HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1.
BACKGROUND
Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1.


METHODS
HIV-1-based lentiviral vector stocks were prepared in 293T cells by the established calcium phosphate transfection method. Human monocytes were isolated from donors' blood by Ficoll-Paque separation and cultured in vitro. To establish an effective technique for vector-mediated gene transfer, primary cultures of human MDM were transduced at varying multiplicities of infection (MOI) and at a range of time points following initial isolation of cells (time-in-culture). Transduced cells were then examined for transgene (green fluorescent protein (GFP)) expression by fluorescent microscopy and reverse transcription polymerase chain reaction (RT-PCR). These cultures were then exposed to wild-type HIV-1, and viral replication was quantitated by p24 assay; production of neurotoxic effector molecules by the transduced MDM was also examined, using indicator neurons.


RESULTS
We have demonstrated that primary human MDM could be efficiently transduced (>50%) with concentrated HIV-1-based defective lentiviral vectors (DLV). Furthermore, DLV-mediated gene transduction was stable, and the transduced cells exhibited no apparent difference from normal MDM in terms of their morphology, viability and neurotoxin secretion. Challenge of DLV-transduced MDM cultures with HIV-1(Ba-L) revealed a 4- to 5-fold reduction in viral replication, as measured by p24 antigen production. This effect was associated with the mobilization of the GFP-expressing DLV construct by the wild-type virus.


CONCLUSIONS
These data demonstrate the inhibition of HIV-1 replication in primary MDM, by a DLV vector that lacks any anti-HIV-1 transgene. These findings lay the initial groundwork for future studies on the ability of DLV-modified monocytes to introduce anti-HIV-1 genes into the CNS. Lentiviral vector-mediated gene delivery to the CNS by monocytes/macrophages is a promising, emerging strategy for treating neuro-AIDS.
Zeng
Lingbing
L
Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
Planelles
Vicente
V
Sui
Ziye
Z
Gartner
Suzanne
S
Maggirwar
Sanjay B
SB
Dewhurst
Stephen
S
Ye
Linbai
L
Nerurkar
Vivek R
VR
Yanagihara
Richard
R
Lu
Yuanan
Y
D002478:Cells, Cultured; D003673:Defective Viruses / Q000235:genetics* / Q000378:metabolism; D005240:Feasibility Studies; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000821:virology; D008957:Models, Genetic; D014161:Transduction, Genetic / Q000379:methods*; D014779:Virus Replication
D049452:Green Fluorescent Proteins