Severe Combined Immune Deficiency Patients Research Articles

Human Pro-T-Cell Manufacturing in Vitro Is a Safe Procedure for Hematopoietic Stem Cell Transplantation with Delayed T-Cell Reconstitution: Interim Results from 2 Different Clinical Trials

Introduction: Alternative donor Hematopoietic stem cell transplantation (HSCT) strategies are followed by a prolonged period of immune deficiency due to delayed T-cell reconstitution. Either Umbilical Cord (CB) blood transplant or CD34+ selected haplo-transplant provided similar T-cell lymphocytes (CD3+ and CD4+) recoveries at any time point, and recipients had significantly fewer CD4+ T-cells during the first six months compared to HLA-matched donors. The GMP generation of Human T lymphoid Progenitors (HTLP) that are competent to rapidly become in vivo differentiated functional and thymic-educated T-cells may overcome the post-transplant delayed immune reconstitution. Method: HTLP safety and efficacy to accelerate immune reconstitution is currently evaluated in 2 multicentric and single-arm clinical trials: after umbilical cord blood in adult hematologic malignancy (Eudract 2019-004883-23) and after partially HLA compatible CD34+ immune-selected allogeneic hematopoietic stem cell transplantation in Severe Combined Immune Deficiency (SCID) patients (Eudract 2018-001029-14). Results: We enrolled and treated six patients, 4 SCID and 2 adults with malignancy, with an average follow-up of 8.00 (range, 1.60 to 37.7) months. HTLP cell product was infused between D7 to D14 post-HSCT for SCID patients after ensuring the decrease of the ATG level while meaning for the adult patient; the expanded pro-T cells were injected at D0. The Pro-T manufacturing process allows to robustly expand CD34+ cells to HTLP with a median of 79.7% (range 54.6; 98.9) of differentiation (CD7+ CD3-) and a median fold of proliferation of 14.7 (range 8.80; 18.5). No chromosome instability was induced during the expansion. Patients received increasing doses ranging from 0.1 to 0.5 10 6 HTLP/kg. The amount of CD3+/kg was less than 11.2 (range 0; 588)/ kg. No early or mid-term toxicity due to the administration of HTLP has been reported for evaluable patients (6/6). One SCID patient died on D53 because of severe VOD. Primary engraftment was confirmed in 4 out of 5 patients. Interestingly, despite the gradual increase of doses, there was no aGVHD over grade >2. Preliminary efficacy results suggest that circulating CD3+ CD4+ TCRαβ+ T cells > 50/μl before M4 depended on the increased infused dose of HTLP and an intrathymic paracrine effect. Notably, there was no viral infection disease or relapse during the outcome. Discussion conclusion: First infusions of HTLP in both clinical trials confirmed the safety and reproducible GMP manufacturing. HLTP offers an exciting perspective for improving immune reconstitution in alternative hematopoietic transplants.

Preventing Transmission of Vaccine-Associated Viral Infections from a Patient With Severe Combined Immune Deficiency

Background: The transmissibility of vaccine-strain viruses from immunocompromised patients, such as those with severe combined immune deficiency (SCID) is unknown. The infection control management of a patient diagnosed with SCID and infected with vaccine-strain varicella zoster virus (VZV) and measles virus is described below. A previously healthy, full-term boy was vaccinated at 14 months with measles mumps rubella varicella (MMR) vaccine. He had received prior vaccinations, including rotavirus, without adverse effects. During the 6 weeks after vaccination, the patient developed signs and symptoms clinically consistent with chicken pox and measles. An immune work-up revealed SCID. Methods: The Alberta Health Services (AHS) SCID protocol was followed to manage the patient upon admission at 17 months of age. Multiple meetings with various stakeholders were held to ensure appropriate precautions were followed to minimize the risk of pathogen transmission. Results: The patient was placed on airborne and contact precautions in a negative-pressure room. The pressure differential of the room to the corridor was continually monitored and displayed at the entry to the room. Staff known to be immune to VZV or measles were not required to wear an N95 respirator. All intrahospital movement of the patient was coordinated with the respective care teams and departments, including infection prevention and control, facilities maintenance and engineering, respiratory therapy, and diagnostic imaging. A mask was placed on the patient when movement outside the room was required. VZV testing was positive for the Oka/vaccine strain on all samples tested (ie, nasopharyngeal, skin, blood, and cerebrospinal fluid). Nasopharyngeal swabs and blood were PCR positive for measles genotype A/vaccine strain virus. Both viruses were persistently positive in spite of treatment with acyclovir, valganciclovir, varicella zoster immune globulin, and intravenous immune globulin. Conclusions: There is currently no documented transmission of measles vaccine-strain virus, and transmission of VZV vaccine-strain virus is rare. According to the AHS SCID protocol, the use of airborne and contact precautions for a patient identified with measles and/or VZV supersedes the use of a positive-pressure room for patients identified with SCID. Newborn screening for SCID was implemented in Alberta in June 2019. As a result, more SCID patients will be diagnosed earlier in their course, and therefore prior to most routine vaccinations. However, newborn screening will not pick up some types of combined immune deficiencies. Some children may still be at risk of vaccine-associated illnesses due to undiagnosed underlying immune deficiencies.Funding: NoneDisclosures: None

