Subjects. Ten subjects between the ages of 3 months and 15 years (median, 11.5 months) were enrolled between 2009 and 2012 (Figure 1 and Table 1). Four subjects were diagnosed between 1 and 15 months of age (median, 9 months) when they were hospitalized for failure to thrive or infections; 5 subjects were diagnosed in utero or at birth because a sibling or other family member had ADA deficiency; 1 subject was diagnosed by positive newborn screening for SCID, which had been recently implemented in the US in California. Conditions for enrollment included confirmed absence of ADA enzyme activity in peripheral blood or fetal cells and evidence of immune deficiency (absolute lymphocyte count [ALC] < 200 cells/mm3 or T cell lymphoblastic response to phytohemagglutinin [PHA] with Δ c.p.m. < 5000) prior to initiation of ERT. Genetic mutations in the ADA gene were documented in 9 of 10 subjects (and 1 was not tested). Subjects had been treated with ERT from diagnosis until enrollment in the study (birth to more than 14 years), which included discontinuation of ERT within 1 week of the bone marrow harvest for the gene transfer. All subjects remain alive, at this writing, with normal growth for their age (except for subject 401, who had already neared maximal height at 15 years old, Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI90367DS1) and are 42 to 84 months (median 57 months) after infusion.

Figure 1 Diagram of participant flow and number of subjects entered, followed, and analyzed. TNC, total nucleated cells.

Busulfan myelosuppression. RIC was achieved using a standard busulfan dose on day –3 of transplant of 90 mg/m2 of subject’s body surface area, which corresponded to 2.65 to 5.23 mg busulfan/kg body weight (Table 2). Exposure to busulfan was determined by calculating AUC, and levels ranged widely, from 2427 to 6714 μmol/l per min (median 5344 μmol/l per min, Table 2). The thrombocytopenia followed a consistent pattern among subjects (Supplemental Figure 2A) with nadirs between days 19 and 27 after busulfan and subsequent recovery without need for platelet transfusions. Neutropenia nadir occurred between 15 days and 32 days after busulfan dosing (Supplemental Figure 2B) with increasing absolute neutrophil count (ANC) thereafter. Subject 407 experienced prolonged neutropenia (ANC, 250–350 cells/mm3) beyond 42 days after transplant and received 1 course of granulocyte CSF (G-CSF) for treatment at 10-month follow-up. Transient transaminitis was observed in 3 subjects (subjects 405, 406, 407) beginning on day 22 after busulfan dosing (and approximately 1 month after ERT withdrawal) and spontaneously resolving by day 60 after transplant (Supplemental Figure 2, C and D).

Table 2 Infusion of the transduced cell product

CD34+ cell dosages and transduction efficiency. Subjects received between 0.6 and 8.4 million (median, 6.8 million) CD34+ cells per kg of body weight at the time of infusion (day 0, Table 2), which was dependent upon the initial cell collection from the bone marrow harvest and cell recovery after Ficoll-Hypaque mononuclear enrichment, CD34+ purification, and culture and transduction processes. Vector copy number (VCN) in the transduced product ranged from 0.18 to 2.68 copies per cell and ADA enzyme activity from 40 to 614 units (nmol/min/108 cells). Typically, the younger subjects received larger cell doses (Supplemental Figure 3, as a result of higher starting cell numbers from the bone marrow harvest, data not shown), with the exception of subject 402, where there was poor recovery of the mononuclear cells during the Ficoll gradient centrifugation step. Additionally, there was a manufacturing problem with the cell product for subject 407, which resulted in 2 products being given 5 days apart without additional conditioning. There were no toxicities associated with the cell infusion in any of the 10 subjects.

Safety. No subjects experienced serious adverse events (SAEs) attributable to the gene-transfer vector or cell product. Replication-competent retrovirus (RCR) was not detected in any subject. There were no leukoproliferative events, although several subjects do have stable persistent clones with vector integrations adjacent to MECOM or LMO2 loci (Aaron R. Cooper, unpublished observations), which are common integration loci that have been documented in other γ-retroviral GT trials (16–19). At this writing, all subjects remain alive and well and without restricted activity.

Adverse events. Three subjects (subjects 403, 407, and 410) had prolonged neutropenia (grades 2 to 3) lasting 10 to 18 months after engraftment of gene-modified cells (data not shown). This neutropenia may reflect the abnormal myelopoiesis that may occur with ADA deficiency (20) rather than from the GT procedure. Five subjects developed infectious complications requiring initial or prolonged hospitalization at 2 days to 5 months after transplant that resolved with therapy and did not require restarting ERT (Supplemental Table 1). Subject 410 was hospitalized for 5 days at approximately 20 months after transplant for observation of rash and fever associated with vaccination and antibiotic therapy. No other hospitalizations have been recorded to date.

