Eyal Grunebaum

Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency

Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed outcome of HCT in 106 patients with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family donors (MSDs, MFDs) had significantly better overall survival (86% and 81%) in comparison with HCT from matched unrelated (66%; P < .05) and haploidentical donors (43%; P < .001). Superior overall survival was also seen in patients who received unconditioned transplants in comparison with myeloablative procedures (81% vs 54%; P < .003), although in unconditioned haploidentical donor HCT, nonengraftment was a major problem. Long-term immune recovery showed that regardless of transplant type, overall T-cell numbers were similar, although a faster rate of T-cell recovery was observed after MSD/MFD HCT. Humoral immunity and donor B-cell engraftment was achieved in nearly all evaluable surviving patients and was seen even after unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and show that, if patients survive HCT, long-term cellular and humoral immune recovery is achieved.

Omenn syndrome is associated with mutations in DNA ligase IV

2003;197:527-35. 5. Sun X, Becker-Catania SG, Chun HH, Hwang MJ, Huo Y, Wang Z, et al. Early diagnosis of ataxia-telangiectasia using radiosensitivity testing. J Pediatr 2002; 140:724-31. 6. Chun HH, Sun X, Nahas SA, Teraoka S, Lai CH, Concannon P, et al. Improved diagnostic testing for ataxia-telangiectasia by immunoblotting of nuclear lysates for ATM protein expression. Mol Genet Metab 2003;80:437-43. 7. Nahas SA, Butch AW, Du LT, Gatti RA. Rapid-flow cytometry-based SMC1 phosphorylation assay for identification of ataxia-telangiectasia homozygotes and heterozygotes. Clin Chem 2008. In press. 8. Bener A, Hussain R, Teebi AS. Consanguineous marriages and their effects on common adult diseases: studies from an endogamous population. Med Princ Pract 2007;16:262-7. 9. Nowak-Wegrzyn A, Crawford TO, Winkelstein JA, Carson KA, Lederman HM. Immunodeficiency and infections in ataxia-telangiectasia. J Pediatr 2004;144: 505-11. 10. Elzaouk L, Moelling K, Pavlovic J. Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model. Exp Dermatol 2006;15:865-74.

Defining combined immunodeficiency

BACKGROUND Although the extreme condition of typical profound T-cell dysfunction (TD), severe combined immunodeficiency (SCID), has been carefully defined, we are currently in the process of better defining less typical T-cell deficiencies, which tend to present with autologous circulating T-cell combined immunodeficiency (CID). Because autologous cells might interfere with the outcome of bone marrow transplantation, protocols usually include conditioning regimens. Therefore it is important to define the numbers of autologous cells usually detected in patients with CID versus those with SCID. OBJECTIVES We sought to determine the number of circulating T cells in patients with SCID as opposed to those with CID, to study their function, and to evaluate their possible detection during newborn screening using T-cell receptor excision circle (TREC) analysis. METHODS Numbers of circulating CD3(+) T cells (as determined by means of flow cytometry), in vitro responses to PHA, and TREC levels, all measured at presentation, were compiled from the research charts of the entire cohort of patients followed prospectively for T-cell immunodeficiency at the Hospital for Sick Children. Clinical data were ascertained retrospectively from the patients hospital charts. RESULTS One hundred three patients had CD3(+) determinations, and 80 of them had a genetic diagnosis. All patients considered to have typical SCID had CD3(+) T-cell counts of fewer than 500 cells/μL. Some variability was observed among different genotypes. In vitro responses to PHA were recorded in 88 patients, of whom 68 had a genetic diagnosis. All patients with low CD3(+) T-cell numbers (<500 cells/μL) also had markedly decreased responses to PHA (typical SCIDs). However, responses ranged widely in the groups of patients with TD who had more than 500 CD3(+) autologous circulating T cells per microliter. Although patients with Omenn syndrome and ζ chain-associated protein, 70 kDa (ZAP70), and purine nucleoside phosphorylase (PNP) deficiencies had low responses, patients with the p.R222C mutation in the IL-2 receptor γ(IL2RG) gene as well as IL-10 receptor and CD40 ligand deficiencies had normal or near-normal mitogen responses. Finally, 51 patients had TREC levels measured. All patients with typical SCID, Omenn syndrome, and ZAP70 deficiency had low TREC levels. In contrast, patients with mutations in forkhead box protein 3 (FOXP3), CD40 ligand (CD40L), and IL-10 receptor α(IL10RA), as well as patients with the p.R222C mutation in the IL2RG gene, had normal TREC levels. CONCLUSION Patients with typical SCID can be defined as having fewer than 500 circulating CD3(+) T cells. Most patients with autologous T cells still have profound TD, as defined by reduced in vitro function and thymus output. Some patients with conditions including TD have normal TREC levels and will therefore not be detected in a TREC-based newborn screening program.

