Fig. 1 Selective advantage of genetically modified PBL with restoration of ADA activity and T-cell functions following PEG-ADA discontinuation. a, From top to bottom. PEG-ADA and gene therapy: PEG-ADA dosages () and infusions of transduced PBL (). The patient presented at 3 mo of age with pneumonia, pulmonary subatelectasia, hepatomegaly, lymphopenia (130 cells/µl), low Ig levels and absent proliferative responses to mitogens, and PEG-ADA was started after the diagnosis of ADA-SCID. The patient is affected by a frameshift ADA mutation (462delG) that generates a mutated protein with no detectable activity in E. coli10. The ADA-SCID gene therapy protocol was approved by the Ethical Committee of Scientific Institute HS Raffaele in 1990 and by the National Committee for Bioethics in 1991 (ref. 11). The retroviral vector GIADAl encodes the ADA cDNA and the NeoR marker gene, and has an increased titer and stability of expression over the ones used previously1 (data not shown). Gene transfer into PBL, cryopreservation, quality controls and infusions were performed as described1. Starting at 19 mo, the patient received 2.06× 109 engineered PBL in 13 infusions until PEG-ADA tapering-down (3 y, 10 mo). Frequency of transduced PBL: proportion of vector-positive PBL measured by quantitative real time PCR for NeoR (). PBL ADA: Intracellular ADA activity measured in PBL () by high-performance capillary electrophoresis (HPCE). BM MNC ADA activity also raised from 20–90 to 370 U (nmol/mg/h) after discontinuation, and ADA activity in PB T-cell lines was in normal range. RBC dAXP: dAXP toxic metabolites in RBCs measured by HPCE (). CD3+ lymph counts: absolute counts of CD3+ T cells () in PB; the dashed middle line indicates the average counts observed in the previous 3 y of PEG-ADA therapy±sd (dotted lines). Anti-CD3 responses: PBL proliferative responses to soluble anti-CD3 mAb () expressed as counts per minute (cpm). Before discontinuation, responses to anti-CD3 mAb (stimulation index (SI): 10) and PHA (SI: 24) were severely depressed. After discontinuation, responses to anti-CD3 mAb alone (SI: 370), or in combination with anti-CD28 mAb (SI: 550), PWM (SI: 129), PHA (SI: 311), and alloantigens (SI: 94), were all in normal range and the patient showed a modest but significant in vitro response to TT, after an in vivo boost (medium: 900±200 cpm; TT: 2100±400). b, Antibody responses to bacteriophage ΦX174. Immunization with bacteriophage (arrows) was performed 3 times as described6. Patient’s antibody responses () are represented as Kv (titer). Anti-ΦX174 antibodies were of the IgM isotype after the first dose. The proportion of IgG isotype antibodies within total anti-ΦX174 antibodies, after the second and third immunization, is shown above the graphs. The range of responses of normal controls is indicated by the dashed lines. Two historical ADA-SCID patients who underwent HLA-identical (O) and HLA-non identical (❇) BMT (ref. 6) are shown for comparison. a b