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Objective: We here describe treatment outcomes in two adenosine deaminase (ADA)-deficiency patients (pt) who received stem cell gene therapy (SCGT) with no cytoreductive conditioning. As this protocol has features distinct from those of other clinical trials, its results provide insights into SCGT for ADA deficiency., Patients and Methods: Pt 1 was treated at age 4.7 years, whereas pt 2, who had previously received T-cell gene therapy, was treated at age 13 years. Bone marrow CD34(+) cells were harvested after enzyme replacement therapy (ERT) was withdrawn; following transduction of ADA cDNA by the γ-retroviral vector GCsapM-ADA, they were administered intravenously. No cytoreductive conditioning, at present considered critical for therapeutic benefit, was given before cell infusion. Hematological/immunological reconstitution kinetics, levels of systemic detoxification, gene-marking levels, and proviral insertion sites in hematopoietic cells were assessed., Results: Treatment was well tolerated, and no serious adverse events were observed. Engraftment of gene-modified repopulating cells was evidenced by the appearance and maintenance of peripheral lymphocytes expressing functional ADA. Systemic detoxification was moderately achieved, allowing temporary discontinuation of ERT for 6 and 10 years in pt 1 and pt 2, respectively. Recovery of immunity remained partial, with lymphocyte counts in pts 1 and 2, peaked at 408/mm(3) and 1248/mm(3), approximately 2 and 5 years after SCGT. Vector integration site analyses confirmed that hematopoiesis was reconstituted with a limited number of clones, some of which were shown to have myelo-lymphoid potential., Conclusions: Outcomes in SCGT for ADA-SCID are described in the context of a unique protocol, which used neither ERT nor cytoreductive conditioning. Although proven safe, immune reconstitution was partial and temporary. Our results reiterate the importance of cytoreductive conditioning to ensure greater benefits from SCGT.

In this case report, we describe successful BMT with RIC in a patient with delayed-onset ADA deficiency. A three-yr-old Japanese boy was diagnosed with delayed-onset ADA deficiency because of recurrent bronchitis, bronchiectasia, and lymphopenia. In addition, autoimmune thyroiditis and neutropenia were present. At four yr of age, he underwent BMT with a RIC regimen, including busulfan and fludarabine, from an HLA-identical healthy sister. Engraftment after BMT was uneventful without GVHD. Decreased ADA levels in blood immediately increased following BMT, and the patient was disease-free 13 months after BMT. These results suggest that BMT with RIC may sufficiently restore immune regulation in delayed-onset ADA deficiency. A longer follow-up period is needed to confirm these observations., (© 2012 John Wiley & Sons A/S.)

A four year-old boy with adenosine deaminase (ADA-) deficient severe combined immunodeficiency(SCID) receiving PEG-ADA was treated under a gene therapy protocol targeting peripheral blood lymphocytes (PBLs) in 1995. After eleven infusions of autologous PBLs transduced with retroviral vector LASN encoding ADAcDNA, he exhibited increased levels of the CD8+ T lymphocytes, serum immunoglobulin, specific antibodies and delayed type hypersensitivity skin tests. Follow-up studies also provided evidence of long-term persistence and function of transduced PBLs with improvement in the immune function. However, the therapeutic effect of this gene therapy has been difficult to assess because of the concomitant treatment of PEG-ADA. Two ADA-SCID patients have been currently treated with autologous bone marrow CD34+ cells engineered with a retroviral vector GCsapM-ADA after discontinuation of PEG-ADA. The restoration of intracellular ADA enzymatic activity in lymphocytes and granulocytes resulted in correction of the systemic toxicity and liver function in the absence of PEG-ADA treatment. Both patients are at home where they are clinically well, and they do not experience adversed effect, with follow up being 12 months after CD34+ cells gene therapy.

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.

Introduction: Adenosine deaminase (ADA) deficiency is an ultrarare inherited purine metabolism disorder characterized by severe combined immunodeficiency. Elapegademase‐lvlr is a new pegylated recombinant bovine ADA used in enzyme‐replacement therapy (ERT) for ADA deficiency. Therefore, replacement with the new drug may eliminate the infectious risks associated with the currently used bovine intestinal‐derived product, pegademase. Methods: We conducted a multicenter, single‐arm, open‐label, phase 3, and postmarketing clinical study of elapegademase for patients with ADA deficiency. The following biochemical markers were monitored to determine an appropriate dose of elapegademase: the trough deoxyadenosine nucleotide (dAXP) level ≤0.02 μmol/mL in erythrocytes or whole blood and the trough serum ADA activity ≥1100 U/L (equivalent to plasma levels ≥15 μmol/h/mL) indicated sufficient enzyme activity and detoxification as efficacy endpoints and monitored adverse events during the study as safety endpoints. Results: A total of four patients (aged 0–25 years) were enrolled. One infant patient died of pneumonia caused by cytomegalovirus infection whereas the other three completed the study and have been observed in the study period over 3 years. The infant patient had received elapegademase at 0.4 mg/kg/week until decease and the others received elapegademase at maximum doses of 0.3 mg/kg/week for 164–169 weeks. As a result, all four patients achieved undetectable levels of dAXPs together with sufficient enzyme activity, increased T and B cell numbers, and slightly elevated and maintained IgM and IgA immunoglobulin levels. Serious adverse events occurred in three patients, all of which were assessed as unrelated to elapegademase. Conclusions: This study showed that elapegademase had comparable safety and efficacy to pegademase as ERT for ADA deficiency by demonstrating stable maintenance of sufficient ADA activity and lowering dAXP to undetectable levels, while no drug‐related adverse events were reported (Trial registration: JapicCTI‐163204). [ABSTRACT FROM AUTHOR]