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

Defects in purine nucleoside phosphorylase (PNP) enzyme activity result in abnormal nucleoside homeostasis, severe T cell immunodeficiency, neurological dysfunction, and early death. Protein transduction domain (PTD) can transfer molecules into cells and may help restore PNP activity in cases of PNP deficiency. However, long-term use of PTD to replace enzymes in animal models or patients has not previously been described. We fused human PNP to the HIV-TAT PTD and found that the fusion with TAT changed the retention and distribution of PNP in PNP-deficient mice. TAT induced rapid intracellular delivery of PNP into tissues, including the brain, prevented urinary excretion of PNP, and protected PNP from neutralizing antibodies, resulting in significant extension of the enzymes biological activity in vivo. Frequent TAT-PNP injections in PNP-deficient mice corrected the metabolic disorder and immune defects with no apparent toxicity. TAT-PNP remained effective over 24 weeks of treatment, resulting in continued improvement in immune function and extended survival. Our data demonstrate that TAT changes the properties of PNP in vivo and that long-term intracellular delivery of PNP by TAT corrects PNP deficiency in mice. We provide evidence to promote further use of PTD to treat diseases that require repeated intracellular enzyme or protein delivery.

Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency

Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34(+)-enriched cell fraction that contained CD34(+) cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3(+), CD4(+), and CD8(+)), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and childrens growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

Recent advances in understanding and managing adenosine deaminase and purine nucleoside phosphorylase deficiencies.

Purpose of the reviewTo review the recent advances in the understanding and management of the immune and nonimmune effects of inherited adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies. Recent findingsAbnormal thymocyte development and peripheral T-cell activation in ADA-deficient and PNP-deficient patients cause increased susceptibility to infections and immune dysregulation. The impaired purine homeostasis also damages many other cell types and tissues. Animal studies suggest that defects in surfactant metabolism by alveolar macrophages cause the pulmonary alveolar proteinosis commonly seen in ADA-deficient infants, while toxicity of purine metabolites to cerebellar Purkinje cells may lead to the ataxia frequently observed in PNP deficiency. Patients’ outcome with current treatments including enzyme replacement and stem cell transplantations are inferior to those achieved in most severe immunodeficiency conditions. New strategies, including intracellular enzyme replacement, gene therapy and innovative protocols for stem cell transplantations hold great promise for improved outcomes in ADA and PNP deficiency. Moreover, newborn screening and early diagnosis will allow prompt application of these novel treatment strategies, further improving survival and reducing morbidity. SummaryBetter understanding of the complex immune and nonimmune effects of ADA and PNP deficiency holds great promise for improved patients’ outcome.

