Characteristics of the Patients

Data through June 2, 2017, are presented for all 22 treated patients with β-thalassemia who were enrolled in the two studies. The duration of follow-up after transplantation ranged from 15 to 42 months.

Table 1. Table 1. Characteristics of the Patients and Cellular Products.

In HGB-204, a total of 23 patients between the ages of 12 and 35 went through the consenting process. Of these patients, mobilization was initiated in 19, and 18 received the infusion. All the treated patients met the criteria for transfusion dependence and included 8 patients with a β0/β0 genotype, 6 patients with a βE/β0 genotype, and 4 patients with other β-thalassemia genotypes (Table 1 and Figure 1, and Table S1 in the Supplementary Appendix). Patients had started receiving red-cell transfusions at a median age of 3.5 years, and 6 had undergone splenectomy. In the 2 years before study enrollment, the median annual red-cell transfusion volume was 164 ml per kilogram per year (range, 124 to 261), which included 183 ml per kilogram per year in the 8 patients with a β0/β0 genotype and 147 ml per kilogram per year in the 10 patients with other genotypes.

In HGB-205, 4 patients between the ages of 16 and 19 years went through the consenting process and received an infusion of the drug product. Of these patients, 3 had a βE/β0 genotype, and 1 was homozygous for the IVS1-110 mutation, a β+ genotype with only trace endogenous βA-globin expression (0.9 g per deciliter in this patient), a severe clinical presentation equivalent to that seen in patients with a β0/β0 genotype (Table 1 and Figure 1). Three of the patients had started receiving red-cell transfusions before the age of 3 years, and 3 had undergone splenectomy. In the 2 years before enrollment, the median red-cell transfusion volume was 182 ml per kilogram per year (range, 139 to 197).

Drug Product Characteristics and Pretransplantation Conditioning

Patients underwent conditioning with a single agent, intravenous busulfan. Because complete myeloablation is essential for high-level engraftment with ex vivo transduced hematopoietic stem cells27,28 and increased busulfan metabolism had been reported in some patients with β-thalassemia,29 plasma busulfan pharmacokinetic analysis was performed in all the patients, either after the first busulfan injection (in HGB-204) or daily (in HGB-205).30 Such analysis was followed by dose adjustments to achieve appropriately targeted drug exposure. The average daily plasma busulfan area-under-the-curve values ranged from 3029 to 4714 μM per minute in HGB-204 (estimated values) and from 4670 to 5212 μM per minute in HGB-205 (actual values).

Among the HGB-204 study patients, the median dose of the drug product was 11.0 million CD34+ cells per kilogram (range, 6.1 million to 18.1 million) in patients with a β0/β0 genotype and 7.1 million CD34+ cells per kilogram (range, 5.2 million to 13.0 million) in those with other genotypes. The vector copy number in the drug products ranged from 0.3 to 1.5 (Table 1). In HGB-205, the dose was 8.8 million CD34+ cells per kilogram in the IVS1-110 homozygote and 12.0 million CD34+ cells per kilogram (range, 8.9 million to 13.6 million) in the 3 patients with a βE/β0 genotype. The vector copy number in the drug products ranged from 0.8 to 2.1 (Table 1).

Safety

No safety issues were attributed to the BB305 vector in either study. In HGB-204, five mild adverse effects (all grade 1) were characterized as related or possibly related to the drug product. Other than the hematologic alterations that commonly occur after busulfan conditioning, all adverse events of grade 3 or higher that occurred in two or more patients are listed in Table S2 in the Supplementary Appendix. Nine serious adverse events were reported, including two episodes of veno-occlusive liver disease attributed to busulfan conditioning. In HGB-205, no serious adverse events were considered to be related to the drug product. All nonhematologic adverse events of grade 3 or higher are listed in Table S3 in the Supplementary Appendix. In the two studies, all adverse events were treated with standard measures.

