Adenosine deaminase (ADA) is a ubiquitous enzyme that is crucial for the metabolism of adenosine and 2-deoxyadenosine. Impaired function of ADA leads to accumulation of purine metabolites that are particularly toxic to the rapidly proliferating bone marrow cells and thymocytes. Inherited ADA defects account for 15–20% of all causes of SCID, and some Canadian populations such as the Mennonite and Canadian First Nations seem to have increased frequency of ADA deficiency [
7]. The implementation of newborn screening (NBS) for severe immune deficiency, and the inclusion of ADA among the conditions tested in some of the NBS algorithms, such as the one implemented in Ontario, are expected to reveal the true incidence of ADA deficiency. T, B and Natural Killer (NK) cells dysfunction is often present in ADA-deficient patients in infancy. In addition, patients may suffer from alveolar proteinosis [
8], diverse neuro-developmental abnormalities [
9] as well as bone and cartilage malformations [
10]. Since the mid-1980, weekly injections of polyethylene glycol-modified bovine ADA (PEG-ADA) have been used to remove the toxic purine metabolites, improve T and B cell functions and correct for some of the non-immunologic abnormalities [
11]. However, PEG-ADA does not cure ADA deficiency, as it is effective in only 80% of patients and the immune recovery often diminishes over time [
12]. Moreover, the cost of PEG-ADA treatment (>$US 100,000/year) restricts its availability for many patients. Nevertheless, health Ministries of some Canadian provinces, such as Ontario and British Columbia have reluctantly sponsored PEG-ADA treatment. HSCT from an unaffected HLA identical sibling donor without any chemotherapy has been shown to restore immunity in ADA deficient patients, and is currently considered the treatment of choice [
13]. However, HLA identical sibling donors are available for only a minority of patients while the use of HLA-mismatched related or unrelated donors is associated with significant morbidity and mortality [
14]. These disappointing results of HSCT, together with the early availability of the ADA gene sequence, prompted the investigation of GT for ADA deficiency.
Initial GT trials for ADA deficiency, performed already in early 1990, delivered the ADA gene into T lymphocytes or umbilical cord blood/bone marrow progenitor cells using the murine gamma-retroviral vector. Similar to the protocols used for allogeneic HSCT, autologous GT were done in ADA-deficient patients without the use of cytoreductive conditioning. This was based on the assumption that ADA-proficient cells would have a “survival advantage” over the original ADA-deficient cells. Yet, for ethical reasons, patients enrolled in this trial were given PEG-ADA, which negated the survival advantage of the gene-corrected cells. Hence, despite detection of ADA-corrected cells in the host, inadequate amount of cells persisted to confer significant clinical benefit. It took almost a decade until the group in Italy, led by Drs. Aiuti and Naldini reintroduced non-myeloablative doses of busulfan or melphalan without PEG-ADA, into the GT trials for ADA deficiency [
15]. Together with improved gene transduction techniques and the use of MLV-derived replication-deficient vector to deliver the ADA cDNA into cells, the Milan group was able to achieve lasting ADA expression in cells. This resulted in humoral and cellular immune reconstitution, decrease in susceptibility to infections [
16] and correction of the bone abnormalities [
17]. One Canadian patient who participated in this study is now almost 10 years after receiving GT and is clinically well. Subsequent studies at Great Ormond Street in the UK, at The National Human Genome Institute, the Children’s Hospital Los Angeles, and later the UCLA Mattel Children’s Hospital as well as Japan demonstrated the critical role of non-myeloablative pre-transplantation conditioning in gene therapy for ADA SCID [
18‐
20]. Recently, long-term follow-up (range, 2.3–13.4 years) of the 18 ADA-deficient patients who received ADA GT in Milan revealed that all survived [
21]. PEG-ADA was resumed in 3 patients, of which 2 later received HSCT from HLA identical sibling donors that were not available prior to GT. The relatively short follow-up of the ADA-deficient patients who received GT in England and North America precludes direct comparison with the Milan outcome, yet the overall results and safety of all these studies are encouraging. Indeed the success of the Milan ADA GT led to commercialization of the viral vector by GlaxoSmithKline (GSK) as Strimvelis™, which recently received marketing authorization in Europe. The impact of such move on ADA GT practicalities, including cost for patients and availability in North America are still not clear. Impressively, and in contrast to GT trials for other PID described below, all ADA-deficient patients who received GT in the USA and Europe survived, and none experienced abnormal clonal expansions or leuko/lympho-proliferative disorders. Although analyses of retroviral vector integrations in patients’ cells demonstrated insertion near proto-oncogenes sites (including LMO2) similar to those found in other PID trials, there was no skewing of the T cell repertoire or clonal selection/expansion in vivo. Despite the lack of insertional genotoxicity with gamma-retroviruses in ADA GT, concerns regarding leukemogenesis have led to the development of SIN LV vectors. Studies using these vectors for ADA deficiency are currently being completed in England (ClinicalTrials.gov Identifier: NCT01380990) and the USA (ClinicalTrials.gov Identifier: NCT01852071). Deletion of proteins from the vector packaging plasmids and the SIN mechanism have made their use safer. Moreover, as LV vectors can transduce non-dividing cells, such as quiescent hematopoietic stem cells, it is postulated that the efficacy of gene delivery into the very early stem cells will be improved. Interestingly, based on experiments in murine models, the current ADA SIN LV trials continue the administration of PEG-ADA for 30 days after the GT. More than 30 ADA-deficient patients have been treated with the SIN LV vector. Immune reconstitution has been achieved with no vector-related complications, although follow-up period for most patients is still short (<3 years). Several ADA-deficient patients from Quebec and Ontario, who lacked HLA-matched sibling donors, have already received GT under this protocol. Although GT is still very expensive (more than $ US 200,000/patient), the cost is less than the life-long continuation of PEG-ADA and possibly even less than an HLA-mismatched HSCT that is often associated with prolonged admissions and complications. Accordingly, the Ministry of Health in several Canadian provinces have approved the out-of-county expenses. After the control of infections and PEG-ADA administration, and coordination by the Canadian referring team with the centers performing the GT, patients typically spend 7–10 days at the GT center. During this period, the patients’ bone marrow cells are harvested, CD34 expressing cells are selected and transduced with the viral vector, busulfan is administered, and the gene-corrected cells are infused. Patients who are clinically well can return to the referral center prior to the development of chemotherapy-induced neutropenia. Close monitoring and frequent follow-ups are coordinated between the referring teams and the GT centers. In the future, shipping the patients’ bone marrow to designated centers might prevent the need for them to commute, further simplifying GT and reducing its costs. Indeed, researchers in the US and UK have began investigating the effects of cryopreservation of the cells on the success of LV GT for ADA-deficient patients (NCT02999984).