovascular disease, metabolic disorders, neurocognitive abnormalities, liver and renal disease, bone disorders, malignancy and frailty . Since HAART does not cure HIV, further research is needed. Next to drug design, also vaccine development and gene therapy are explored as possible ways to control the current pandemic. In a first section of this work, we investigated the potential of LEDGF/p75 asa target for HIV gene therapy. We evaluated the effect on HIV replication of LEDGF/p75 KD, LEDGF325-530 overexpression and the combination of both in primary CD4+ T-cells. Overexpression of LEDGF325-530 resulted in the most potent inhibition of HIV-1 replication in vitro . Hence, this approach was evaluated in vivo in a NSG mouse model for HIV infection. Overexpression of LEDGF325-530 rendered primary T-cells more resistant to HIV infection compared to cells expressing an interaction-deficient D366N control, as illustrated by up to 30% engraftment and a 3-fold reduction of the p24 antigen in the circulating blood. In our setting not all transplanted cells were transgenic (70% transduced cells) and hence protection from HIV infection was not complete. Still, our results validate LEDGF/p75 as a novel antiviral target in HIV gene therapy thereby preventing the virus to become a stable, heritable element of the infected cell. LEDGF/p75 and gene therapy Being the tethering factor of HIV integration, LEDGF/p75 also plays a pivotal role in gene therapy, where it determines the integration site distribution of lentiviral vectors that are frequently employed to correct genetic disorders. Trials for treatment of several blood cell diseases demonstrated that long-term gene correction is feasible and in some patients gene therapy may provide equal clinical benefit with less risk than standard treatments. The property of retroviral vectors to integrate the transgene into the genome of the cell is both an advantage and a hurdle for gene therapy. With the therapeutic benefits of continuous transgene expression has also come the recognition of real clinical risks, such as leukemogenesis in gene therapy trials using γ -retroviral vectors . Undesirable consequences of non-specific integration could be avoided by finding a way to redirect lentiviral vector integration. Initial attempts include fusion proteins containing a DNA binding domain coupled to HIV IN . Although some of these fusions provided proof-of-concept, the fusion with the DNA binding domain severely affected the IN function and functionality was solely shown in vitro . Even if the IN fusions had in vivo activity, vector titers remained low and theefficiency of retargeting difficult to assess . In a second part of this dissertation, we engineered LEDGF/p75 to retarget lentiviral integration in the genome. An alternative chromatin bindingdomain, the heterochromatin protein 1β (CBX1 or HP1 β ) was fused to the C-terminal portion of LEDGF/p75 (aa 325-530, LEDGF325-530). CBX1 recognizes and binds di- and tri-methyled H3K9 on the chromatin. These marks associate with pericentric and heterochromatin, which is generally disfavored for lentiviral integration. Fusing a new chromatin binding module to LEDGF325-530 changed the behavior of the latter from a potent integration-inhibitor into an efficient cofactor. We characterized proviral integration sites using 454 pyrosequencing. Analysis of the LV integration distribution demonstrated that the CBX1-LEDGF325-530 fusion retargeted lentiviral integration away from RefSeq transcription units towards regions high in CBX1 binding sites. LEDGF fusions were stably overexpressed in LEDGF KD cells, minimizing the competition with the endogenous LEDGF/p75, and demonstrating proof-of-principle that artificial LEDGF-fusions can be employed to retarget viral vector integration in the human genome. As the cellular role of LEDGF/p75 remains largely unknown, stable KD of the protein might interfere with its function and alsooverexpression of the IN-binding C-terminal end of LEDGF/p75, might affect downstream pathways because of its binding to several other cellularproteins. Furthermore, two extra retroviral vectors were needed to create stable LEDGF/p75 KD and CBX1-LEDGF325-530 overexpression augmenting the risk for insertional mutagenesis. In a next step towards safer gene therapy we demonstrated retargeting of lentiviral vectors following transient overexpression of CBX1-LEDGF325-530 using mRNA electroporation in wild-type cells, expressing endogenous LEDGF/p75. In addition, we proved in a cell culture model for X-CGD that functional rescue can be achievedafter retargeting of the therapeutic vector using this technology. Altogether, this work demonstrates that retargeting of lentiviral vector integration is feasible upon transient expression of a LEDGF-chimerain WT cells and that gene expression after retargeting can rescue a disease phenotype. These results are of great importance for gene therapy with integrating vectors where insertional mutagenesis remains a major concern.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Virology and Gene Therapy
Faculty of Medicine, Campus Kulak Kortrijk
Biochemistry, Kulak (-)

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