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Clinical Trial
. 2014 Mar 6;370(10):901-10.
doi: 10.1056/NEJMoa1300662.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV

Pablo Tebas  1 , David Stein, Winson W Tang, Ian Frank, Shelley Q Wang, Gary Lee, S Kaye Spratt, Richard T Surosky, Martin A Giedlin, Geoff Nichol, Michael C Holmes, Philip D Gregory, Dale G Ando, Michael Kalos, Ronald G Collman, Gwendolyn Binder-Scholl, Gabriela Plesa, Wei-Ting Hwang, Bruce L Levine, Carl H June
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Clinical Trial

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV

Pablo Tebas et al. N Engl J Med. .
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Abstract

Background: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.

Methods: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.

Results: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.

Conclusions: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Figures

Figure 1. Lymphocyte Values
In Panel A, median total lymphocyte, CD4 T-cell, and CD8 T-cell values for all study participants are shown (see Fig. S1 in the Supplementary Appendix). The increase in total lymphocyte count is due to an increase in the number of CD4 T cells, since changes in CD8 counts are negligible. In Panel B, the change in CD4 T-cell count from baseline is plotted for all participants, and the median change is plotted for each cohort. In Panel C, the median ratios of CD4 T cells to CD8 T cells are plotted for all participants.
Figure 2. CCR5-Modified CD4 T Cells in the Circulation and Mucosal Tissues
Panel A shows that the median absolute number of CCR5-modified circulating CD4 T cells was similar in participants with adequate CD4 T-cell recovery after highly active antiretroviral therapy (HAART) (cohort 1) and in those with inadequate CD4 T-cell recovery after HAART (cohort 2). Panel B shows CCR5-modified cell traffic to rectal mucosal tissues. Patients in cohort 1 (green triangles) underwent a rectal biopsy at baseline (blue circle) and on days 21 and 112, and those in cohort 2 (brown squares) underwent biopsies at baseline (blue circle) and on days 42 and 252. Box plots show the 25th percentile (lower edge of the box), mean (dotted line in the box), median (solid line in the box), 75th percentile (upper edge of the box), and 90th percentile (whisker). CCR5-modified CD4 T-cells constituted a mean of 0.6%, 1.1%, 0.4%, and 0.4% (and a median of 0.8%, 0.4%, 0.2%, and 0.2%) of rectal mucosal mononuclear cells on days 21, 42, 112, and 252, respectively.
Figure 3. Changes in Viremia during Treatment Interruption
Panel A depicts HIV viral loads (HIV RNA) for the six patients in cohort 1. SB-728-T was infused on day 0. A 12-week (84-day) treatment interruption (shaded area) was initiated on day 28 and terminated on day 112. The treatment interruption was terminated prematurely, on day 84 (week 8 of the interruption period), in Patients 204 and 251. The dotted lines indicate reinstitu-tion of highly active antiretroviral therapy. The historical HIV-RNA set point for each patient is also shown. The limit of detection (LOD) for the viral-load assay is plotted at 50 copies. Patient 205 was heterozygous for CCR5 delta32. Panel B shows the median CD4 T-cell, CD8 T-cell, and CCR5-modi-fied T-cell counts in cohort 1 during the treatment interruption, as well as the viral load.
Figure 4. CCR5-Modified CD4 T Cells during Treatment Interruption
The median count for total CD4 T cells, unmodified CD4 T cells, and CCR5-modified CD4 T cells for all patients in cohort 1 is plotted in Panel A. CCR5-modified CD4 T cells in blood are shown as a fraction of CD4 cells (Panels B and D) and as a fraction of white cells (Panels C and E).
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