Treatment Of Severe Combined Immunodeficiency Research Articles

Disruption of cyclin D1 degradation leads to the development of mantle cell lymphoma

Cyclin D1 has been recognized as an oncogene due to its abnormal upregulation in different types of cancers. Here, we demonstrated that cyclin D1 is SUMOylated, and we identified Itch as a specific E3 ligase recognizing SUMOylated cyclin D1 and mediating SUMO-induced ubiquitination and proteasome degradation of cyclin D1. We generated cyclin D1 mutant mice with mutations in the SUMOylation site, phosphorylation site, or both sites of cyclin D1, and found that double mutant mice developed a Mantle cell lymphoma (MCL)-like phenotype. We showed that arsenic trioxide (ATO) enhances cyclin D1 SUMOylation-mediated degradation through inhibition of cyclin D1 deSUMOylation enzymes, leading to MCL cell apoptosis. Treatment of severe combined immunodeficiency (SCID) mice grafted with MCL cells with ATO resulted in a significant reduction in tumor growth. In this study, we provide novel insights into the mechanisms of MCL tumor development and cyclin D1 regulation and discover a new strategy for MCL treatment.

Diagnosis and treatment of severe combined immunodeficiency

Diagnosis and treatment of severe combined immunodeficiency

Advances in the treatment of severe combined immunodeficiency

Severe Combined Immunodeficiency (SCID) is the most profound inborn error of immunity affecting cellular and humoral immunity. Hematopoietic stem cell transplantation has been a curative treatment since 1968. Huge progress has been made since then in understanding the underlying genetics, improving outcomes from transplant, and introducing gene therapy in particular for adenosine deaminase deficient- and IL2 receptor gamma-deficient SCID. Newborn screening has been widely introduced across the world to enable definitive treatment before infection occurs. This article aims to review the latest evidence on how to achieve curative treatment with minimal short- and long-term toxicity, normal immune reconstitution and good quality of life.

When Screening for Severe Combined Immunodeficiency (SCID) with T Cell Receptor Excision Circles Is Not SCID: a Case-Based Review.

Newborn screening efforts focusing on the quantification of T cell receptor excision circles (TRECs), as a biomarker for abnormal thymic production of T cells, have allowed for the identification and definitive treatment of severe combined immunodeficiency (SCID) in asymptomatic neonates. With the adoption of TREC quantification in Guthrie cards across the USA and abroad, typical, and atypical SCID constitutes only ~ 10% of cases identified with abnormal TRECs associated with T cell lymphopenia. Several other non-SCID-related conditions may be identified by newborn screening in a term infant. Thus, it is important for physicians to recognize that other factors, such as prematurity, are often associated with low TRECs initially, but often improve with age. This paper focuses on a challenge that immunologists face: the diagnostic evaluation and management of cases in which abnormal TRECs are associated with variants of T cell lymphopenia in the absence of a genetically defined form of typical or atypical SCID. Various syndromes associated with T cell impairment, secondary forms of T cell lymphopenia, and idiopathic T cell lymphopenia are identified using this screening approach. Yet there is no consensus or guidelines to assist in the evaluation and management of these newborns, despite representing 90% of the patients identified, resulting in significant work for the clinical teams until a diagnosis is made. Using a case-based approach, we review pearls relevant to the evaluation of these newborns, as well as the management dilemmas for the families and team related to the resolution of genetic ambiguities.

Orphan designation: Allogeneic cultured postnatal thymus-derived tissue, Treatment of severe combined immunodeficiency due to FOXN1 deficiency

Orphan designation: Allogeneic cultured postnatal thymus-derived tissue, Treatment of severe combined immunodeficiency due to FOXN1 deficiency

Orphan designation: Allogeneic cultured postnatal thymus-derived tissue, Treatment of severe combined immunodeficiency due to FOXN1 deficiency

Orphan designation: Allogeneic cultured postnatal thymus-derived tissue, Treatment of severe combined immunodeficiency due to FOXN1 deficiency

Orphan designation: Autologous CD34+ cells transfected with retroviral vector containing adenosine deaminase gene, Treatment of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency

Orphan designation: Autologous CD34+ cells transfected with retroviral vector containing adenosine deaminase gene, Treatment of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency

Outcomes for Umbilical Cord Blood Transplantation in Severe Combined Immunodeficiency Disorders: Ten-Year Experience.

