Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5

Abstract

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

FIG. 1.

Effects of the method of T-cell activation on Ad5/F35 vector transduction and expression efficiency. Primary CD4 T cells were activated overnight with 5 μg/ml phytohaemagglutinin (PHA) or anti-CD3/28 antibody-conjugated beads at a 3:1 bead:cell ratio and then infected at a multiplicity of infection (MOI) of 600 with Ad5/F35 vector. (A) Ad5/F35 vector-expressing green fluorescent protein (GFP) was used to measure transduction efficiency by fluorescence activity at Days 5 to 11 post-stimulation. The percent of cells expressing GFP are shown, as well as the mean fluorescence intensity (MFI), which reflects the level of GFP expression in each cell. (B) Ad5/F35 vector expressing the CCR5-specific ZFN, SB-728, was used to measure nuclease activity after transduction. Boxes above the gel show the method of T-cell stimulation, ZFN treatment, and the day of culture. Nuclease activity was measured by using the surveyor nuclease assay on CCR5. Cleavage fragments are indicated by the arrows to the right of the gel. Percent disruption was determined by image analysis as described in the methods and is indicated at the bottom of the gel.

FIG. 2.

Human serum inhibition of Ad5/F35 transduction in T cells. Primary CD4 T cells were activated overnight with anti-CD3/28 antibody-conjugated beads at a 3:1 bead:cell ratio and then transduced at an MOI of 30, 100, or 300 with Ad5/F35 vector-expressing GFP in the presence of 5% pooled human serum or in the absence of human serum. GFP was measured on Day 3 after initial activation. The percent of cells expressing GFP and the MFI of cells gated as positive are shown.

FIG. 3.

Ad5/F35 CCR5 ZFN vector titration and T-cell expansion. To evaluate the best balance of MOI for efficient transduction and preservation of robust cell expansion, CD4 T cells were activated overnight with CD3/28 beads as previously described and then transduced with Ad5/F35 vector-encoding CCR5-ZFN at MOI's of 0 or mock transduced, 400, 600, 800, 1200, and 2000. (A) Cells were monitored for growth and population doubling level calculated based on starting number of cells and number of cells continued in culture at each time point after sampling: Mock transduced (black line, open square symbols), MOI 400 (gray line, filled square symbols), MOI 600 (black line, open triangle symbols), MOI 800 (gray line, closed triangle symbols), MOI 1200 (black line, open circle symbols), and MOI 2000 (gray line, closed circle symbols). (B) Gene disruption at CCR5 and the closely related CCR2 was measured by the SURVEYOR nuclease assay: MOI 400 (white), MOI 600 (diamond hatch), MOI 800 (gray), MOI 1200 (diagonal hatch), and MOI 2000 (black).

FIG. 4.

Validation of large-scale ex vivo clinical process for T-cell expansion and CCR5 modification incorporating a dynamic perfusion bioreactor. (A) Cells from an HIV-1 infected donor were stimulated ex vivo and transduced as described in the Methods section. Research scale untransduced, solid line and diamond symbols; research scale transduced, dotted line and square symbols; clinical scale transduced, dashed line and triangle symbols. (B) The production of IL-2, TNF-α, and Interferon-γ in supernatants are shown—research scale untransduced, research scale CCR5-ZFN transduced, and clinical scale CCR5-ZFN-transduced cultures. Expanded T cells from the culture in (A) were harvested, washed, resuspended in fresh media, and restimulated with fresh anti-CD3/CD28 mAb-coated beads. Supernatants were collected 24 hr later. Research scale untransduced, black bars; research scale transduced, gray bars; clinical scale transduced, white bars. An additional HIV-1 infected donor (C) was compared for production of cytokines as well as β-chemokines as in (B). Spontaneous secretion of cytokines and β-chemokines (production in the absence of restimulation) was also assayed as a measure potency of final product cells for infusion. Cells were harvested at the end of culture, and beads were removed and resuspended in fresh culture media. Supernatants were collected 24 hr later. Shown is the production of IL-2, TNF-α, MIP-1α, MIP-1β, GM-CSF, RANTES, and Interferon-γ. Research scale untransduced and unstimulated (spontaneous), white; research scale untransduced and restimulated with fresh anti-CD3/anti-CD28 mAb beads, light gray; clinical scale transduced and unstimulated (spontaneous), dark gray; clinical scale transduced and restimulated with fresh anti-CD3/anti-CD28 mAb beads, black.

FIG. 5.

T-cell receptor (TCR) Vβ repertoire of CCR5-ZFN T cells produced with large-scale ex vivo clinical process. TCR Vβ chain repertoire before and after a control research-scale expansion or after a clinical-scale expansion of CCR5-ZFN-transduced cells from a representative HIV+ donor. T cells from a total of three donors (one healthy, two HIV+) were analyzed. Three-dimensional plot of the complementary determining region 3 (CDR3) length distribution for the 26 TCR Vβ family of genes was analyzed as a ratio of the Vβ to hypoxanthine-guanine phosphoribosyltransferase transcript (HPRT). X-axis, TCR Vβ family; y-axis, TCR Vβ/HPRT ratio; z-axis, CDR3 length. Calculated TCR Vβ percentages of alteration are listed in Supplementary Table 1.

FIG. 6.

NOD/scid/γ_c^null (NSG) mouse–human xenograft biotoxicity study design. The days on study and the procedures at each timepoint are shown. See Methods for a detailed description of study procedures. (A) Study 1 utilized three unique human donors. CD4 T cells were transduced (24 mice/donor) or mock transduced (8 mice/donor) with Ad5/F35 vector-encoding CCR5-ZFN, and infused along with autologous peripheral blood mononuclear cells (PBMC) to support engraftment. Study 1 duration was restricted to 8 weeks due to onset of xenogeneic graft-versus-host disease (xGVHD). (B) Study 2 (38 mice) was performed using a single donor and without co-injection of autologous PBMC to delay xGVHD onset and allow a longer study observation period. Engraftment was supported by IL-2 injections concomitant with CCR5-ZFN-modified CD4+ T cells.