New considerations in hematopoietic stem cell transplantation for severe combined immunodeficiency: how did newborn screening change our field, and can we finally brake the glass ceiling for haploidentical transplantation?

Pioneered in 1968, hematopoietic stem cell transplantation (HSCT) first cured a patient with severe combined immune deficiency (SCID) transplanted from a matched sibling, bringing hope for this previously fatal disease. Since then, HSCT has become the standard of care treatment for SCID with thousands of patients transplanted successfully worldwide. Initially successful mainly in patients with a matched sibling donor and in specific easier to transplant types of SCID, nowadays, most patients with SCID undergo successful transplantation due to HSCT technique advances. These include refined human leukocyte antigen (HLA)-tissue typing, use of alternative donors, availability of new stem cell sources such as umbilical cord blood, less toxic chemotherapeutic conditioning, as well as improved graft-versus-host disease (GvHD) prophylaxis. Other factors contributing to the success of transplantation include the improvement of supportive care by molecular detection of viral infections, enabling preemptive antiviral treatment before organ damage occurs. Increased awareness for primary immunodeficiency disorders (PID), leading to earlier diagnosis and referral to specialist centers, has been another important factor in successfully transplanting SCID patients. A major game changer in the last decade has been the implementation of neonatal screening for SCID. This increased early diagnosis, allowing for this disease to be almost universally diagnosed soon after birth in countries which included this test in their newborn screening program. As a result, early and optimal transplant timing and conditions could be achieved. However, very early diagnosis also raised new questions regarding SCID patients with a “leaky” phenotype, as well as dilemmas regarding transplant and conditioning regimens in very young infants. With improved diagnosis and treatment options, overall survival has increased to over 90% for SCID babies with a genoidentical donor and similar results are emerging for matched unrelated donor HSCT. Due to new advances, we hope to achieve similar results for those given HSCT from haploidentical donors as well. This review will focus on the new considerations in HSCT seen in recent years, and examines the effect they have had on treatment options and outcomes for SCID patients. Statement of novelty: The field of HSCT has advanced considerably since the first successful SCID bone marrow transplant in 1968. However, success rates have been limited due to delayed diagnosis and poor outcome of patients for which a HLA-matched donor could not be found. This review will discuss recent advances occurring in the last decade in HSCT for SCID, and our hopes to bring cure to this once fatal disease.

Survey of Infection Control Precautions for Patients with Severe Combined Immune Deficiency.

Severe combined immune deficiency (SCID) is caused by an array of genetic disorders resulting in a diminished adaptive immune system due to impaired T lymphocytes. In these patients, active infection at the time of hematopoietic transplantation has been shown to increase morbidity and mortality. To prevent transmission of infections in SCID patients, standardized infection control precautions should be implemented. An online survey regarding SCID-specific protocols was distributed through several immunodeficiency organizations. Seventy-three responses were obtained, with the majority (55%) of responses from the USA, 15% from Canada, and the remainder from 12 other countries. Only 50% of respondents had a SCID-specific infection control protocol at their center, and while a majority of these centers had training for physicians, a small minority had training for other healthcare workers such as nursing and housekeeping staff. Significant variability of infection control practices, such as in-patient precautions, required personal protective equipment (PPE), diet restrictions, visitor precautions and discharge criteria, was found between different treatment centers. There is a paucity of evidence-based data regarding the safest environment to prevent infection in SCID patients. Institutional protocols may have significant impact on infection risk, survival, family well-being, child development and cost of care. From these results, it is evident that further multi-center research is required to determine the safest and healthiest environment for these children, so that evidence-based infection control protocols for patients with SCID can be developed.