The treatment of severe combined immunodeficiency (SCID) is immune reconstitution using hematopoietic stem cell (HSC) transplantation early in life. HLA-identical related donors are the preferred source of HSCs. Since sibling donors are available in <30% of patients, other sources of HSCs are considered-mismatched related donor, umbilical cord blood (UCB), and matched unrelated donor bone marrow. We report the outcome of 10 patients with SCID or combined immunodeficiency (CID) 10 years after UCB transplantation (UCBT) at our institution using a retrospective chart review. Eight patients were alive 10 years post-transplantation. This was the second transplant for 2 patients due to initial transplant engraftment failure. Immunologic reconstitution was demonstrated after transplant with presence of memory T cells at 3 months, naive T cells at 12 months, B cells at 3 months, and normal tetanus/diphtheria toxoid antibody responses at 2 years. Immune response remained robust 10 years post-transplantation. Eight patients developed stage I acute graft-versus-host disease (GvHD), 2 patients developed grades 2-4 GvHD, and 1 child developed chronic GvHD with bronchiolitis obliterans. UCB should be considered as an alternative HSC source for patients with SCID and CID because of its robust and sustained recovery of immune function, low risk of severe GvHD, and accessibility.

Treatment for severe combined immunodeficiency (SCID) in the era of newborn screening (NBS)

Abstract SCID is associated with excellent survival when treated with allogeneic hematopoietic cell transplantation (HCT), gene therapy (GT), or enzyme replacement therapy (ERT) within 3–4 months of birth or prior to infection. NBS for SCID results in the diagnosis by 2–3 weeks of age. This retrospective analysis describes therapy and immune reconstitution of 28 infants diagnosed by NBS in California since 2010 and treated at the University of California, San Francisco at a median of 56 (24–217) days of age. HCT with alkylating chemotherapy (N=6), serotherapy alone (N=6), or no conditioning (N=12) was used in 24 infants, and ERT followed by definitive GT at University of California, Los Angeles, was used in 4 ADA-deficient infants. Overall survival at &amp;gt;2 years was 93% with 1 death from HHV6 and 1 from CMV. The median time to CD4 T cell recovery (&amp;gt;200 T cells/uL) was 73 (10–96) days for ERT patients prior to GT, and 138 days (14–220) for HCT patients. Time from first appearance of donor chimerism to CD4 recovery was evaluable for 14 HCT survivors who lacked pre-HCT maternal chimerism or whose donors were not their mothers. After donor chimerism appeared in peripheral blood at a median of 21.5 (14–169) days, appearance of T cells took 32 (0–84) more days. Second transplant or stem cell boost was required for 10 of 24 patients at a median of 77 (35–382) days following first HCT; for patients eligible for analysis (N=15), need for 2nd therapy was significantly higher in those without detectable donor chimerism by 28 days following therapy (0% vs 83%, p=0.04). NBS for SCID allowed rapid institution of anti-infective and definitive treatment to optimize outcomes, and early detection of donor chimerism was associated with successful T cell recovery.

Investor Outlook: Rising from the Ashes; GSK's European Approval of Strimvelis for ADA-SCID.

GlaxoSmithKline's (GSK) and partner San Raffaele Telethon Institute for Gene Therapy's recent positive European approval for Strimvelis for treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) represents the second EU-approved gene therapy and the first γ-retrovirus and first ex vivo gene therapy. In this article we discuss the significance and implications of this historic approval for the broader gene therapy field.

A protocol for matched unrelated donor hematopoietic stem cell transplantations for severe combined immunodeficiency

Hematopoietic stem cell transplantation using HLA-mismatched related donors for the treatment of severe combined immunodeficiency has led to disappointing outcomes at our institution. This created an impetus to consider other donor sources when an HLA-matched related donor was not available. In 1988, a new protocol using HLA-matched unrelated donors was developed at our institution and continues to be used to date. This has contributed to improved outcomes and change in practice for patients with severe combined immunodeficiency. Statement of novelty: This report describes in detail the protocol for hematopoietic stem cell transplantations using HLA-matched unrelated donors for patients with severe combined immunodeficiency at our institution. This protocol is published for those centres wishing for guidance in setting up procedures for hematopoietic stem cell transplantation.

Clinical, immunologic, and genetic characteristics of RAG mutations in 15 Chinese patients with SCID and Omenn syndrome.

