Memet Aker

Gene Therapy for immunodeficiency due to Adenosine Deaminase Deficiency

BACKGROUND We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)

Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

T-cell-depleted allogeneic bone marrow transplantation for acute leukaemia using Campath-1 antibodies and post-transplant administration of donor's peripheral blood lymphocytes for prevention of relapse

One hundred and forty‐six patients with acute leukaemia (81 with ANLL and 65 with ALL) received allogeneic bone marrow transplantation from their fully matched siblings. 121 patients underwent T‐cell depletion (TCD) using Campath 1 monoclonal rat anti‐human lymphocyte (CDw52) antibodies; 67 with Campath 1M and 54 with Campath 1G isotypes. Patients were conditioned for transplant using either total body irradiation combined with chemotherapy (125 patients) or busulfan and cyclophosphamide (21 patients). 112 recipients of T‐cell depleted allografts received in addition total lymphoid irradiation (TLI) for prevention of rejection. Engraftment of neutrophils (>0.5 × 109/l) and platelets (>25 × 109/l) occurred on days 15 and 18, and on days 18 and 20 in recipients of Campath 1M and Campath 1G treated marrows respectively. Rejection was documented in 6.8% of T‐cell depleted transplants. Leukaemia relapse‐free survival at 2 years was 83% for patients transplanted in first CR, 76% in second CR (P2= 0.34) and 42% in advanced leukaemia (P2= 0.009). 81 marrow recipients, 38 with Campath 1M and 43 with Campath 1G treated marrow, received post‐transplant graded increments of donors peripheral blood lymphocytes (PBL) to induce graft‐versus‐leukaemia (GVL) effects. Administration of donors PBL was associated with clinically significant GVHD and with decreased relapse rate especially in patients with ALL. Our data suggest that in patients receiving marrow allografts depleted of T cells by Campath 1 monoclonal antibodies, rejection can be reduced by adequate pregrafting immunosuppression. In patients with advanced disease, post‐transplant cell‐mediated immunotherapy (CMI) using donors PBL may be beneficial; however, further studies are needed to define the optimal schedule of CMI for safe and effective prevention of relapse following TCD bone marrow transplantation in malignant haematological diseases.

Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation

OBJECTIVE Matched unrelated bone marrow transplantation (BMT) for patients with hematological malignancies is associated with a high incidence of transplant-related complications due to high doses of chemoradiotherapy administered pre-BMT to ensure engraftment. The aim of this study was to investigate the feasibility of low-intensity conditioning for BMT from matched unrelated donors. MATERIALS AND METHODS Sixteen patients with hematologic malignancies underwent non-T-cell-depleted BMT following a low-intensity conditioning regimen consisting of fludarabine monophosphate 30 mg/m(2)/day for 6 days, busulfan 4 mg/kg/day for 2 days, anti-T lymphocyte globulin 10 mg/kg/day for 4 days. Seven of the patients suffered from chronic myelogenous leukemia, four from acute lymphoblastic leukemia, four from acute myelogenous leukemia, and one from Ki-1 non-Hodgkins lymphoma. Three of the patients had secondary leukemia and two were post-autologous BMT (ABMT). All patients were transplanted from fully matched unrelated donors. RESULTS Fifteen of the 16 patients had 100% donor chimerism; no graft rejection was observed. None of the patients developed >Grade II veno-occlusive disease, sepsis, multiorgan failure, or renal or pulmonary toxicity. Four patients died posttransplant; one of thrombocytopenia and severe hemorrhagic cystitis, one of central nervous system toxicity, one of Grade IV graft-vs-host disease, and one following relapse (9 months post-BMT). Survival and disease-free survival at 36 months are 75% (95% confidence interval 46-90%) and 60% (95% confidence interval 30-80%), respectively. CONCLUSION These results indicate that low-intensity conditioning is sufficient to ensure stable engraftment of bone marrow grafts in a matched unrelated setting.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

Significance Telomeres protect the ends of eukaryotic chromosomes. Telomeres shorten with age and serve as a biological clock that limits cell proliferation. Excessive telomere shortening accelerates aging, but telomere elongation may facilitate cancer. We found inherited mutations in the regulator of telomere elongation helicase 1 (RTEL1), which cause Hoyeraal–Hreidarsson syndrome, a fatal disease characterized by accelerated telomere shortening, immunodeficiency, and developmental defects. Introducing a normal RTEL1 gene into affected cells prevented telomere shortening and extended their lifespan in culture. The telomere defects, genomic instability, and growth arrest observed in RTEL1-deficient cells help in our understanding the central roles of telomeres in aging and cancer. Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.

