Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils

Abstract

Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.

Figure 1

Study design. Patients' cells were collected and stored on a separate protocol. Day 0 indicates the day of cell infusion. Cells were thawed on day −5, and transduction was begun on day −4. The busulfan was infused on days −3 and −2. Patients 2 and 3 received palifermin given on days −6 to −4 and −1 to 1. Patient 3 also started rapamycin on day −1 and continued it for 30 days after transplantation.

Figure 2

Results from patient 1 during his first gene therapy trial in 1998. Patient 1 from our trial was enrolled previously on a gene therapy trial that used the same vector but did not include conditioning. The patients received 2 separate infusions of cells as marked by the arrows and the number of cells given × 10⁴. Marking was detectable early and increased after each infusion but eventually was undetectable after 1 year.

Figure 3

Effect of busulfan on neutrophil and platelet counts. (A) The absolute neutrophil count for each patient is plotted beginning before transplant (baseline) to day 50 after transplantation. Cell infusion was day 0 and the busulfan was given days −3 and −2. (B) The platelet count for each patient is plotted beginning before the busulfan and until all patients no longer required any transfusions, day 48.

Figure 4

Percentage of DHR⁺ cells after cell infusion (day 0). Flow cytometric analysis was performed at various time points for each patient to assess for oxidase-positive cells in the peripheral blood. Each patient had undetectable DHR before beginning the therapy. The percentage is plotted on a logarithmic scale with each patient having a different range starting at 0.1 for patient 1, 0.001 for patient 2, and 0.01 for patient 3.

Figure 5

Flow cytometry panel showing DHR analysis for patient 1 at 2 years after gene therapy. Patient 1 who had the best results continues to have approximately 1% DHR-positive cells in the peripheral blood. The MFI for this patient's cells is consistently in the same range as the normal control run concurrently, indicating a close to normal level of oxidase production on a per cell basis.

Figure 6

Patient 1 clonal tracking PCR. Primers were designed based on inserts identified from the LAM PCR from patient 1, and a seminested PCR was performed on samples obtained at various time points to determine their presence in the various lineages.

Figure 7

Clinical results for patient 1. Shown here are the CT scans obtained before gene therapy and 6 months after gene therapy for patient 1 who had biopsy-proven Staphylococcal aureus liver abscesses as indicated by the arrow. These disappeared, leaving only some scarring and regenerating liver as shown in the corresponding post films.