Mutations in Recombination Activating Genes (RAG1 and RAG2) are common genetic causes of severe combined immunodeficiency (SCID) and Omenn syndrome (OS). The clinical, immunologic, and genetic characteristics of RAG mutations in Chinese patients with SCID or OS have not been studied in detail. In this research, 22 RAG mutations were identified in 15 Chinese patients, including 10 novel mutations in RAG1 (R108X, M630T, E510X, S666P, E669K, C730Y, A857V, K847E, L922PfsX7, and L1025FfsX39) and 4 in RAG2 (R73C, I427GfsX12, P432L, and 311insL). L1025FfsX39 is a potential RAG1 hot-spot mutation in the Chinese population. The distribution of RAG1 mutations rather than mutation type seemed to differ between SCID and OS patients. The thymic output of T lymphocytes, TCR rearrangement, and T cell proliferation were severely impaired in RAG mutant patients. These findings will contribute to the early diagnosis and treatment of SCID and OS to a certain extent.

Hematopoietic Stem Cell Transplantation for Severe Combined Immunodeficiency.

Hematopoietic stem cell transplantation (HSCT) is an effective approach for the treatment of severe combined immunodeficiency (SCID). However, SCID is not a homogeneous disease, and the treatment required for successful transplantation varies significantly between SCID subtypes and the degree of HLA mismatch between the best available donor and the patient. Recent studies are beginning to more clearly define this heterogeneity and how outcomes may vary. With a more detailed understanding of SCID, new approaches can be developed to maximize immune reconstitution, while minimizing acute and long-term toxicities associated with chemotherapy conditioning.

A TRECs screening assay and its combination with IL2RG gene analysis for diagnosis of severe combined immunodeficiency

Objective To establish a newborn screening method suitable for clinical application, combined with Sanger sequencing assay for related gene mutation, and thereby to provide early screening and diagnosis basis for severe combined immunodeficiency (SCID). Methods Using Taqman real-time PCR technology, the quantitative detection method for T-cell receptor rearrangement excision circles (TRECs) from the dried blood spots was developed and the methodology evaluation was performed. From January 2013 to June 2014, a total of 30 suspected SCID cases in the Children′s Hospital of Chongqing Medical University were enrolled. TRECs copies were measured in DNA isolated from the dried blood spots. IL2RG gene mutations were also tested in DNA from peripheral blood mononuclear cells by sequencing. Compared with the confirmed clinical diagnosis, the clinical coincidence rate of the above two methods was evaluated, respectively. Results The detection limit of Taqman real-time PCR technique was 103 copies/ml for TRECs from the dried blood spots. The intra-and inter-coefficients of variations were less than 4.7% and less than 9.1% respectively. Among 30 suspected SCID cases, 17 cases, which were confirmed as SCID based on the clinical presentation, were observed that TRECs copies were below normal reference interval. The coincidence rate of Taqman real-time PCR technique and the confirmed diagnosis was 17/17. Among 17 confirmed SCID cases, all were boys. IL2RG gene mutations were identified in 16 cases, including 7 frameshift mutations, 6 missense mutations, 2 silent mutations and 1 splice mutation. Additionally, a compound heterozygosity mutation in RAG1 gene was found in a confirmed SCID case without the IL2RG gene mutation. Conclusion TRECs-based newborn screening, and combination with IL2RG gene analysis technology, may provide technical support for early identification, diagnosis, and selection of treatment for SCID. (Chin J Lab Med, 2015, 38: 21-26) Key words: Severe combined immunodeficiency; Neonatal screening; Interleukin receptor common gamma subunit; Gene rearrangement, T-lymphocyte

Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency

Patients with severe combined immunodeficiency disease who have matched sibling donors (MSDs) can proceed to hematopoietic cell transplantation (HCT) without conditioning chemotherapy. We sought to determine whether the results of HCT without chemotherapy-based conditioning from matched unrelated donors (URDs), either from volunteer adults or umbilical cord blood, are comparable with those from MSDs. We performed a multicenter survey of severe combined immunodeficiency transplantation centers in North America, Europe, and Australia to compile retrospective data on patients who have undergone unconditioned HCT from either URDs (n = 37) or MSDs (n = 66). Most patients undergoing URD HCT (92%) achieved donor T-cell engraftment compared with 97% for those with MSDs; however, estimated 5-year overall and event-free survival were worse for URD recipients (71% and 60%, respectively) compared with MSD recipients (92% and 89%, respectively; P < .01 for both). URD recipients who received pre-HCT serotherapy had similar 5-year overall survival (100%) to MSD recipients. The incidences of grade II to IV acute and chronic graft-versus-host disease were higher in URD (50% and 39%, respectively) compared with MSD (22% and 5%, respectively) recipients (P < .01 for both). In the surviving patients there was no difference in T-cell reconstitution at the last follow-up between the URD and MSD recipients; however, MSD recipients were more likely to achieve B-cell reconstitution (72% vs 17%, P < .001). Unconditioned URD HCT achieves excellent rates of donor T-cell engraftment similar to that seen in MSD recipients, and reconstitution rates are adequate. However, only a minority will have myeloid and B-cell reconstitution, and attention must be paid to graft-versus-host disease prophylaxis. This approach might be safer in children ineligible for intense regimens to spare the potential complications of chemotherapy.

Newborn Screening for Severe Combined Immunodeficiency

Severe combined immunodeficiency (SCID) is a rare disorder that is lethal in childhood if not diagnosed and treated properly. Recently, a number of states have begun neonatal screening programs for SCID to facilitate diagnosis and improve outcomes. Here we review the pathogenesis and treatment of SCID. We also review the rationale for statewide screening programs, the molecular basis for the screening test, and discuss the experience to date of statewide screening in Wisconsin, the first state to implement such a program.

Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies

This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

Development of Safer Gene Delivery Systems to Minimize the Risk of Insertional Mutagenesis-Related Malignancies: A Critical Issue for the Field of Gene Therapy

Integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. The use of a retroviral-derived vector system was responsible for the development of leukemia in five children, who participated in various clinical trials for the treatment of severe combined immunodeficiency (SCID-X1) in France and in the United Kingdom. Unfortunately, the hematological malignancy claimed the life of one patient in 2004, who was enrolled in the French clinical trial. In addition, adeno-associated-viral-(AAV-) mediated gene transfer induced tumors in animal models, whereas the Sleeping Beauty (SB) DNA transposon system was associated with insertional mutagenesis events in cell culture systems. On these grounds, it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells. This paper discusses the latest achievements that have been reported in the field of vector design.

In Vitro Expansion and Transplantation of Intestinal Crypt Stem Cells

Adult tissue stem cells hold great promise for regenerative medicine strategies. Major hurdles in their clinical application persist and include the identification of such stem cells, their isolation and in vitro expansion, and—finally—their transplantation. These hurdles have recently been overcome for stem cells of the intestinal tract. Thus, exciting avenues are now opening up to apply these stem cells for a variety of therapeutic strategies. Herein, we highlight recent developments in intestinal stem cell biology and discuss this progress from the perspective of clinical application. Adult tissue maintenance and repair involves cell production by somatic stem cells and their transit-amplifying (TA) progenitor cells, followed by lineage commitment and terminal differentiation. The intestinal epithelium is the most vigorously regenerating tissue and is renewed every 3‐5 days. This rapid cell renewal, in combination with its modular architecture of crypts and villi, makes it an ideal model to study stem cell biology. The intestinal stem cells reside at the bottoms of crypts in the small intestine and colon. Their TA daughters occupy the remainder of the crypts. Differentiated cells reside on the villi in the small intestine and in crypt tops and in the flat surface epithelium of the colon. The differentiated cell types of the small intestine include the absorptive enterocytes and multiple secretory lineages (Paneth cells, goblet cells, enteroendocrine cells, and tuft cells), as well as the M cells of the Peyer’s patches. 1 Intestinal homeostasis is tightly controlled by wellcharacterized signaling pathways. In particular, the Wnt-, Bmp-, epidermal growth factor (EGF), and Notch pathways have been shown to be major players in this regulation. Wnt signaling constitutes the key pathway to maintain the stem cells and drive proliferation at crypt bottoms. Mice lacking Tcf7l2/Tcf4, a key transcription factor in this pathway, lose all proliferation in crypts. 2 Conversely, colon cancer is caused by loss of the Apc gene, which encodes a key negative regulator of the Wnt pathway. In addition to Wnt signals, Notch signaling is required to maintain crypt cells in the undifferentiated state. 3 EGF signals maintain the proliferative state of the crypt cells. Bmp and Tgf- signaling are activated in villus cells and are believed to regulate differentiation. Bmp ligands are expressed in villi, whereas Bmp antagonists such as noggin and gremlins are expressed at crypt mesenchyme to block Bmp signals from the crypts.4 All these pathways cross-talk with each other, forming complex molecular networks to govern tissue homeostasis.

Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Leukemia in a Murine Bone Marrow Transplantation Model

Abstract 671▪▪This icon denotes a clinically relevant abstractGene therapy with gammaretroviral and lentiviral vectors has shown its potential for the treatment of inherited hematopoietic disorders. However, the occurrence of severe adverse events, namely the induction of leukemias in clinical trails for the treatment of severe combined immunodeficiency (SCID)-X1, chronic granulomatous disease and Wiskott Aldrich Syndrome due to insertional activation of proto-oncogenes by the integrated vector raised safety concerns. Lentiviral vectors are assumed to be safer due to their integration bias to transcription units in comparison to gammaretroviral vectors that have an integration preference to promoter regions and CpG islands. However, the recent report on the development of clonal dominance in a clinical trial for b-thalassemia mediated by deregulated gene expression due to alternative splicing after lentiviral insertion into an intron of HMGA2 highlights potential risks of lentiviral gene therapy.Here we report a case of B-cell lymphoblastic leukemia in a murine bone marrow (BM) transplantation model 199 days post transplantation of cells transduced by a lentiviral vector expressing eGFP from the human glycoprotein-I-b-alpha (GPIba) promoter (-286 to +309 relative to the transcription start site). The mouse developed leukocytosis (179×103/μl white blood cells counts) and leukemic infiltrations were found in the BM, spleen, liver and lung. The leukemia was clonal as determined by Southern blot analysis, derived from the donor cells as determined by the CD45.1 chimerism marker and reinitiated disease in secondary animals (n=7). Leukemic cells had the morphology of lymphoid blasts/progenitors, stained positive for the markers B220, CD43, and partially CD19 and were negative for IgM. We therefore concluded that the cells were arrested and the PrePro-B to Pro-B cell stage.Two lentiviral integrations were identified in the leukemic clone of which one was mapped to intron 8 of the Early B-cell factor 1 (Ebf1) gene, a major regulator of B-cell development. The other insertion site was detected in the first intron of the Nance-Horan-Syndrome-gene that has no known function in hematopoiesis and we could not detect altered expression of this gene. No further genomic alterations were identified by spectral karyotyping and Array CGH analysis. Because the vector contained various splice sites in its backbone and the internal GPIba promoter, we next analyzed potential splice products and detected mRNAs formed from splice events between Ebf1 exons 8 and 9 to the integrated vector.Some of these could result in early termination of the mRNA which would generate a truncated Ebf1 protein containing only the DNA-binding domain. However, the overall contribution of this early stop mRNAs was low (5% of total Ebf1 message) and we could not detect a truncated Ebf1 protein by Western blot. Overexpression of the truncated Ebf1 protein in a B-cell differentiation assay on OP9 cells in vitro did not interfere with B-cell development.In contrast, we found the Ebf1 full length mRNA to be ∼4-fold downregulated and the EBf1 protein in leukemic cells to be reduced compared to stage matched BM B-cell progenitors (CD19+CD43+). Genome wide expression analysis of leukemic cells compared to B-cell progenitors revealed downregulation of Ebf1 target genes and definite signs of transdifferentiation to the myeloid lineage as it was described upon loss of Ebf1 or its downstream target Pax5 in other studies. In addition, Gene Set Enrichment and Gene Ontology analyses suggested upregulation of genes implicated in JAK-STAT signalling. Indeed, we observed strong upregulation of Flt3 and high levels of STAT5 activation, in line with recent reports of B-cell leukemia development in Ebf1+/− mice overexpressing constitutively active STAT5 (Heltemes-Harris et al., J. Exp. Med 2011). STAT5 activation is also found in a large subset of human B-ALL and the occurrence of mono- or bi-allelic EBF1 deletions in human B-ALL is known (Mullighan et al., Nature, 2007). As the lentiviral integration was an early event in the leukemia development in our experiments, haploinsufficiency of Ebf1 will have been most likely the leukemia initiating event.Taken together our results highlight the risk of intragenic lentiviral vector integration of vectors containing splice sites that can induce alternative splicing, which, as shown here, may lead to inactivation of haploinsufficient tumour suppressor genes. Disclosures:Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.