Fludarabine-based protocol for human umbilical cord blood transplantation in children with Fanconi anemia.

PURPOSE A novel conditioning regimen of fludarabine monophosphate (FLM), anti-T-lymphocyte globulin (ATG), and low-dose cyclophosphamide with no irradiation for human umbilical cord blood transplantation (HUCBT) for the treatment of Fanconi anemia (FA) is described. PATIENT AND METHODS A 12-year-old girl with FA received a human umbilical cord blood transplant from a fully matched sibling donor. After the HUCBT, the patient was given granulocyte colony stimulating factor in combination with erythropoietin. Pretransplant conditioning consisted of FLM (30 mg/m2/d) from day -10 to day -5, cyclophosphamide (10 mg/kg/d) on day -7 and -6, and rabbit ATG (ATG-Frasenius, 10 mg/kg/d) from day -4 to day -1. Cyclosporin A (3 mg/kg/d) was administered from day -1 as graft-versus-host disease prophylaxis. Cord blood from a sibling donor was used as a source of hematopoietic stem cells. RESULTS Engraftment was normal and sustained. The regimen was well tolerated with very mild toxicity and no major transplant-related complications or >grade II graft-versus-host disease. Chimerism was 100% donor origin as determined by restriction fragment length polymorphism. CONCLUSIONS It is possible to achieve sustained engraftment and only mild toxicity in FA after HUCBT with a conditioning regimen of FLM, ATG, and cyclophosphamide with no irradiation. These preliminary results with this novel conditioning protocol are encouraging and should be evaluated in a larger group of patients with FA undergoing HUCBT.

Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for β-thalassemia major

PURPOSE Donor lymphocyte infusion (DLI) was used to reverse relapse after allogeneic bone marrow transplantation (BMT) in a patient with beta-thalassemia major. PATIENTS AND METHODS The patient with unstable mixed chimerism after BMT was treated with graded increments of donor lymphocytes (10(5) T cells/kg to 5 x 10(7) T cells/kg) to displace residual hematopoietic host cells. RESULTS DLI resulted in complete donor-derived reconstitution of the hematopoietic compartment. The patient developed mild graft-versus-host disease (GVHD) that could be controlled by steroid treatment. CONCLUSIONS This case report shows that DLI can effectively eradicate host stem cells in mixed chimeras after BMT in nonmalignant hematopoietic diseases.

Allogeneic stem cell transplantation for severe acquired aplastic anaemia using a fludarabine-based preparative regimen.

We reviewed our experience in the treatment of 13 patients with severe acquired aplastic anaemia, using a newly developed non‐myeloablative regimen consisting of fludarabine (total dose 180 mg/m2), cyclophosphamide (total dose 120 mg/kg), and antithymocyte globulin (total dose 40 mg/kg). All except one patient received multiple transfusions and had failed prior immunosuppressive treatment. Twelve out of 13 patients achieved sustained engraftment. One patient was not evaluable for engraftment because of early death on day +10. None of the patients developed graft failure. Mucositis of mild‐to‐moderate severity was the only observed regimen‐related toxicity. The cumulative incidence of acute graft‐versus‐host disease (GvHD) grade II–IV and III–IV was 8·3% and 0%, respectively. With a median follow‐up period of 45 months, the 5‐year overall survival probability was 84%. Eight out of 11 surviving patients have been followed for more than 1 year and only one developed limited chronic GvHD. All patients enjoy a normal life style, with a Karnofsky score of 100%, and all except three, followed for 3, 5 and 6 months respectively, are free of any immunosuppressive medication. The results of this study look promising, while prospective clinical trials may be required to confirm the benefits of this regimen as an alternative to existing protocols.

Kindlin-3 is required for the stabilization of TCR-stimulated LFA-1:ICAM-1 bonds critical for lymphocyte arrest and spreading on dendritic cells

Kindlin-3 is a key lymphocyte function-associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3-null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3-null T lymphocytes failed to trigger the robust LFA-1-mediated T-cell spreading on ICAM-1 and ICAM-1-expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3-null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, β I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1-driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3-null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.