Figure 2. Figure 2. Genomewide Mapping of Vector Unique Integration Sites after Sustained Hematopoietic Reconstitution. Shown are the 10 most represented unique integration sites (UIS) in vector-bearing peripheral-blood mononuclear cells in analyses performed 2 years after gene therapy in two representative patients each in the HGB-204 study and in the HGB-205 study, as compared with samples obtained at 2 years and 8 years from Patient 1003 in the previous LG001 study.20 The samples were analyzed by means of DNA pyrosequencing of ligation-mediated products of polymerase-chain-reaction assay. Each of the 10 sites is indicated by a different color, with gray indicating the cumulative proportion of all the other integration sites. The total number of sites is indicated at the top of each column (see also Fig. S2 in the Supplementary Appendix). In the HGB-204 and HGB-205 studies, no clonal dominance was detected in any patient at any time point. In contrast, Patient 1003 showed a very small total number of UIS, and clonal dominance was observed at the HMGA2 locus (as indicated by an asterisk) at year 2, a dominance that was progressively replaced by other sites by year 8.

No replication-competent lentivirus has been detected in the patients in either study, and serial monitoring of vector integration sites in blood samples has consistently shown polyclonal profiles of unique integration sites without dominant clones. At 12 months after infusion, the median number of unique integration sites was 1646 per patient (range, 202 to 5501) in HGB-204 and 5322 (range, 756 to 8685) in HGB-205. Integration data for representative patients are shown in Figure 2, and in Figure S2 in the Supplementary Appendix.

These integration data include a comparison with findings in Patient 1003, who was enrolled in the previous LG001 study20 and had initial partial clonal dominance at the HMGA2 locus. This clonal expansion was followed to determine whether its presence predicted any adverse event. At the time of this report (year 12), the HMGA2 locus was no longer dominant in Patient 1003 and had not been associated with serious adverse events. After receiving the drug product, Patient 1003 had received no red-cell transfusions during year 2 through year 8 while maintaining a total hemoglobin level of approximately 8 g per deciliter. During year 9 through year 12, the patient resumed sporadic transfusions, although the HbAT87Q levels had remained stable above 2 g per deciliter since month 1820 (data not shown).

Hematopoietic Recovery and Gene Marking of Blood Cells

After the intravenous infusion of the thawed LentiGlobin drug product, neutrophil engraftment occurred within a median of 18.5 days (range, 14.0 to 30.0) in HGB-204 and 16.5 days (range, 14.0 to 29.0) in HGB-205. Platelet engraftment occurred within a median of 39.5 days (range, 19.0 to 191.0) in HGB-204 and 23.0 days (range, 20.0 to 26.0) in HGB-205, during which time there were no bleeding complications resulting in serious adverse events.

Figure 3. Figure 3. Kinetics of Engraftment with Vector-Transduced Hematopoietic Stem Cells. Shown is the average vector copy number (VCN) per diploid genome in peripheral-blood mononuclear cells (PBMCs) after drug product infusion in the HGB-204 and HGB-205 studies among patients with a non–β0/β0 genotype (Panel A) and in those with a β0/β0 genotype or a genotype of equivalent clinical severity (homozygous IVS1-110) (Panel B). The data for Patient 1121, the only patient with a non–β0/β0 genotype who had not yet stopped receiving red-cell transfusions at the last study visit, are indicated with a dotted line in Panel A. Data for three patients with clinically severe genotypes who had stopped receiving transfusions — Patient 1106 (blue squares) and Patient 1123 (blue triangles) with a β0/β0 genotype and Patient 1203 (red triangles) who was homozygous for the IVS1-110 mutation — are shown in Panel B. In a pooled analysis, there was significant correlation between the VCN in the drug product and the VCN in PBMCs at 6 months (Panel C).

The average vector copy numbers in PBMCs over time after drug product infusion in the two studies are shown in Figure 3. The median vector copy number at 15 months was 0.3 copies per diploid genome (range, 0.1 to 0.9) in HGB-204 and 2.0 copies per diploid genome (range, 0.3 to 4.2) in HGB-205. In a pooled analysis, there was significant correlation between the vector copy number in PBMCs at 6 months and the initial vector copy number in the drug product (r2=0.69, P<0.001) (Figure 3C).

