Francesca Ferrua

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.

Update on gene therapy for adenosine deaminase-deficient severe combined immunodeficiency

Purpose of reviewThe present review describes the recent progress in the treatment of adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) using autologous gene-modified hematopoietic stem cells, comparing immune reconstitution with respect to allogeneic transplant and discussing differences with gene therapy for SCID-X1. Recent findingsSince 2000, more than 30 ADA-SCID patients have been treated with gene therapy worldwide, with successful outcome in most cases, consisting of progressive immune reconstitution, efficient systemic detoxification, and long-term multilineage engraftment. Gene therapy resulted in restoration of thymic activity and T-cell functions, although the kinetic of reconstitution was slower compared with standard bone marrow transplant. Unlike allogeneic transplant from alternative donors, survival after gene therapy is excellent. In comparison with SCID-X1, ADA-SCID gene therapy presents a better safety profile and engraftment of multilineage transduced stem/progenitor cells, thanks to the use of nonmyeloablative preconditioning. New approaches using safer integrating vectors are being developed, which may lead to safer and effective gene therapy for ADA-SCID and other genetic disorders. SummaryIn the last decade, gene therapy has been developed as a successful and safe alternative strategy for patients affected by ADA-SCID lacking a compatible sibling donor. The application of innovative vector technology might further improve its efficacy and safety profile.

Absence of VOD in paediatric thalassaemic HSCT recipients using defibrotide prophylaxis and intravenous Busulphan.

Hepatic veno‐occlusive disease (VOD) is a common complication of haematopoietic stem cell transplantation (HSCT), with reported incidences of 5–40% in children. Recently, defibrotide (DF) has been successfully used as prophylaxis and treatment of VOD. This study reports data on 63 human leucocyte antigen‐matched HSCT performed in 57 children affected by beta thalassemia at very high risk for developing VOD (liver fibrosis, iron overload, hepatitis C virus infections, busulphan‐based conditioning, methotraexate + ciclosporine). All patients received a busulphan‐based conditioning regimen, either orally (four HSCT) or intravenously (59 HSCT). All patients received oral DF (40 mg/kg per day, final dose) as VOD prophylaxis from median day −9 to median day +29. In order to overcome the lack of oral paediatric formulations, a galenic formulation was administered. DF was well tolerated. Only one patient fulfilled Seattle Criteria for VOD diagnosis. This patient had discontinued DF 6 d prior to VOD onset, due to high risk of haemorrhage. We concluded that oral defibrotide prophylaxis and i.v. busulphan safely abated VOD incidence in high‐risk patients who had undergone HSCT. A galenic preparation of oral DF also permits this treatment in low‐weight patients. Costs of DF prophylaxis are acceptable considering the reduced incidence of VOD.

Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID

Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of hematopoietic stem/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen has been a crucial factor in achieving stable engrafment of hematopoietic stem cells and therapeutic levels of ADA-expressing cells. Recent studies have demonstrated that gene therapy for ADA-SCID has favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Stem cell gene therapy combined with appropriate conditioning regimens might be extended to other genetic disorders of the hematopoietic system.

B-cell reconstitution after lentiviral vector–mediated gene therapy in patients with Wiskott-Aldrich syndrome

Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene–corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell–activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21−CD35− and CD21low B cells and a reduction in B cell–activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. Conclusions We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency.

BACKGROUND Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies. OBJECTIVES Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells. METHODS Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT). RESULTS We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles. CONCLUSION These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.

Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID

The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.

Gene therapy for Wiskott-Aldrich Syndrome.

The Wiskott-Aldrich Syndrome (WAS) is a monogenic X-linked primary immunodeficiency characterised also by thrombocytopenia, eczema, and a high susceptibility to develop tumours and autoimmunity. WAS patients have a severely reduced life expectancy, unless they undergo a successful HLA-matched haematopoietic stem cell (HSC) transplantation. However, several WAS patients lack a compatible donor and complications, such as autoimmunity, can arise in a significant fraction of HSC transplanted patients. Administration of WAS gene-corrected autologous HSC represents an alternative therapeutic approach, potentially applicable to all WAS patients. To this aim, several gene therapy approaches for WAS using initially γ-retroviral vectors (RVs) and subsequently HIV-based lentiviral vectors (LVs) have been developed. In the present review, we will first describe the results of the preclinical studies conducted in the murine model of WAS and then discuss the outcome of different phase I/II clinical trials using RV or LV- transduced HSC. Both gene therapy approaches led to restored WASP expression, correction of functional defects and clinical improvement. While RV-mediated gene therapy was associated with a high occurrence of leukaemia, results obtained in the first patients treated with LV-based HSC gene therapy indicate a safer risk-benefit profile.

Gene Therapy for Adenosine Deaminase Deficiency: A Comprehensive Evaluation of Short- and Medium-Term Safety

Loss of adenosine deaminase activity leads to severe combined immunodeficiency (ADA-SCID); production and function of T, B, and natural killer (NK) cells are impaired. Gene therapy (GT) with an autologous CD34+-enriched cell fraction that contains CD34+ cells transduced with a retroviral vector encoding the human ADA cDNA sequence leads to immune reconstitution in most patients. Here, we report short- and medium-term safety analyses from 18 patients enrolled as part of single-arm, open-label studies or compassionate use programs. Survival was 100% with a median of 6.9 years follow-up (range, 2.3 to 13.4 years). Adverse events were mostly grade 1 or grade 2 and were reported by all 18 patients following GT. Thirty-nine serious adverse events (SAEs) were reported by 15 of 18 patients; no SAEs were considered related to GT. The most common adverse events reported post-GT include upper respiratory tract infection, gastroenteritis, rhinitis, bronchitis, oral candidiasis, cough, neutropenia, diarrhea, and pyrexia. Incidence rates for all of these events were highest during pre-treatment, treatment, and/or 3-month follow-up and then declined over medium-term follow-up. GT did not impact the incidence of neurologic/hearing impairments. No event indicative of leukemic transformation was reported.