SIVMAC Vpx Improves the Transduction of Dendritic Cells with Nonintegrative HIV-1-Derived Vectors
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Gene Therapy (2009) 16, 159–163 & 2009 Macmillan Publishers Limited All rights reserved 0969-7128/09 $32.00 www.nature.com/gt SHORT COMMUNICATION SIVMAC Vpx improves the transduction of dendritic cells with nonintegrative HIV-1-derived vectors G Berger1,2,3, C Goujon1,2,3, J-L Darlix1,2,3 and A Cimarelli1,2,3 1LaboRetro, Department of Human Virology, Ecole Normale Supe´rieure de Lyon, Lyon, France; 2INSERM, U758, Lyon, France and 3University of Lyon, Lyon1, IFR128 BioSciences Lyon-Gerland, Lyon-Biopole, Lyon, France Lentiviral vector (LV)-mediated gene therapy bears an removes a specific restriction to lentiviral infection in these intrinsic risk of insertional mutagenesis following integration cells. Here, we describe that the transduction efficiency of into the host genome. Nonintegrative LVs may offer an DCs with nonintegrative HIV-1 vectors can also be improved alternative avenue at least in nondividing cells where via Vpx-VLPs that promote the accumulation of complete episomal viral DNA persists stably. Owing to their central and episomal viral DNA. In this setting, Vpx increases both role in immune system functions, differentiated dendritic cells the number of transduced cells and the levels of transgene (DCs) offer an interesting cell target for these vectors. We expression. Thus, these results describe a simple procedure have previously described that the transduction of DCs with by which transduction of differentiated DCs can be achieved wild-type HIV-1-derived vectors can be considerably im- at low viral inputs with safer LVs to improve both the number proved by providing DCs with noninfectious virion-like of transduced cells and the levels of transgene expression. particles (VLPs) carrying Vpx (Vpx-VLPs), a nonstructural Gene Therapy (2009) 16, 159–163; doi:10.1038/gt.2008.128; protein coded by members of the SIVSM/HIV-2 lineage that published online 31 July 2008 Keywords: nonintegrative; HIV-1 lentiviral vectors; DCs; Vpx One of the major interests of lentiviral vectors (LVs) in susceptible to lentiviral infection.11,12 We have previously gene therapy is their ability to provide long-lasting reported that members of the SIVSM/HIV-2 lineage code transgene expression following integration into the host for a protein, Vpx, are capable of relieving the restriction genome. This very property may lead to severe con- that is responsible for the poor susceptibility of DCs to sequences on the cell’s physiology owing to viral integra- lentiviral-mediated modification.13,14 Indeed, Vpx aug- tion into genes that deregulate the normal cellular ments the efficacy of infection of DCs with integration- transcriptional program.1 Given the propensity of lentivir- competent HIV-1 vectors when it is added in trans onto al integrases (IN) for active transcriptional units,2 DCs via noninfectious virion-like particles (VLPs) de- this represents a non-negligible issue. To circumvent rived from SIVMAC (Vpx-VLPs). In this setting, Vpx this problem, several groups have evaluated the use of promotes the accumulation of viral DNA. Thus, we integration-incompetent LVs.3–8 Gene expression from studied whether similar effects could be observed these vectors is driven from viral DNA molecules that following modification of DCs with nonintegrative remain in an episomal form after their entry into the HIV-1-based LV (as depicted in Figure 1a). nucleus as 1 or 2 long terminal repeat circles (1- or 2-LTRs, Viral integrases (IN) exert several functions during the respectively). These episomes are considered as a dead- viral life cycle, ranging from viral particle formation to end product in the normal retroviral infection process, but reverse transcription, and possibly nuclear import. Thus, they are a functional substrate for the transcriptional IN mutants have been divided into two classes according machinery.9 As they lack an origin of replication, these to the specific or pleiotropic defects they impose on forms are transient in dividing cells, but remain stably mutant viruses (class I or II, respectively18). Mutants in present as episomes in the nucleus of nondividing cells. the catalytic site of HIV-1 IN belong to the former. We Dendritic cells (DCs) are an interesting cell type for first constructed an integration defective HIV-1 LV by gene therapy purposes in light of their master role in the introducing a single point mutation in the catalytic site of regulation of immune system responses.10 Although viral the IN (IN-D116A). This mutation did not impair viral infection of DCs can be obtained with lentiviral-derived production and yielded similar infectious titers as vectors, the efficacy of this modification is generally low compared to wild-type (WT) on HeLa cells, if transduced unless high viral inputs are used, because DCs are poorly cells were examined 3 days after infection. However, a sharp loss of green fluorescent protein (GFP)-expressing cells was specifically observed in cells transduced with