Blood HbAT87Q Levels and Changes in Transfusion Requirements

Figure 4. Figure 4. Changes in Transfusion Requirements after Gene Therapy. Panel A shows the patients in the HGB-204 study (blue) and the HGB-205 study (red) who stopped red-cell transfusions after receipt of the LentiGlobin drug product. The horizontal bars show the interval between the drug product infusion and the patient’s independence from red-cell transfusion (lighter color) and the interval since receipt of the last transfusion (darker color), including the number of months without transfusion at the time of the data analysis. The total hemoglobin level for each patient at the last study visit is shown on the right. The 12 patients with a non–β0/β0 genotype who discontinued red-cell transfusions are listed above the horizontal black line, and the 2 patients with a β0/β0 genotype (Patients 1106 and 1123) and Patient 1203 with two copies of the IVS1-110 mutation are listed below the line. Among the 6 patients with a β0/β0 genotype and 1 patient with a non–β0/β0 genotype (Patient 1121) who were still receiving red-cell transfusions, most of the patients were receiving a lower annualized red-cell volume (Panel B) and a lower annualized number of transfusions (Panel C) than before gene therapy. In Panels B and C, the value before gene therapy is indicated in blue, and the value after gene therapy in gray.

Of the 22 patients who were treated, 13 had a non–β0/β0 genotype. Of these 13 patients, all but 1 stopped receiving red-cell transfusions after gene therapy. At the last study visit (12 to 36 months after infusion), the median HbAT87Q level was 6.0 g per deciliter (range, 3.4 to 10.0), and the median total hemoglobin level was 11.2 g per deciliter (range, 8.2 to 13.7) (Figure 4A). Patient 1121, who had a vector copy number of 0.3 in the drug product, had a blood vector copy number of 0.10 at the last follow-up and continued to receive red-cell transfusions. In addition, Patient 1118 received a single transfusion 13 months after drug product infusion during an acute viral illness.

Table 2. Table 2. Summary of Outcomes in the 22 Study Patients.

At the last study visit, of the 9 patients with a β0/β0 genotype or homozygosity for the IVS1-110 mutation, 6 had a median HbAT87Q level of 4.2 g per deciliter (range, 0.4 to 8.7) and continued to receive transfusions. However, there was a median reduction of 74% (range, 7 to 100) in the annual number of transfusions and a 73% reduction (range, 19 to 100) in the annual transfusion volume, as compared with transfusion support in the 2 years before enrollment (Figure 4B and 4C). The remaining 3 patients with a β0/β0 genotype or two copies of the IVS1-110 mutation had not received transfusions for 14 to 20 months (Figure 4A). At the most recent follow-up (12 to 30 months), the patients’ HbAT87Q level ranged from 6.6 to 8.2 g per deciliter, and the total hemoglobin level ranged from 8.3 to 10.2 g per deciliter. Additional characteristics of this subgroup of patients are summarized in Table S5 in the Supplementary Appendix. Table 2 provides a summary of the outcomes in the 22 patients.

Figure 5. Figure 5. Kinetic Analysis of HbAT87Q in Blood. Shown is the average level of vector-derived hemoglobin A with a T87Q substitution (HbAT87Q) in blood samples obtained from patients in the HGB-204 and HGB-205 studies among those with a non–β0/β0 genotype (Panel A) and in those with a β0/β0 genotype or a genotype of equivalent clinical severity (homozygous IVS1-110) (Panel B). The only patient with a non–β0/β0 genotype who had not yet stopped receiving red-cell transfusions (Patient 1121) is indicated with a dotted line in Panel A. The two patients with a β0/β0 genotype (Patient 1106 [blue squares] and Patient 1123 [blue triangles]) and the patient with a IVS1-110 mutation (Patient 1203 [red triangles]) who had stopped receiving transfusions are indicated in Panel B. Blood HbAT87Q levels correlated with VCN values in PBMCs at 6 months (Panel C).

In the two studies, blood HbAT87Q levels correlated with blood vector copy number levels (r2=0.75, P<0.001) (Figure 5C). Other factors, such as age, genotype, and splenectomy status, did not appear to correlate with gene expression. The studies were not powered to conclusively assess determinants of response. The hemoglobin fractions that contributed to total hemoglobin levels over time and the timing of red-cell transfusions in representative patients with β0/β0, βE/β0, and other non–β0/β0 genotypes are shown in Figure S1 in the Supplementary Appendix. Hemoglobin fractions in erythroid burst-forming units in the four patients in HGB-205 are shown in Table S4 in the Supplementary Appendix.