Adenosine deaminase deficiency can present with features of Omenn syndrome

However, FIP1L1-PDGFRA–positive patients with CEL may also respond to anti–IL-5 antibody treatment, perhaps because the FIP1L1-PDGFRA gene fusion alone is not sufficient and may require IL-5 to induce CEL. To understand whether the eosinophil differentiation process can be stopped by neutralizing IL-5, we administrated 4 intravenous injections of an anti–IL-5 antibody (mepolizumab, 750 mg each) with intervals of 2 weeks. The patient did not respond with a detectable decrease of his eosinophil count as a consequence of this treatment (Fig 1, B), suggesting that the neutralization of IL-5 by mepolizumab was not sufficient to stop the eosinophil differentiation process. The antibody treatment had also no influence on the relative distribution of T subpopulations and B cells or the expression of markers indicating their activation. Although single imatinib treatment had no effect (Fig 1, A), we thought a combination of mepolizumab and imatinib mesilate might be able to reduce eosinophil numbers. Therefore, we treated the patient with 400 mg imatinib daily, starting 2 weeks after the last mepolizumab infusion. At this time, mepolizumab still exhibits antieosinophil activity in hypereosinophilic patients. Again, no effect of imatinib on eosinophil numbers or symptoms was seen (Fig 1, B). The patient was subsequently substituted with 25 mg hydrocortisone. He died of heart failure in 2006. Because imatinib resistance might be caused by mutations, we analyzed the primary structure of the entire FIP1L1-PDGFRA fusion gene. To this end, the complete FIP1L1-PDGFRA cDNA was amplified by RT-PCR and sequenced. We identified 2 mutations in PDGFRA sequence (accession no. M22734: T1949C and T2034C), resulting in 2 amino acid changes within the kinase domain: S601P and L629P (Fig 1, C and D). No homologous mutations have been described in the BCR-ABL fusion oncoprotein causing imatinib resistance in chronic myeloid leukemia. Therefore, the exact molecular mechanisms through which these newly identified mutations likely cause imatinib resistance remain to be investigated. It should be noted that no mRNA was available before the patient received the drug for the first time. Therefore, the possibility exists that the imatinib treatment may have influenced the mutation. Nevertheless, mutations within the kinase domain of oncoproteins are common causes of drug resistance. Taken together, patients with CEL with the FIP1L1-PDGFRA gene fusion may be resistant to imatinib mesilate because of mutations within the tyrosine kinase domain of the chimeric protein, and increased IL-5 levels in blood do not predict an antieosinophil response to anti–IL-5 antibody therapy. The identification of novel drug-resistant variants may help to develop the next generation of target-directed compounds for CEL and other leukemias.

Pulmonary alveolar proteinosis in patients with adenosine deaminase deficiency

BACKGROUND Inherited defects in the function of adenosine deaminase (ADA) cause severe combined immunodeficiency (SCID) and affect many other cells and tissues. OBJECTIVES We sought to characterize the frequency and features of pulmonary alveolar proteinosis (PAP) in patients with ADA deficiency. METHODS Clinical and laboratory features of all patients with SCID caused by ADA deficiency in a single center were analyzed. Bronchoalveolar lavage (BAL) fluid and lung biopsy specimens were stained with hematoxylin and eosin and periodic acid-Schiff, visualized by means of electron microscopy, and studied for associated infections. As a control group, BAL fluid and biopsy specimens from 22 patients with SCID caused by other genetic abnormalities were similarly assessed. RESULTS Among 16 consecutive patients with ADA deficiency, 7 had BAL fluid containing periodic acid-Schiff-positive surfactant-like material with macrophages engulfing degenerating lamellar bodies and/or lung biopsy specimens with alveolar spaces filled with homogeneous granular eosinophilic material and large macrophages. The lung pathology was typical of PAP. Identification of various pathogens coincided with PAP in 3 of these patients. We have diagnosed PAP among patients with ADA deficiency more commonly in the last 10 years than previously (P= .05), likely reflecting increased awareness of this condition. There were no significant differences in the clinical or immunologic characteristics between patients with ADA deficiency with or without PAP. Similar findings of PAP were not found among patients with SCID caused by other genetic abnormalities (P= .001). ADA coupled to polyethylene glycol or allogeneic hematopoietic stem cell transplantation rapidly corrected this pulmonary complication. PAP seems to have contributed to the death of only 1 patient with ADA deficiency. CONCLUSIONS ADA deficiency predisposes to the development of PAP, which could be reversed after enzyme replacement or transplantation.

Two novel mutations in a purine nucleoside phosphorylase (PNP)-deficient patient

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive disease, which presents clinically as severe combined immunodeficiency (SCID). We report here two novel mutations in the PNP gene that result in SCID phenotype, in a single patient. The maternal‐derived allele carries a C to T transition in exon 2 resulting in a premature stop codon at amino acid 57. The paternal‐derived mutation is a G to A transition at position +1 in intron 3, causing a complete skipping of exon 3 and a reading frameshift at the exon 2–exon 4 junction. The predicted polypeptide encoded by the aberrantly spliced mRNA terminates prematurely after only 89 amino acids. Both mutations predict severely truncated proteins resulting in a complete deficiency of PNP enzymatic activity, yet the development of profound immunodeficiency in this patient is greatly delayed.