Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial

Abstract

X-linked chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by a defect in the gp91(phox) gene. In an effort to treat X-CGD, we investigated the safety and efficacy of gene therapy using a retroviral vector, MT-gp91. Two X-CGD patients received autologous CD34(+) cells transduced with MT-gp91 after a conditioning regimen consisting of fludarabine and busulfan. The level of gene-marked cells was highest at day 21 (8.3 and 11.7% in peripheral blood cells) but decreased to 0.08 and 0.5%, respectively, 3 years after gene transfer. The level of functionally corrected cells, as determined by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, reached a peak at day 17 (6.5% patient 1 (P1) and 14.3% patient 2 (P2) of total granulocytes) and declined to 0.05% (P1) and 0.21% (P2), 3 years later. Some retroviral vectors were found to have integrated within or close to the proto-oncogenes MDS1-EVI1, PRDM16, and CCND2; however, no abnormal cell expansion or related hematological malignancy was observed. Overall, the gene transfer procedure did not produce any serious adverse effects and was able to convert a significant fraction of blood cells to biologically functional cells, albeit for a short period of time.

Figures

Figure 1

Overall flow of retroviral gene therapy for CGD. Day 0 indicates the day of cell infusion. G-CSF mobilized peripheral blood CD34+ cells were collected, and half of them were transduced with monocistronic γ-retroviral vector expressing gp91phox. CGD, chronic granulomatous disease; G-CSF, granulocyte-colony stimulating factor.

Figure 2

Hematopoietic and lymphoid reconstitution. (a) White blood cell counts (WBC, black circle) and absolute neutrophil counts (ANC, white triangle) of P1 and P2 are indicated before and after gene therapy. GT means the infusion of retrovirally transduced CD34+ cells (Day 0). (b) Counts of helper T cells (CD4+, black circle), cytotoxic T cells (CD8+, white circle), B cells (CD19+, white square), and NK cells (CD16/56+, black square) are shown. To compare the kinetics of transduced cells (as determined by DHR assay) with that of other immune cells, the percentage of NADPH oxidase-positive cells is shown in a white triangle. For the detailed kinetics of transduced cells, see Figure 4a. DHR, dihydrorhodamine; GT, gene therapy; NADPH, nicotinamide adenine dinucleotide phosphate; NK cells, natural killer cells.

Figure 3

Functional reconstitution. (a) Kinetics of gp91-positive cells in peripheral blood. The percentage of the NADPH oxidase-positive granulocytes (black circle), vector genome-positive cells (white square), and gp91phox protein-positive granulocytes (white triangle) are shown. These were measured by DHR assay, quantitative real-time PCR, and flow cytometry, respectively. (b) Percentage of vector-containing CFU-GM colonies derived from bone marrow. The presence of a vector in CFU-GM colonies was assayed by PCR using two different primer sets. The location of primers in the vector is indicated in the figure. (c) Quantification of vector-positive cells in bone marrow. The percentage of gene-modified cells was analyzed by quantitative real-time PCR. CFU-GM, colony forming unit-granulocyte/macrophage; DHR, dihydrorhodamine; FACS, fluorescence-activated cell sorting; NADPH, nicotinamide adenine dinucleotide phosphate, Q-PCR; quantitative real-time PCR.

Figure 4

LAM-PCR analysis of vector integrants. LAM-PCR analysis of ex vivo transduced CD34+ cells before transplantation (pre) as well as at various weeks (w) after transplantation derived from (a) P1 and (b) P2. -control, negative control; IC, internal control of LAM-PCR; LAM-PCR, linear amplification-mediated PCR; M, 100 bp Marker.

Figure 5

Contribution of individual integration sites to gene-corrected hematopoiesis. Depicted is the retrieval frequency of individual integration sites in sequenced polyclonal cell samples from (a) P1 and (b) P2. The clonal contribution of individually gene-corrected cells was assessed by counting identical integration site sequences after 454 pyrosequencing of LAM-PCR amplicons. The relative contribution of individual amplicons is given as the percentage of all insertion flank sequence reads encountered in the particular sample. The ten most frequent retroviral integration sites (RIS) are ranked from 1 to 10, according to the retrieval frequency. Genes highlighted in grey were associated with the ten strongest RIS at more than one time point. LAM-PCR, linear amplification-mediated PCR; Pre, pretransplantation sample; w, weeks after gene therapy.