Long-Term Outcome of Adenosine Deaminase-Deficient Patients-a Single-Center Experience.

Inherited defects in the adenosine deaminase (ADA) enzyme can cause severe combined immune deficiency (SCID) and systemic abnormalities. Management options for ADA-deficient patients include enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and gene therapy (GT). Here, we describe the long-term benefits of these treatments. Survival, infections, systemic sequelae, and laboratory assessments were recorded for all ADA-deficient SCID patients, managed at a single center since 1985, who survived 5 or more years following treatment. Of 20 ADA-deficient patients, the 8 (40%) who survived 5 or more years (range 6-29.5years, median 14years) were included in the study. Among the long-term survivors, two patients were treated exclusively with ERT, five underwent HSCT (three from HLA-matched sibling donors, two from HLA-mismatched related donors), and one received GT. The long-term survivors often suffered from recurrent respiratory infections; however, opportunistic infections occurred in only one patient. Systemic sequelae included lung disease such as bronchiectasis and asthma (four patients), neurologic abnormalities (six patients), metabolic disturbances (two patients), allergy and autoimmunity (six patients), and neoplasms (three patients). Normal CD4+ T cell numbers and function, as well as antibody production, were usually observed after HSCT and GT, but not after ERT. Late deaths occurred in two patients at 15 and 25years after HSCT, respectively, and were attributed to respiratory failure. ADA-deficient patients commonly suffer from long-term complications, emphasizing the need for improved management and for multi-disciplinary follow-up.

Outcome of Children with Primary Immune-Deficiencies (PIDs) Enrolled in a Phase I-II Trial Based on the Infusion of BPX-501 Donor T Cells Genetically Modified with a Novel Suicide Gene (inducible Caspase 9, iC9) after T-Cell Depleted HLA-Haploidentical Allogeneic Stem Cell Transplantation (haplo-HSCT)

▪Background: We previously reported that haplo-HSCT after depletion of α/β T cells / CD19 B cells is a suitable option for patients with PIDs, with a high success rate (Bertaina et al., Blood 2014). However, in many patients, we observed a delay in the recovery of adaptive immunity, sometimes resulting in life-threatening or even fatal events, providing the rationale for exploring an innovative approach based on the post-transplantation infusion of donor T cells, genetically modified with the novel suicide gene iC9 (BPX-501 cells), with the aim of accelerating reconstitution of adaptive immunity. As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood.Patients and study design: In this multicenter, prospective phase I-II trial (enrolling both malignant and non-malignant diseases; sponsored by Bellicum Pharmaceuticals®, ClinicalTrials.gov identifier: NCT02065869) 20 children with PIDs have been enrolled. Nine children had Severe Combined Immune Deficiency (SCID), 5 Wiskott-Aldrich syndrome (WAS), 2 Chronic Granulomatosis Disease (CGD) and one each of 4 other PIDs detailed in Table 1, which also reports relevant patient-, donor- and transplant- characteristics. All patients were transplanted after depletion of donor α/β T cells and CD19 B cells, employed to prevent graft-versus-host disease (GvHD) and post-transplant lymphoproliferative disorders (PTLD). Details on cell subsets infused with the graft are reported in Table 1. No patient was given any post-transplantation GvHD prophylaxis. The phase I portion of the trial consisted of a classical 3+3 design with 3 cohorts, with escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. Patients in the phase II portion received the highest dose identified during the phase I portion (1x106 cells/kg).Results: All patients engrafted and no secondary graft failure was recorded. The median time to neutrophil and platelet recovery was 16 (range 11-35) and 10 days (range 7-14), respectively. BPX-501 cells were infused at a median time of 15 days (range 13-56) after the allograft. Four patients were enrolled in the phase I portion of the study; one received 2.5x105 and 3 received 1x106 cells/kg. The remaining 16 children were treated in the phase II part, with a dose was 1x106 cells/kg. Five children experienced Grade I (3 patients) or Grade II (2 children) acute GvHD, which resolved with either topical or systemic steroids in 3 patients. The other 2 cases resolved after the infusion of Rimiducid (AP1903) which activated the iC9 suicide gene. The cumulative incidence of acute GvHD is shown in Figure 1A. Two of the 17 patients at risk (i.e. those with a follow-up longer than 100 days after transplantation) developed mild (skin only) chronic GvHD. The median time to discharge was 36 days (range 26-180). Eight patients (40%) experienced one episode of re-hospitalization after initial discharge. After a median follow-up of 10 months (range 50 days-20 months), all patients are alive and disease-free. Details on expansion/persistence of BPX-501 cells, as well as on recovery of CD3+ cells are shown in Figure 1B and 1C, respectively. Ig serum levels of SCID patients at 6 and 12 months after transplant are depicted in Figure 1D. The median follow-up for WAS patients is 510 days (124-531 days) and their median platelet count at 1, 3 and 12 months after haplo-HSCT was 136 (range 48-168), 204 (range 98-280) and 143 (117-365) x109/L, respectively. One SCID patient who developed BCG bacterial dissemination after the allograft resolved this infection and 3 patients (2 with SCID and one with WAS) cleared cytomegalovirus (2 children) or Adenovirus (one child) infections pre-existing at time of conditioning regimen.Conclusions: These results indicate that haplo-HSCT, after depletion of α/β T cells and B cells followed by adoptive infusion of donor BPX-501 cells, is an effective alternative for those children with PIDs in need of an urgent allograft or lacking a suitable HLA-matched donor. BPX-501 cells expand in vivo and persist over time, contributing to fasten the recovery of adaptive T-cell immunity and to clear infections. In view of the absence of fatalities and of the low rate of mild chronic GvHD, we conclude that infusion of BPX-501 T cells with the iC9 cell-suicide system may potentially render haplo-HSCT a first-line option for children with PIDs. [Display omitted] [Display omitted] DisclosuresQasim:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership, Research Funding; Catapult: Research Funding; Calimmune: Research Funding. Slatter:Medac: Other: Travel grants. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Isolation protocol for patients with severe combined immune deficiency

