Thus, we tested whether we could produce LVs containing a mutation in one of the packaging vectors that disabled the integrase protein in the lentivirus particle (and thus prevented integration of the provirus reverse-transcribed DNA) [14] and [15]. We demonstrated that ID-LVs could be produced at high titers and were effective at transducing human monocytes. Upon
terminal differentiation of the iDCs, expression of the transgenes (cytokines and antigen) persisted for 3 weeks. The constitutive and robust expression of cytokines (GM-CSF/IFN-α for SmyleDC and GM-CSF/IL-4 for SmartDC) enabled generation of stable and functional iDCs that could self-differentiate in vitro or in vivo. Since we noticed a modest (10–15%) gene marking of monocytes transduced with ID-LV-GFP, it is possible that ID-LVs expressing the cytokines needed for iDC differentiation and maintenance provide a selective this website mTOR inhibitor advantage for the transduced monocytes for about 3 weeks. A previous report demonstrated
that differentiated human APCs (DCs and macrophages maintained in culture for 4 and 8 days, respectively) could be transduced with ID-LVs [20], but the current work is the first demonstration that monocytes can also be effectively transduced with ID-LVs prior to their differentiation, which then maintain the DCs functional and alive. The capability of ID-LVs to infect monocytes seems to reflect what occurs during natural HIV-1 infection, as monocytes are one of the relevant HIV-1 reservoirs [32]. During initial infection (i.e., in non-activated
monocytes and CD4+ T cells), most of the viral cDNA exists as unintegrated linear DNA form (for which fewer copies eventually integrate), or nuclear circular forms, which can lead to “abortive” defective integrations or are ultimately degraded (for a review, see [33]). Nevertheless, prior to HIV-1 integration, transcription of and translation of viral genes present in the unintegrated DNA forms is observed which initiate a rapid sequence of events to shut-down the antigen-presentation machinery (such as down-regulation of MHC class I expression via Nef [34]). Ironically to the natural biology of HIV-1 hindering DC differentiation, HIV-1-derived ID-LVs co-expressing combinations of cytokines were actually able to potently induce DC differentiation. Other groups had previously reported the transduction of monocytes by LVs, but since these studies lacked of the 8 h GM-CSF/IL-4 pre-conditioning step to activate the monocytes prior to LV transduction [35] and [36], the gene transfer efficiency was usually low. Co-expression of GM-CSF/IFN-α or GM-CSF/IL-4 was readily observed in the monocytes preconditioned with cytokines and transduced with ID-LVs which induced their prompt differentiation into highly stable and immunologically competent APCs.
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