Engraftment and Gene Expression

Figure 1. Figure 1. Engraftment with Transduced Cells and Therapeutic Gene Expression in the Patient. Panel A shows vector copy number values in blood nucleated cells and the short-lived CD15+ (neutrophils) fraction thereof over 15 months after infusion of transduced CD34+ cells. Initial values in transduced cells before the infusion are shown. Panel B shows total hemoglobin levels and calculated levels of each hemoglobin fraction based on high-performance liquid chromatography measurements of globin chains. The percent contribution of hemoglobin fractions at month 15 is also indicated. The hemoglobin A (HbA) levels are derived from the regular red-cell transfusions received by the patient before gene therapy and briefly thereafter (the last red-cell transfusion occurred on day 88). HbA 2 is an alternative adult hemoglobin that is not derived from transfused blood. HbF denotes fetal hemoglobin, and HbS sickle hemoglobin.

Neutrophil engraftment was achieved on day 38 after transplantation, and platelet engraftment was achieved on day 91 after transplantation. Figure 1A shows the trajectory of vector copy numbers and Figure 1B shows production of HbAT87Q. Gene marking increased progressively in whole blood, CD15 cells, B cells, and monocytes (Fig. S2 in the Supplementary Appendix), stabilizing 3 months after transplantation. Increases in levels of vector-bearing T cells were more gradual.

HbAT87Q levels also increased steadily (Figure 1B) and red-cell transfusions were discontinued, with the last transfusion on day 88. Levels of HbAT87Q reached 5.5 g per deciliter (46%) at month 9 and continued to increase to 5.7 g per deciliter (48%) at month 15, with a reciprocal decrease in HbS levels to 5.5 g per deciliter (46%) at month 9 and 5.8 g per deciliter (49%) at month 15. Total hemoglobin levels were stable between 10.6 and 12.0 g per deciliter after post-transplantation month 6. Fetal hemoglobin levels remained below 1.0 g per deciliter.

Safety

The patient had expected side effects from busulfan conditioning. Grade 3 and 4 events included grade 4 neutropenia, grade 3 anemia, grade 3 thrombocytopenia, and grade 3 infection with Staphylococcus epidermidis (with positive results on blood culture), all of which resolved with standard measures. After the patient was discharged from the hospital, four grade 2 adverse events were reported: lower limb pain 3 months after treatment and transient increases in alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase between 5 and 8 months after treatment. All these events resolved spontaneously.

No adverse events related to the LentiGlobin BB305–transduced stem cells were reported (Table S1 in the Supplementary Appendix). Test results for the presence of replication-competent lentivirus were uniformly negative. Serial monitoring of integration sites in peripheral-blood samples showed a consistently polyclonal profile without detection of a dominant clone (defined as a single clone accounting for >30% of unique integration events) through month 12 (Fig. S3 in the Supplementary Appendix).

Clinical and Biologic Measures

The patient was discharged on day 50. More than 15 months after transplantation, no sickle cell disease–related clinical events or hospitalization had occurred; this contrasts favorably with the period before the patient began to receive regular transfusions. All medications were discontinued, including pain medication. The patient reported full participation in normal academic and physical activities. Magnetic resonance imaging (MRI) of the head at 8 months showed unchanged punctate subcortical white-matter hypodensities. Lower limb MRI at 14 months showed no recent bone or tissue damage.

Table 1. Table 1. Key Laboratory Values before Gene Therapy (at Screening) and at 3-Month Intervals after Infusion of Transduced CD34+ Cells.

Changes in sickle cell disease–related biologic measures are shown in Table 1. Complete blood counts were stable, reticulocyte counts decreased substantially (Fig. S4 in the Supplementary Appendix), and circulating erythroblasts were not detected. Laboratory values, including urinary microalbumin levels, indicated normal renal and liver functions. Although iron chelation was discontinued before transplantation, the ferritin levels decreased to 363 μg per liter at month 15, and MRI of the liver 1 year after treatment showed a low iron load (level of mobilizable circulating iron, relaxation rate R2*=117 Hz; and iron level, 3.1 mg per gram vs. 54 Hz and 14.6 mg per gram before gene therapy). Plasma levels of total bilirubin and lactate dehydrogenase normalized. Soluble transferrin receptor levels improved and were 3.4 times as high as normal values at screening and 1.5 times as high at months 12 and 15, indicating progressive normalization of erythropoiesis.

Figure 2. Figure 2. Results of Sickle Cell Disease–Specific Red-Cell Assays. Panel A shows rates of red-cell sickling under normoxic conditions (20% oxygen saturation) and Panel B shows rates of red-cell sickling under hypoxic conditions (10% oxygen saturation) in the patient at 6 months and 12 months after gene therapy and among control patients from whom red-cell samples were obtained: two patients with heterozygous A/S “sickle trait” (Controls 1 and 2; Control 1 is the patient’s mother) and three patients with sickle cell disease (Controls 3, 4, and 5). Similar results were obtained at 7% and 5% oxygen saturation rates (data not shown). T bars indicate standard errors. Panel C shows oxygen dissociation curves for red cells 12 months after gene therapy in the patient and in the patient’s heterozygous (A/S) mother (Control 1). These analyses were performed simultaneously, under identical conditions. The mean red-cell deoxygenation curve (solid black line) and the mean red-cell reoxygenation curve (dashed black line) for 15 untreated patients with sickle cell disease are also shown. Panel D shows red-cell deformability 12 months after gene therapy in the patient as compared with his heterozygous (A/S) mother (Control 1) and another patient with sickle cell disease (Control 6). The gray zone demarcates the range within which 95% of non–sickle cell disease red cells fall, and the black curve is the mean curve for healthy participants. The elongation index was calculated as the ratio of the length (A) and width (B) of a cell, where (A−B) was divided by (A+B), and the result was expressed as a decimal between 0 and 1. Panel E shows the red-cell density profile 12 months after gene therapy in the patient, obtained with the use of a phthalate gradient. We measured 10 samples (indicated with the numbers 1 through 10 on the black curve) at various phthalate densities. Red lines demarcate three different densities of cells: low (<1.086 mg per milliliter), medium (1.086 to 1.096 mg per milliliter), and high (>1.096 mg per milliliter). Orange lines indicate limits of a normal profile. The values for the patient are shifted to the left because of the associated single α-globin gene deletion. Cells denser than 1.110 mg per milliliter of phthalate solution are considered to be dense cells.

Because the patient received a regular transfusion regimen for 4 years before this study and because of the exchange transfusion before transplantation, meaningful comparative studies before and after transplantation could not be conducted. However, the proportions of sickled red cells in the patient’s blood at months 6 and 12 were significantly lower than those in untreated patients with sickle cell disease (βS/βS) (Figure 2A). At month 12, the sickling rate in hypoxic conditions was not significantly different from that of the patient’s asymptomatic, heterozygous (βA/βS) mother (Figure 2B). Oxygen dissociation studies, which quantify oxygen saturation relative to the partial pressure of oxygen, showed that results in the patient at month 12 and results in a heterozygous (βA/βS) control were similar (Figure 2C and 2D).