Infants with severe combined immune deficiency (SCID) typically present in the first few months of life with severe, recurrent, opportunistic infections, and without definitive treatment the condition is invariably fatal (Gaspar et al. 2013). Many centres believe that protective isolation is required for treatment of SCID patients once diagnosed, and the isolation protocol varies across institutions. This paper describes the isolation protocol for SCID requiring hematopoietic stem cell transplant (HSCT) that has been utilized at our institution for over the last 25 years. We believe that the profound immunodeficiency in SCID patients warrants a more restrictive treatment to limit the morbidity and mortality associated with HSCT. With this protocol, we have seen a very low infection rate in and have a very good survival rate for our SCID population.

Congenital defects in V(D)J recombination.

The V(D)J recombination is a DNA rearrangement process that generates the diversity of T and B lymphocyte immune repertoire. It proceeds through the generation of a DNA double-strand break (DNA-DSB) by the Rag1/2 lymphoid-specific factors, which is repaired by the non-homologous end joining (NHEJ) DNA repair pathway. V(D)J recombination also constitutes a checkpoint in the lymphoid development. V(D)J recombination defect results in severe combined immune deficiency (SCID) with a lack of T and B lymphocytes. The V(D)J recombination represents one of the few programmed molecular events leading to DNA-DSBs that strictly relies on NHEJ. Two NHEJ factors, Artemis and XLF/Cernunnos, were identified through the molecular studies of SCID patients. Mutations in PRKDC and DNA Ligase IV genes also result in SCID. Studies in mice have demonstrated that XLF/Cernunnos is dispensable for V(D)J recombination in lymphoid cells but not for the repair of genotoxic-induced DNA-DSBs, which raises the question of the implication of Rag1/2 factors in the DNA repair phase of V(D)J recombination. New factors of NHEJ, such as PAXX, are being identified. Patients with NHEJ deficiency (XRCC4) without immune deficiency were recently reported. We, therefore, may not have yet the complete picture of DNA-DSB repair in the context of V(D)J recombination.

Overview of 15-year severe combined immunodeficiency in the Netherlands: towards newborn blood spot screening.