Correspondence: Dr A Cimarelli, LaboRetro, Department of Human the IN-D116A mutant when cells were analyzed 11 days Virology, ENS-Lyon INSERM, U758, 46 Alle´e d’Italie, 69364 Lyon, after infection (Figure 1b). This result is not surprising France. E-mail: [email protected] for dividing cells transduced with a nonintegrative LV, as Received 6 May 2008; revised 23 June 2008; accepted 7 July 2008; episomal DNA is expected to dilute over cell divisions. published online 31 July 2008 Indeed, when the amount of integrated viral DNA was Vpx in the transduction of DCs by nonintegrative HIV-1 LVs G Berger et al 160 Figure 1 Schematic representation of the lentiviral vectors (LVs) used in the study and characterization of a nonintegrative LV. (a) Integration-deficient LVs were engineered by introducing a single point mutation in the catalytic site of integrase (IN-D116A) in the context of an HIV-1-based packaging construct (8.2).15 LVs were obtained after transfection of 293 T cells with the packaging construct coding gag-pro-pol and all the accessory proteins (Tat, Rev, Vif, Vpr and Nef; not marked for clarity), a miniviral genome bearing a CMV-GFP expression cassette and a construct coding the vesicular stomatitis virus G envelope protein, as described.15 Noninfectious virion-like particles (referred to as Vpx-VLPs) were similarly produced by transfection of an SIVMAC-based construct coding gag-pro-pol and viral accessory proteins (SIV3+, Tat, Rev, Vif, Vpx, Vpr and Nef16). No viral genome was transfected in this case. We have previously documented that Vpx is the protein responsible for the positive effect of VLPs on the infectivity of incoming HIV-1 LVs.13 Virions were purified by ultracentrifugation on a double step sucrose cushion (45–25%) and normalized by protein content against standards of known infectivity, or by their infectious titers on HeLa cells, as previously described.17 Infections were carried out on 105 cells for 2 h prior to extensive cell washing. The percentage of transduced GFP-positive cells was determined 3 days after infection by flow cytometry. When indicated, Vpx- VLPs were provided together with GFP-coding vectors at a multiplicity of infection (MOI) of 2. (b) HeLa cells were transduced with normalized amounts of wild-type (WT) and IN-D116A LVs and analyzed by flow cytometry 3 and 11 days after infection. Control infections were performed in the presence of the reverse-transcriptase inhibitor azidothymidine (AZT) at 10 mg/ml. (c) The presence of integrated provirus was analyzed on transduced cells infected at MOI 10 by Alu-PCR, as described previously.9 Briefly, the first round of PCR was carried out for 20 cycles with a primer specific for the proviral DNA and a second round specific for Alu sequences in the genome. Fivefold dilutions of the first PCR were then used as the template for a second semiquantitative PCR using two internal primers specific for viral DNA. Amplified products were transferred onto a nylon membrane and hybridized with 32P-labeled specific probes prior to Phosphorimager quantification. A representative gel is shown here together with a graph presenting data obtained from three independent experiments. CMV, cPPT, central polypurine tract; GFP, green fluorescent protein; p.i., post-infection; RRE, Rev-responsive element; *, self-inactivating. determined by Alu-PCR, none was observed in cells effect was observed on stimulated PBLs. Of note, transduced with the IN-D116A mutant (Figure 1c). transduction with the IN-D116A mutant virus yielded a Next, the ability of normalized WT and IN-D116A substantially lower percentage of transduced cells than mutant LVs was analyzed on primary DCs, macrophages WT, even if analysis was carried out 3 days post- and phytohemagglutinin-stimulated primary blood lym- infection, that is, prior to substantial dilution of episomal phocytes (PBLs) (Figure 2a). As we had previously DNA following cell division. We believe that this result reported, Vpx-VLPs increased by over 10-fold the can be due to lower transcriptional activity from the percentage of GFP-positive DCs following infection with episomal DNA in PBLs as opposed to the integrated WT HIV-1 vectors.13 When compared to WT, similar forms. In addition to their positive effect on the amounts of GFP-expressing cells were also obtained percentage of HIV-1-LVs transduced cells, Vpx-VLPs following transduction of DCs with the IN-D116A also increased the median fluorescence intensity of GFP- mutant. Similar to WT, the addition of Vpx-VLPs during expressing cells (MFI; Figure 2b), suggesting that in cells IN-D116A LV infection dramatically increased the per- in which they exert a positive effect on viral infection, centage of transduced cells at all viral inputs tested (by Vpx-VLPs also act positively on the levels of transgene 10- to 20-fold). Accordingly, the percentage of macro- expressed intracellularly. Modified DCs could be kept in phages transduced with the IN-D116A mutant in the culture for over a month, retaining stable expression of presence of Vpx-VLPs increased, although to a lower the transgene (Figure 2c).