Specific transduction of HIV-susceptible cells for CCR5 knockdown and resistance to HIV infection: a novel method for targeted gene therapy and intracellular immunization

Abstract

HIV-1 gene therapy offers a promising alternative to small molecule antiretroviral treatments and current vaccination strategies by transferring, into HIV-1-susceptible cells, the genetic ability to resist infection. The need for novel and innovative strategies to prevent and treat HIV-1 infection is critical due to devastating effects of the virus in developing countries, high cost, toxicity, generation of escape mutants from antiretroviral therapies, and the failure of past and current vaccination efforts. As a first step toward achieving this goal, an HIV-1-susceptible cell-specific targeting vector was evaluated to selectively transfer, into CCR5-positive target cells, an anti-HIV CCR5 shRNA gene for subsequent knockdown of CCR5 expression and protection from HIV-1 infection. Using a ZZ domain/monoclonal antibody-conjugated Sindbis virus glycoprotein pseudotyped lentiviral vector, here we demonstrate the utility of this strategy for HIV-1 gene therapy by specifically targeting HIV-1-susceptible cells and engineering these cells to resist HIV-1 infection. CCR5-positive human cells were successfully and specifically targeted in vitro and in vivo for transduction by a lentiviral vector expressing a highly potent CCR5 shRNA which conferred resistance to HIV-1 infection. Here we report the initial evaluation of this targeting vector for HIV-1 gene therapy in a preexposure prophylactic setting.

FIGURE 1

CCR5 shRNA lentiviral vector: A third-generation lentiviral vector, CCLc-x-PGK-EGFP, containing an EGFP reporter gene was used to generate the CCR5 shRNA construct. The CCR5 shRNA was expressed under the control of the human polymerase-III U6 small RNA promoter and was inserted upstream of the EGFP reporter gene.

FIGURE 2

CCR5-positive cell-specific targeting of Ghost-R5-X4-R3 cultured cells: A mixed culture of Ghost-R5-X4-R3 and HEK-293T cells was transduced with the CCR5-targeting vectors, EGFP-ZZ and CCR5shRNA-ZZ. Seventy-two hours posttransduction, the cells were stained with a PE-conjugated anti-human CD4 antibody. Cells were analyzed by FACS for CD4 and EGFP expression.

FIGURE 3

CCR5-positive cell-specific targeting of human primary PBMCs: Freshly isolated PBMCs were transduced with the CCR5-targeting vectors. Seventy-two hours posttransduction, cells were stained with either an APC-conjugated antihuman CD3 (T cells), PE-conjugated antihuman CD14 monocyte/macrophages), or a PE-conjugated antihuman CD19 (B cells) antibody and analyzed by FACS.

FIGURE 4

Downregulation of CCR5 protein and mRNA levels in transduced Ghost-R5-X4-R3 cells and PBMCs: A mixed culture of Ghost-R5-X4-R3 and HEK-293T cells was transduced with the CCR5 targeting vectors, EGFP-ZZ and CCR5shRNA-ZZ. A, Seventy-two hours posttransduction, the cells were stained with a PE-conjugated antihuman CCR5 antibody and analyzed by FACS for CCR5 and EGFP expression. B, Ghost-R5-X4-R3 cells were also analyzed by QRT-PCR for intracellular CCR5 mRNA. C, Human PBMCs were transduced with the CCR5-targeting vectors, EGFP-ZZ and CCR5shRNA-ZZ, and analyzed by FACS for CCR5 and EGFP expression.

FIGURE 5

HIV-1 challenge of CCR5 shRNA vector–transduced Ghost-R5-X4-R3 cells and PBMCs: Mixed culture Ghost-R5-X4-R3/HEK-293T cells transduced with the EGFP-ZZ (◆) or CCR5shRNA-ZZ (■) vector were challenged with an R5-tropic BaL-1 strain of HIV-1, MOI 0.05, and analyzed for (A) p24 antigen by ELISA and (B) quantitated for infectious virus by a Ghost cell assay. PBMCs, non-transduced (◆), EGFP-ZZ (■), or CCR5shRNA-ZZ (▲) vector-transduced were challenged with HIV-1 BaL-1 at an MOI of 0.01. Cell culture supernatants were assayed for (C) p24 antigen by ELISA and (D) quantitated for infectious virus by a Ghost cell assay.

FIGURE 6

In vivo CCR5-positive cell-specific targeting: Adult NOD-SCID-IL2r-γ knockout mice were injected RO with fresh PBMCs after 2 weeks later by injection with the CCR5-targeting vector. Cells from various lymphoid organs were analyzed for cell transduction by staining for (A) T cells, (B) CCR5, (C) B cells, (D) macrophages and EGFP by FACS.