Severe combined immune deficiency (SCID) is a fatal primary immunodeficiency usually presenting in the first months of life with (opportunistic) infections, diarrhea, and failure to thrive. Hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are curative treatment options. The objective of the study was to assess the morbidity, mortality, and diagnostic and therapeutic delay in children with SCID in the Netherlands in the last 15 years. These data may help to judge whether SCID should be considered to be included in our national neonatal screening program. In the period 1998–2013, 43 SCID patients were diagnosed in the Netherlands, 11 of whom were atypical SCID (presentation beyond the first year). The median interval between the first symptom and diagnosis was 2 months (range 0–1173 months). The total mortality was 42 %. In total, 32 patients were treated with HSCT of whom 8 were deceased. Nine patients died due to severe infectious complications before curative treatment could be initiated. Conclusion: Because of a high mortality of patients with SCID before HSCT could be initiated, only a national newborn screening program and pre-emptive HSCT or GT will be able to improve survival of these patients.“What is known”• SCID is a fatal disease if a curative hematopoietic stem cell transplantation cannot be performed in time.• Newborn screening for SCID enables early diagnosis in the asymptomatic phase.“What is new”• Nine out of 43 SCID patients in the Netherlands died due to severe infectious complications before curative treatment could be initiated.• Only newborn screening and pre-emptive curative therapy will improve survival of children with SCID in the Netherlands.

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Cancer: Darwinian tumour suppression.

Competition for access to a survival factor has been found to explain why incoming cells from the bone marrow replace resident cells in the thymus. Reducing this competition can cause tumours to form. See Article p.465 In the thymus, T cells develop from precursor cells that are constantly replaced by newly arriving bone marrow progenitor cells. Hans-Reimer Rodewald and colleagues show that this is the result of competition between the 'old' and 'new' cells. In the absence of cell competition, when the influx of new bone marrow progenitor cells is blocked in mice, old cells reacquire the ability to self-renew and eventually become transformed, leading to the development of T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease. At the same time there are changes in gene expression and the appearance of genetic mutations often also found in human T-ALL. Thus cell competition can act as a tumour-suppressor mechanism. This work may also help to explain the widely discussed T-cell leukaemia seen in X-linked severe combined immune deficiency patients after therapy with gene-corrected autologous progenitors.

Cell competition is a tumour suppressor mechanism in the thymus.

Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

Hematopoietic cell transplantation for treatment of primary immune deficiencies.

Hematopoietic cell transplantation (HCT) has the potential to cure primary immune deficiency syndromes (PIDS) that are a group of disorders primarily affecting a single lineage, e.g., lymphoid or myeloid lineage. Generally, implementation of various conditioning regimens depends the type of IDS. Some syndromes that cause profound immune deficiency may not require a conditioning regimen. There appears to be a barrier even in cases of severe combined immune deficiency (SCID), particularly in the situation of HLA mismatched or haploidentical grafts. For example, donor B cell chimerism is less likely in γ-chain deficiency (X-SCID), as host cells persistently occupy the B lymphocyte niche, than in syndromes without B cells such as adenosine deaminase (ADA) deficiency. The immune defect may be corrected by partial reconstitution of normal immune cells, in other words full donor chimerism of the affected cell subset may not be required. This concept may add further rationale to limiting the intensity of the conditioning regimen.SCID encompasses a broad range of inherited defects that individually cause a profound immune deficiency of both T and B cell function. The individual genetic defects give rise to various phenotypes, and, since the goal of HCT is to restore both T and B cell function, the SCID phenotype must be taken into consideration in addition to the degree of recipient-donor mismatch. Other biologic factors associated with the SCID phenotype may influence the barrier to engraftment, such as host NK cells, which may survive intensive conditioning regimens. One of the difficulties in analyzing outcome of HCT in SCID patients is the relative rarity of the condition, thus needing large multicentric studies. Recent studies show that the most important factor for improved survival after an HLA-identical sibling graft was younger age at time of HCT. Factors significantly associated with improved survival after haploidentical transplants were B+ SCID phenotype, protected environment, and lack of pulmonary infections before HCT. The advent of neonatal screening and in utero diagnosis has allowed early detection of SCID and therefore prompt intervention at an early age.Primary T cell immunodeficiency (PTCD) syndromes may be differentiated from SCID by virtue of reduced but not completely absent T cell function, or absent T cell function with the presence of B lymphocyte or NK cell function. Allogeneic marrow transplantation remains the only curative therapy available for these disorders. Worse outcomes were seen in patients with PTCD compared to other types of immune deficiencies, regardless of donor. Although life-threatening infections may be less common early in life, children with PTCD often develop organ damage from chronic infections, particularly lung disease, prior to HCT.In Wiskott-Aldrich syndrome, HCT offers significantly improved survival chances for patients. Achieving full donor chimerism was shown to be a favorable factor. In general, however, the studies suggest that low intensity regimens offer the potential for achieving donor cell engraftment with less morbidity than standard regimens, an important consideration for patients who currently may consider the risks of conventional transplants unacceptably high.