ADA enzyme activity and ERT. ERT was discontinued approximately 7 days before bone marrow harvest. The oldest subject at time of treatment (subject 401) restarted ERT at 6 months after GT because his peripheral blood mononuclear cell (PBMC) ADA enzyme activity did not reach normal levels and his lymphocyte counts did not recover (Figure 2A and Figure 3A). All other subjects have remained off ERT to the time of this writing (3 to 6 years as of May 2016). Subjects 404, 408, and 410, who were all 3 months old at the time of GT, developed the highest levels of PBMC ADA activity, which was sustained throughout follow-up (Figure 2A). Except for subject 401, all subjects have PBMC ADA activity in the normal range of the assay, although the time to normalization varied from 1 to 24 (median, 6) months after transplant. Endogenous ADA activity from the GT was sufficient to deplete toxic adenosine metabolites (measured by the portion of adenine nucleotides that were deoxyadenosine nucleotides [dAXP] in peripheral blood) to below 6% in all subjects, except for subject 401, who shows 0% dAXP from exogenous ERT (Figure 2B).

Figure 2 ADA enzymatic activity in PBMCs and percentages of deoxyadenine nucleotides in erythrocytes. (A) ADA enzyme activity in PBMCs was measured biochemically. The low and high normal reference range for the ADA enzyme assay in human PBMCs is indicated by the 2 parallel broken lines. (B) Adenine and deoxyadenine metabolites were measured in erythrocytes by high-pressure liquid chromatography and the percentage that were dAXP (dAMP + dADP + dATP) were plotted as %dAXP. The time when ERT was resumed for subject 401 is indicated.

Figure 3 Cell counts and lymphocyte proliferation responses after GT. (A) ALCs plotted from time of GT through subjects’ last recorded follow-up. (B–G) Cell counts at ≥ 12 months after GT through subjects’ last recorded follow-up plotted according to age. Black horizontal bars represent the 10th percentile of normal (33). (B) CD3+ pan T cells. (C) CD4+ T cells. (D) CD8+ T cells. (E) CD19+ B cells. (F) CD16/56+ NK cells. (G) CD4/CD45RA+ naive T cells. (H) Lymphocyte proliferation to PHA expressed as the percentage of lower limit of normal (LLN) for the laboratory that conducted the assay. Black dotted horizontal line is drawn at 100% of normal.

Cell counts. The rate and level of ALC recovery corresponded to ADA activity (Figure 3A). Subjects 408 and 410 had the most rapid rise and recovery of their ALC compared with the remaining subjects. Subjects 401 and 403 (the oldest at time of GT at 15 and 8 years, respectively) had the lowest ALC recovery. Although all subjects who remained off ERT showed increases of their ALC after GT, only subjects 408 and 410 reached low normal (10th percentile) counts in absolute CD3+ and CD4+ T cells, B cells (CD19+), NK cells (CD56+16+), and naive T cells (CD4+/CD45RA+) for their age (Figure 3, B–G, and Supplemental Figure 4) at 12 months of follow-up. Of interest, subject 401, who resumed ERT, did have low normal levels of B and NK cells, although he continued to have very low T cell counts. No subject in the CD8+ T cell subset had normal counts (Figure 3D and Supplemental Figure 4C).

T cell function. T cell activation was assessed by measuring the subjects’ lymphocytes’ ability to respond to the lectin PHA. All but 2 subjects (subjects 401 and 407) had normal responses to PHA (Figure 3H). Despite having low numbers of T cells, subjects 403 and 406 had positive responses, suggesting that even a small number of corrected T cells was sufficient to restore this broad cellular response. Subject 401, despite being on ERT, failed to have a response; this result is consistent with a previous observation that long-term ERT does not fully restore immune function (6).

TCR repertoire. Quantitative spectratyping to identify T cell receptor (TCR) β-variable (TRBV) CDR3 region rearrangement distribution at the molecular level was performed on subjects 401, 402, 404, and 405 at distinct time points after GT (Supplemental Figure 5, A–D). Regular Gaussian distribution of TRBV peak length across all families was observed in subjects 402, 404, and 405, indicating that those subjects had normal TCR diversity by 12 to 24 months after GT. Subject 401 had abnormal-appearing TRBV peak distribution seen in a small number of families (BV04, BV10, BV30), despite having been restarted on ERT for approximately 18 months prior to analysis. The distribution of TRBV families in CD4+ and CD8+ T cells was measured in subjects 403, 406, and 407 by flow cytometry at several time points throughout their follow-up (Supplemental Figure 6). Subjects 407 and 408 also had TCR excision circle (TREC) measured, and only subject 408 had normal TREC levels (10,145 TREC copies/million CD3+ T cells; normal ≥ 6794) for her age. In subject 407, TREC levels were undetectable.

B cell function. The subjects’ ability to make IgM and IgA after GT was assessed by measuring serum immunoglobulin levels. At 12 months after GT, all but 3 subjects (subjects 401, 403, and 407) had normal levels of IgM (Figure 4A), whereas only 3 subjects (subjects 404, 408, and 410) had normal levels of IgA (Figure 4B). This outcome corresponded to the subjects’ B cell counts (subjects 404, 408, and 410 had B cells > 200 cells/mm3), suggesting that correction of humoral function required robust B cell reconstitution.