Effect on Hemolysis and Dyserythropoiesis

In HGB-205, in an exploratory analysis that was performed among patients who had stopped receiving red-cell transfusions after gene therapy, the degree of hemolysis at first stabilized relative to pretransplantation levels and was fully corrected in Patients 1201 and 1202 by 36 months after treatment (Table S6 in the Supplementary Appendix). Because strict adherence to iron chelation therapy was difficult in the 3 patients with a βE/β0 genotype, they underwent regular phlebotomy, in which 200 ml of blood was withdrawn each month. At the time of the last study visit, the patients’ hemoglobin levels were stable, despite a cumulative phlebotomy volume of more than 1 liter per patient. Patient 1203 was receiving deferiprone to remove excess iron. The tissue iron content in the patient’s heart and liver is being followed with the use of magnetic resonance imaging (MRI), and normalization of values was awaited before the discontinuation of iron chelation or phlebotomy.9 Such discontinuation of treatment was reported in Patient 1202, who is no longer receiving iron chelation therapy and who completed phlebotomy 36 months after gene therapy.

Figure 6. Figure 6. Iron Metabolism and Abatement of Dyserythropoiesis in the HGB-205 Study. Shown is the progressive decrease in the plasma level of soluble transferrin receptor (TFR) (Panel A) and increase in the hepcidin:ferritin ratio (Panel B), as indicated by solid circles representing data for three patients — Patient 1201 (P1), Patient 1202 (P2), and Patient 1206 (P4), all of whom had a βE/β0 genotype — before and after gene therapy (GT). Data for Patient 1203 (P3, triangles), who was homozygous for the IVS1-110 mutation, showed no change in either marker. The entire follow-up data for the three patients with a βE/β0 genotype (from 3 to 36 months) are shown with horizontal bars indicating medians. The data were pooled and compared with pooled values at screening with the use of a two-tailed Wilcoxon rank-sum test. Also shown are the correlations between hemoglobin levels and markers of ineffective erythropoiesis and iron homeostasis, including soluble TFR (Panel C), erythroid protoporphyrin (PP) IX (Panel D), hepcidin (Panel E), and the ratio of hepcidin to ferritin (Panel F). In Panels C through F, data for Patient 1201 are indicated by squares, Patient 1202 by gray triangles, Patient 1203 by green triangles, and Patient 1206 by circles. The Spearman test was used to assess correlations between biologic variables. Hemoglobin levels had a negative correlation with soluble TFR levels (Spearman r=−0.63, P<0.001) and erythroid protoporphyrin IX levels in red cells (Spearman r=−0.85, P<0.001) and a positive correlation with hepcidin levels and hepcidin:ferritin ratios (Spearman r=0.57, P=0.01 for both calculations). At values of more than 9.5 g per deciliter of hemoglobin, the erythroid mass was reduced and peripheral iron control was restored by hepcidin. Below 8.5 g per deciliter, ineffective erythropoiesis was maintained and hepcidin repressed, as indicated by the vertical red dotted line separating the two performance levels.

In addition, we investigated biologic markers of dyserythropoiesis (Figure 6, and Table S6 in the Supplementary Appendix).31 Plasma levels of two markers of ineffective erythropoiesis or erythroid expansion, soluble transferrin receptor32 and erythroid protoporphyrin IX,33 were within normal ranges for the 3 patients with a βE/β0 genotype after they had stopped receiving transfusions. In addition, the plasma ratio of hepcidin to ferritin increased substantially over time in these patients. The normalization in markers of dyserythropoiesis correlated with the hemoglobin levels that were achieved. Together, these results point to the elimination of dyserythropoiesis in the 3 patients with a βE/β0 genotype who were treated. In contrast, complete normalization of biologic markers of ineffective erythropoiesis was not observed in Patient 1203 (IVS1-110 homozygote) (Figure 6, and Table S6 in the Supplementary Appendix).