Signalling to transcription: Store-operated Ca 2+ entry and NFAT activation in lymphocytes

In cells of the immune system that are stimulated by antigen or antigen–antibody complexes, Ca 2+ entry from the extracellular medium is driven by depletion of endoplasmic reticulum Ca 2+ stores and occurs through specialized store-operated Ca 2+ channels known as Ca 2+-release-activated Ca 2+ (CRAC) channels. The process of store-operated Ca 2+ influx is essential for short-term as well as long-term responses by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription downstream of Ca 2+ influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin. The importance of the Ca 2+/calcineurin/NFAT signalling pathway for lymphocyte activation is underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC channel function, store-operated Ca 2+ entry, NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defence. We recently used a two-pronged genetic approach to identify Orai1 as the pore subunit of the CRAC channel. On the one hand, we initiated a positional cloning approach in which we utilised genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic region linked to the mutant gene in the SCID family described above. In parallel, we used a genome-wide RNAi screen in Drosophila to identify critical regulators of NFAT nuclear translocation and store-operated Ca 2+ entry. These approaches, together with subsequent mutational and electrophysiological analyses, converged to identify human Orai1 as a pore subunit of the CRAC channel and as the gene product mutated in the SCID patients.

Distinct IL-7 Signaling Outcomes in Normal Human B and T-Lineage Progenitors.

Severe combined immune deficiency (SCID) patients with mutations in the IL-7 receptor (R) alpha chain or the gamma common subunit chain exhibit a severe block in early thymocyte development but have normal numbers of circulating B cells. This phenotype has been interpreted as evidence that human T and B-cell development have different IL-7 signaling requirements. We hypothesize that these apparent differences may reflect activation of different signaling pathways by IL-7, culminating in varying outcomes in survival/proliferation. Thus, the goal of the current study was to test this hypothesis using purified populations of normal human thymic and B-lineage lymphoid progenitors. CD34+ thymocytes (CD1a- multi-lineage lymphoid progenitors and CD1a+ early T-lineage progenitors representing 1–3% of total thymocytes) and CD34− thymocytes (CD4+/CD8+, CD4+/CD8−, and CD4−/CD8+ subpopulations) were purified using anti-CD34 magnetic beads. CD19+ B-lineage cells were purified with anti-CD19 magnetic beads from 4-wk cultures of cord blood CD34+ stem cells plated on MS-5 murine stromal cells. Flow cytometry revealed that the IL-7R was expressed on approx. 95% of CD34+ thymocytes, approx. 50% of CD34− thymocytes, and 30–50% of CD19+ B-lineage cells. Two to 3-day incubation with IL-7 induced proliferation of CD34+ thymocytes, and promoted survival (but not proliferation) of CD34- thymocytes and CD19+ B-lineage cells. Each population was stimulated with log concentrations of IL-7 for 1–20 min, lysed, and Western blotted. CD34+ thymocytes underwent robust phosphorylation (p) of STAT5, ERK1/2, AKT and the AKT substrate GSK3beta within 1 min and peaked at 5–10 min. Signaling was detected with as little as 10 pg/ml IL-7. CD34− thymocytes underwent comparable induction of pSTAT5, but no induction of pERK1/2, pAKT or pGSK3beta at up to 10 ng/ml IL-7. Incubation of CD34+ thymocytes with IL-7 for up to 3 days revealed IL-7 dose-dependent suppression of the cyclin-dependent kinase inhibitor p27Kip1, but no suppression of p27Kip1 was detected in IL-7 stimulated CD34− thymocytes. CD19+ cells exhibited IL-7 dose-dependent induction of pSTAT5, but showed only minimal induction of pERK1/2 and pGSK3beta (and only at 10 ng/ml IL-7), and did not suppress expression of p27Kip1. IL-7 induced pAKT and pERK1/2 in CD34+ thymocytes was blocked by the PI-3 kinase inhibitor LY294002 and the MEK inhibitor U0126, respectively. However, only LY294002 blocked proliferation. IL-7 promoted the expansion of CD19+ B-lineage cells cultured on MS-5 stromal cells, and expansion was blocked by LY294002 but not UO126. These collective results suggest that whereas induction of pSTAT5 is a constant outcome of IL-7 signaling in normal human lymphoid progenitors, activation of PI3-kinase/AKT and subsequent proliferation:occurs in CD34+ thymocytes,does not occur in CD34− thymocytes, andoccurs in CD19+ B-lineage cells; but potentially requires an additional cytokine stimulus derived from MS-5 stromal cells that cooperates with IL-7 to transduce a proliferative signal.These IL-7 signaling outcomes may at least partially explain the differences in T and B lymphocyte development in SCID patients with mutations in the IL-7R alpha chain and the gamma common subunit chain.

