Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5 alpha protein, a CCR5 shRNA, and a TAR decoy

Abstract

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

Figure 1

Combination anti-HIV lentiviral vector and detection of transgene expression. (a) A third-generation lentiviral vector, pCCLc-x-PGK-EGFP, which contains an EGFP reporter gene was used to generate the combination anti-HIV construct. (b) The three transgenes, a chimeric human/rhesus macaque TRIM5α driven by the MNDU3 promoter, a CCR5 shRNA driven by the human polymerase-III small RNA U6 promoter, and a TAR decoy driven by the U6 promoter were inserted upstream of the EGFP reporter gene to derive pCCLc-combination-PGK-EGFP. Ghost-R5-X4-R3 cells were left nontransduced or were transduced with the EGFP alone or combination lentiviral vectors. Total cellular RNA was extracted and analyzed by quantitative real-time PCR for expression of (c) TRIM5α, (d) the CCR5 shRNA, and (e) the TAR decoy using gene-specific primers. (f) U6 snRNA was used as an internal control. Experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; HIV, human immunodeficiency virus; shRNA, short hairpin RNA; TAR, transactivation response element.

Figure 2

Stability of the combination vector in transduced cells. Ghost-R5-X4-R3 cells were left nontransduced or were transduced with the EGFP alone or combination lentiviral vectors. Total genomic DNA was extracted and analyzed by PCR with primers specific for the respective vector transgenes. (a) MNDU3 (forward) and EGFP (reverse), (b) MNDU3 (forward) and TAR decoy (reverse), (c) MNDU3 (forward) and CCR5 shRNA (reverse), (d) TAR decoy (forward) and EGFP (reverse), and (e) albumin (forward and reverse). One kilobase DNA ladder (DLa), log DNA ladder (DLb), combination transfer vector plasmid (lane 1), nontransduced cells (lane 2), EGFP-alone vector transduced (lane 3), and combination vector transduced (lane 4). A schematic of the PCR products is below the panels. EGFP, enhanced green fluorescent protein; kb, kilobase; TAR, transactivation response element.

Figure 3

Downregulation of CCR5 surface expression and mRNA levels in transduced cells. Ghost-R5-X4-R3 cells were transduced with the EGFP alone and combination lentiviral vectors. (a) Seventy-two hours post-transduction, the cells were analyzed for CCR5 expression by fluorescence-activated cell sorting (FACS). (b) Cells were also analyzed by quantitative real-time PCR (QRT-PCR) for intracellular CCR5 mRNA levels. The FACS data are a representative of quadruple experiments. The QRT-PCR experiment was performed in triplicate. EGFP, enhanced green fluorescent protein.

Figure 4

HIV-1 challenge of combination vector–transduced cultured cells. Ghost-R5-X4-R3 cells were transduced with the EGFP alone (closed squares) or the combination (closed triangles) lentiviral vector. Nontransduced (closed diamonds) and vector-transduced cells were subsequently challenged with the R5-tropic BaL-1 strain of HIV-1 at a multiplicity of infection (MOI) of (a) 0.01 and (b) 0.05. Cells were also challenged with the X4-tropic NL4-3 strain of HIV-1 at an MOI of (c) 0.01 and (d) 0.05. On various days postinfection, cell culture supernatants were analyzed for HIV-1 p24 antigen by enzyme-linked immunosorbent assay. The challenge experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; HIV-1, human immunodeficiency virus type 1; PI, postinfection.

Figure 5

HIV-1 challenge of combination vector–transduced CD34⁺ HPC–derived macrophages. CD34⁺ hematopoietic progenitor cells (HPCs) were transduced with the EGFP alone (closed squares) or the combination (closed triangles) lentiviral vector and cultured in a macrophage differentiation medium. Upon development of mature macrophages, nontransduced (closed diamonds) and vector-transduced cells were challenged with the R5-tropic BaL-1 strain of HIV-1. Cell culture supernatants were sampled and analyzed for HIV-1 p24 antigen by enzyme-linked immunosorbent assay (a) [multiplicity of infection (MOI) 0.01] and (b) (MOI 0.05). Challenge supernatants were also analyzed for infectious virus by a Ghost cell assay (c) (MOI 0.01) and (d) (MOI 0.05). The challenge experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; HIV-1, human immunodeficiency virus type 1; PI, postinfection.

Figure 6

Generation of escape mutants after long-term viral challenge. Naive nontransduced (closed diamonds), EGFP alone (closed squares), and combination (closed triangles) vector–transduced ghost-R5-X4-R3 cells were challenged with the day 25 culture supernatants from their respective initial viral challenges. (a) BaL-1 multiplicity of infection (MOI) 0.01, (b) BaL-1 MOI 0.05, (c) NL4-3 MOI 0.01, and (d) NL4-3 MOI 0.05. Cell culture supernatants were analyzed for infectious virus by a Ghost cell assay. Experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; PI, postinfection.

Figure 7

Selective survival advantage and detection of HIV-1 proviral integration of challenged cells. Ghost-R5-X4-R3 cells were transduced with the EGFP alone or the combination lentiviral vector. Cells were mixed at a ratio of 1:1 and 2:1 (transduced:nontransduced) and were challenged with the (a) R5-tropic BaL-1 or (b) X4-tropic strain of HIV-1 at a multiplicity of infection (MOI) of 0.01. Cells were analyzed by fluorescence-activated cell sorting for EGFP expression preinfection and on day 21 postinfection. Detection of HIV-1 integration: ghost-R5-X4-R3 nontransduced, EGFP alone, and combination vector–transduced cells were challenged with the (c) R5-tropic BaL-1 and (d) X4-tropic NL4-3 strain of HIV-1 at MOIs of 0.01 and 0.05. On day 25 postinfection, genomic DNA from virus challenged cells was analyzed by quantitative real-time PCR for integrated HIV-1 provirus using primers specific for the pol gene. Experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; HIV-1, human immunodeficiency virus type 1.

Figure 8

Toxicity of combination vector transduction. (a) CD34⁺ hematopoietic progenitor cells were transduced with the EGFP alone or combination lentiviral vector, and differentiated into mature macrophages. Cells were stained with the macrophage cell surface markers CD14, CD4, and CD68, and analyzed by fluorescence-activated cell sorting (FACS). (b) Peripheral blood mononuclear cells (PBMCs) were transduced with the EGFP alone or combination vectors. On various post-transduction, the transduced cells were analyzed for EGFP expression by FACS or (c) total cell counts. (d) Monocytes and dendritic cells from PBMCs were left unmanipulated or incubated with PHA, LPS, an 11-amino-acid human peptide, or a 13-aa rhesus macaque peptide. Lymphocytes were added back and analyzed for immune cell activation. All experiments were performed in triplicate. EGFP, enhanced green fluorescent protein; LPS, lipopolysaccharide; PHA, phytohemagglutinin.