Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates

Abstract

RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34+ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.

Fig. 1.

Identification of a potent shRNA against huCCR5. (A) CEM-NKR-CCR5 cells were transduced with lentiviral vectors expressing random shRNAs against huCCR5 in 96-well plates, cultured for 3 days, and analyzed by flow cytometry for CCR5 expression in EGFP-expressing populations. Among the 400 shRNAs screened, CCR5 shRNA(1005) reduced CCR5 more efficiently than our previously published CCR5-shRNA (13). The CCR5-shRNA (13) was selected based on the initial criteria by Elbashir et al. (19). (B) Efficient reduction of endogenous CCR5 expression in human primary lymphocytes (huPBLs). PHA/IL-2-activated huPBL were transduced with lentiviral vectors bearing shRNA(1005) and analyzed 8 days after infection by monoclonal antibody staining and flow cytometry for CCR5 expression in the EGFP+ population. Mock, no vector transduction; no shRNA, vector transduction without shRNA expression; N/A, not applicable. The percentage of CCR5-expressing cells within the EGFP+ population (% CCR5 in EGFP+) was calculated and is indicated on the top of each image. The percentage number in each quadrant is also indicated.

Fig. 2.

Reduction of rhCCR5 by an shRNA(1005). An shRNA against rhCCR5(1005) was tested in rhCCR5-expressing 293T cells. Cells were transduced with an SIV-based lentiviral vector bearing shRNA(1005) against rhCCR5 and analyzed for CCR5 and EGFP expression by monoclonal antibody staining and flow cytometry at 4 days after transduction. The rhCCR5 shRNA reduced rhCCR5 expression in rhCCR5–293T cells but did not reduce huCCR5 in huCCR5-expressing CCR5NKRCEM cells because of a single-nucleotide mismatch in target sequence. Similarly, huCCR5 shRNA reduced huCCR5 but not rhCCR5. (B) Reduction of CCR5 in primary rhesus macaque lymphocytes. Before a study of lentiviral vector transduction and transplant of CD34+ cells, PBLs from two rhesus macaques (animal identifications, RQ3570 and RQ5427) were isolated, PHA/IL-2-activated, and transduced with an SIV vector expressing shRNA against rhCCR5(1005) in vitro. CCR5 expression was analyzed by flow cytometry in EGFP+ cells. A vector expressing an shRNA against firefly luciferase was used as a control. Mock, no vector transduction; N/A, not applicable. The percentage of CCR5-expressing cells within EGFP+ population (%CCR5 in EGFP+) was calculated and is indicated on the top of each image. The percentage number in each quadrant is also indicated.

Fig. 3.

In vivo vector marking, CCR5 down-regulation, and siRNA expression. (A) Stable EGFP marking in PB cells in transplanted animals. After CD34+ cell transplant, the percentage of EGFP expression was monitored in granulocyte (■), monocyte (◇), and lymphocyte (▲) populations by flow cytometric analysis. (B) Stable CCR5 reduction in EGFP+ lymphocytes. The percentage of CCR5 expression in EGFP+ (black bar) and in EGFP− (gray bar) cells was monitored by flow cytometry. Control animal 2RC003 was previously transplanted with a lentiviral vector bearing EGFP (20) but no shRNA expression unit. (C) A representative CCR5/EGFP plot at 5 months after transplant. PB from transplanted macaques was stained for CCR5 and CCR5 and EGFP expression in lymphocyte population and analyzed by flow cytometry. Based on the percentage of events in each quadrant (shown in each quadrant), the percentage of CCR5 expression in EGFP+ and EGFP− lymphocyte populations was calculated and is shown on the top of each image. (D) Detection of siRNA in rhesus macaque lymphocytes. The 22-nt antisense-strand siRNA was detected by micro-Northern blot analysis in the small RNA fraction of PHA/IL-2-stimulated lymphocytes from an shRNA-transduced animal (animal identification, RQ3570) but not in cells from control animal (animal identification, 2RC003).

Fig. 4.

Inhibition of SIV replication ex vivo. PBLs from RQ3570 at 13 months after transplant were sorted for EGFP+ and EGFP− population, stimulated with PHA/IL-2 for 2 days and IL-2 for 2 days, and infected with SIVmac239. (A) Mean fluorescent intensity (MFI) of CCR5 expression in EGFP+ sorted (black bar) and EGFP− sorted (gray bar) PHA/IL-2 activated lymphocyte. (B) The percentage of CCR5 expression in EGFP+ and EGFP− sorted lymphocytes. CCR5 expression was monitored during ex vivo culture by flow cytometric analysis and compared between EGFP+ (black bar) and EGFP− sorted (gray bar) lymphocyte populations. (C) SIV p27 production in EGFP+ and EGFP− sorted lymphocytes. After stimulation, 1 × 10⁵ EGFP+ (△) or EGFP− (■) cells were infected with 100 μl of SIVmac239 at a multiplicity of infection of 0.04 (the infectious unit of the virus stock was 4 × 10⁴ ml by titrating on MAGI-CCR5 cells) for 1 h and monitored for p27 production for 11 days in culture supernatant. The infection experiment was done in triplicate. The average p27 production in culture supernatant and error bar (standard deviation) are shown.