A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

1090. Enzyme Replacement Therapy with Pegylated Adenosine Deaminase (PEG-ADA) Does Not Impede Immune Reconstitution Following Transplantation of Gene-Corrected Bone Marrow Cells in the Murine Model of ADA-SCID

In humans, the genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of Severe Combined Immune Deficiency (SCID) and results in severe lymphopenia. The current standard is either an allogeneic bone marrow transplant from an MHC-matched donor or enzyme replacement therapy (ERT) with PEG-ADA; BMT from a donor other than a matched sibling has poor outcome. Because of a postulated selected advantage for corrected T cells, ADA-deficient SCID has been an early candidate disease for gene therapy. Recent studies by Aiuti and co-workers using oncoretroviral vectors to transduce CD34+ cells from the bone marrow of ADA-deficient SCID patients, together with cessation of ERT and moderate cytoreduction with Busulfan show clinical benefit, however, the relative roles of the cytoreduction and the cessation of PEG-ADA have yet to be elucidated. A murine model of ADA-deficient SCID was produced by Rodney Kellems and co-workers (U.Texas, Houston) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19-20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. In these studies, we evaluated the role of ERT as a single variable on the ability of gene-corrected cells to repopulate 10-week old ADA-deficient mice that received a cytoablative dosage of total-body irradiation (900 cGy) prior to receiving ADA-deficient marrow transduced with the retroviral vector MND-MFG-huADA. These mice, previously maintained on ERT, were either continued on the same low dose ERT (n=8) or not (n=8). ADA -/- mice receiving ERT had 89% survival and those not receiving ERT had 75% survival after radiation and BMT. Mice were euthanized between 4-5 months post transplant and were perfused with PBS prior to tissue removal. Proviral marking was assessed by qPCR in the peripheral blood leukocytes, thymus, spleen, bone marrow, lung and liver, as well as in sorted cell populations from the thymus (CD4/8), spleen (CD19) and bone marrow (CD11b). Gene marking was seen in all of the tissues assessed with little difference between those animals receiving ERT and those that did not. Although not significant, marking was slightly higher in the hematopoietic tissues analyzed from mice receiving ERT. Proviral marking (copies/ cell) was highest in the thymus and spleen and lowest in the bone marrow. Cell populations enriched from these organs had marking similar to the organ from which they were isolated. Marking in the lung and liver was similar to that seen in the hematopoietic tissues and may be relevant due to the severe lung and hepatocellular pathology seen in ADA-deficient mice. With or without ERT, ADA- deficient mice receiving gene therapy had improved immune cellularity and function compared to ADA-deficient mice receiving mock transductions and maintained on ERT. In these studies, we have observed no selective lymphoid expansion of the gene-corrected T cells, whether ERT was given or withheld, but nevertheless there was immunologic improvement from host (ADA-deficient) cells.