Figure 4 Serum immunoglobulin levels at 12 months or more after GT through subjects’ last recorded follow-up plotted according to age. Black horizontal bars represent the lower limit of normal range (34). (A) IgM. (B) IgA. (C) IgG. Black symbols represent last IgG level recorded while subject was receiving i.v. Ig RT. Blue symbols represent IgG levels after i.v. Ig RT was discontinued.

Subjects 402 and 404 had immune responses to the neoantigen bacteriophage ϕχ174 tested (21) and B cell studies performed (Table 3 and Supplemental Figures 7 and 8). Subject 402 (who remains on i.v. Ig replacement therapy [RT]) received a lower total CD34+ cell dose than subject 404 and responded to primary challenge with bacteriophage ϕχ174, but did not respond to secondary or tertiary challenge (Supplemental Figure 7A), whereas subject 404 responded to both primary and secondary challenges (Supplemental Figure 7B). When B cell subsets were measured, subject 402 exhibited a relative deficiency in memory B cells that had class switched to IgA and IgG subtypes compared with subject 404 (8.3% vs. 17.1% IgA and 8.3% vs. 43.7% IgG), despite having normal ratios of memory, mature, and immature B cells (Supplemental Figure 8).

Discontinuation of supportive medications. Subjects 402, 404, 405, 408, and 410 were able to discontinue all bacterial and fungal prophylactic medications, and within this group, subjects 404, 408, and 410 were able to also discontinue i.v. Ig RT (Table 3). Additionally, after discontinuing i.v. Ig RT, subjects 404, 408, and 410 were able to maintain total IgG levels at or near the normal range for their ages (Figure 4C). Ability to stop prophylactic medications correlated significantly with higher ADA enzyme activity (P = 0.001) and younger age at treatment (P = 0.003). However, ability to stop prophylactic medications did not significantly correlate with busulfan AUC during RIC (P = 0.110) or with CD34+ cell dose (P = 0.177). Ability to discontinue i.v. Ig RT also correlated significantly with higher ADA enzyme activity (P < 0.0001) and younger age at transplant (P = 0.008) and did not correlate significantly with busulfan AUC (P = 0.425) nor with CD34+ cell dose (P = 0.098). As a result of their good immune reconstitution, subjects 404 and 410 received and responded to immunizations with inactivated vaccines (diphtheria, tetanus, and pertussis [DTaP]; Table 3 and Supplemental Figure 9).

Quantification of VCN in peripheral blood. To assess the engraftment and persistence of ADA gene–modified CD34+ cells, peripheral blood samples were obtained at serial time points, genomic DNA was extracted, and the levels of gene marking in PBMCs and granulocytes (depleted of contaminating lymphocytes) were determined using Droplet Digital PCR (ddPCR, Bio-Rad) to quantify VCN. As expected, the 2 subjects (subjects 408 and 410) with the highest lymphocyte counts also had among the highest gene marking in PBMCs and granulocyte fractions (Figure 5, A and B). Subject 408 had VCN between 0.4 and 0.9 in PBMCs and up to 0.09 VCN in granulocytes (equivalent to 9% of cells, if at single copy/cell). Subject 410 had VCN between 0.5 and 1.1 in PBMCs and up to 0.26 VCN in granulocytes (26% equivalent). Gene marking in granulocytes was significantly higher in subjects who were able to stop prophylactic medications (P = 0.024) and i.v. Ig RT (P = 0.007) compared with subjects who were not. The remaining subjects had gene marking that was 2-fold or more lower in the granulocyte fractions compared with those seen in subjects 408 and 410.

Figure 5 VCN in peripheral blood cells at 6 months or more after GT through subjects’ last recorded follow-up. (A) PBMCs. (B) Granulocytes. Mean is shown as horizontal bars.

IL-7 and hBAFF levels. Lymphocyte homeostasis and survival are regulated, in part, by cytokine levels. For example, IL-7 is involved in T cell lymphopoiesis and homeostasis (22) and B cell–activating factor of the TNF family (BAFF) promotes B cell maturation and survival (23). We previously reported that serum IL-7 levels were inversely proportional to ALCs in subjects of a GT trial (15). Serum IL-7 levels were particularly increased in the first 4 months after GT after PEG-ADA had been withdrawn and prior to reconstitution of peripheral T lymphocytes. In the current trial, we again measured serum IL-7 levels in subjects and found a similar inverse relationship between IL-7 levels and T cell counts (Supplemental Figure 10A).

BAFF levels have been reported to be inversely correlated with peripheral B cell numbers (23). In the current study, we measured serum BAFF levels from subjects and found that BAFF inversely correlated with absolute B cell counts (Supplemental Figure 10B). T cells began to stabilize at approximately 5 months after GT (with concomitant lowering of IL-7 levels), whereas B cells began to stabilize later, at approximately 8 months, after GT (with concomitant lowering of BAFF levels). These results show that ADA-deficient subjects have the ability to respond to lymphopenia by regulating their cytokine levels.