177. Lentiviral Vector Mediated ADA Gene Transfer in ADA-Deficient SCID Mice

Genetic deficiency of adenosine deaminase (ADA) is responsible for ~20% of human severe combined immune deficiency (SCID). While promising results have been obtained by Aiuti and co-workers using oncoretroviral vectors to transduce CD34+ cells from the bone marrow of ADA-deficient SCID patients, lentiviral vectors may lead to higher levels of ADA gene transfer that could extend benefits to older subjects with less transducible bone marrow or allow lower dosages of cytoreductive therapy to be used. We have constructed a SIN HIV-1-based lentiviral vector, SMPU-R-MND-ADA, which was produced at high titer (1 × 10e10 i.u./ml after 100-fold concentration) using VSV-G pseudotype and have shown it to express high levels of ADA enzyme activity in human and murine cell lines in vitro. A model of ADA-deficient SCID was produced by Rodney Kellems and co-workers (U.Texas, Houston) by ADA gene knock-out and rescue of neonatal lethality with an ADA mini-gene expressed in the placenta during gestation. These mice die from a non-infectious pulmonary disease within the first month of life, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. We have determined that transplantation of newborn ADA −/− mice with normal congenic bone marrow allows long-term survival without PEG-ADA and facilitates breeding.

Insertional oncogenesis in gene therapy: how much of a risk?

Insertional mutagenesis is an unavoidable consequence of the transposition of genetic material. Whether it involves an integrating virus, a transposable element, a replicationdefective viral vector or plasmid DNA, the ectopic chromosomal integration of DNA is a mutagenic event that may disrupt chromatin or gene structure, thereby altering gene transcription, regulation and/or coding sequences. The gravest concern associated with such mutations is the risk of cell transformation. The acquisition of the malignant phenotype may follow tumor suppressor gene inactivation, typically a recessive event, or oncogene trans-activation, which is likely to exert a dominant effect. The propensity of transposable elements to induce experimental tumors in animals has long been exploited to study the genetic basis of cancer. The earliest analyses of the therapeutic potential of gene transfer have thus recognized the risk of insertional oncogenesis as inherent to the use of integrating vectors. Yet, there have been extremely few reports documenting insertional oncogenesis in rodent, dog and non-human primate gene transfer studies. Based on these experimental data, gene therapists and their patients have justifiably assumed that the risk of insertional oncogenesis would be very low. This reassuring point of view was challenged last year when the firstever case of iatrogenic malignant transformation was reported in a gene therapy patient treated for Xlinked severe combined immune deficiency (SCID). The consternation brought upon by this serious adverse event (SAE), which shook the medical community and the concerned regulatory agencies, was compounded, but also mitigated, when a second case was reported 6 months later – in the same trial. These two SAEs imposed a reevaluation of the safety concerns associated with gene transfer and of the risk benefit ratio of gene therapy in the context of SCID as well as other diseases. Following a temporary hold on gene therapy trials using retroviral vectors to transduce hematopoietic stem cells, the US Food and Drug Administration and the NIH Recombinant DNA Advisory Committee judiciously concluded that this pair of SAEs did not constitute ground for the cessation of gene therapy investigation. The interrupted trials were therefore allowed to reopen, pending the case-by-case re-evaluation of the risk/benefit ratio for every study and the appropriate revision of the monitoring plans and of the information provided to prospective patients before obtaining informed consent. The prompt resumption of these trials is good news for the field, but the need to reassess the safety of the many gene transfer strategies under development remains. Our understanding of the biological mechanisms that determine, in qualitative and quantitative terms, the risk of clonal expansion and transformation, is still fragmentary. However, two recent reports provide valuable insight into the transforming mechanisms that may have operated in the two SCID patients who developed clonal T-cell expansions. Based on these observations, we can begin to identify possible factors that increase the risk that insertional mutagenesis will turn into oncogenesis. While prospective studies are direly needed to analyze and quantify the role of these risk factors, the reports by HaceinBey-Abina et al and Dave et al provide important clues for taking steps to investigate and reduce the risk of oncogene trans-activation by integrating gene transfer vectors. Gene therapy of X-linked SCID: landmark success and two SAEs

Human severe combined immune deficiency and DNA repair.

Human severe combined immune deficiency (SCID) is the most serious inherited immunological deficit. Recent work has revealed defects in the predominant pathway for double-strand break repair called nonhomologous DNA end joining, or NHEJ. Progress in the biochemistry and genetics of NHEJ and of human SCID has proven to be synergistic between these two fields in a manner that covers the range from biochemical etiology to considerations about possible gene therapy for the B- SCID patients.