Severe Combined Immune Deficiency Disease Research Articles

Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy.

The importance of safer approaches for gene therapy has been underscored by a series of severe adverse events (SAEs) observed in patients involved in clinical trials for Severe Combined Immune Deficiency Disease (SCID) and Chromic Granulomatous Disease (CGD). While a new generation of viral vectors is in the process of replacing the classical gamma-retrovirus–based approach, a number of strategies have emerged based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer. Currently, these methods display lower efficacies than viral transduction although many of them can yield more than 1% engineered cells in vitro. Nuclease-based approaches, wherein an endonuclease is used to trigger site-specific genome editing, can significantly increase the percentage of targeted cells. These methods therefore provide a real alternative to classical gene transfer as well as gene editing. However, the first endonuclease to be in clinic today is not used for gene transfer, but to inactivate a gene (CCR5) required for HIV infection. Here, we review these alternative approaches, with a special emphasis on meganucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential.

Treatment of Adenosine Deaminase Deficiency

Severe combined immune deficiency disease due to a deficiency of the enzyme adenosine deaminase is a rare disease. However, it has been used as a prototype disease for the development of a variety of treatment modalities that are currently applied in more frequent diseases. For example, allogeneic bone marrow transplantation and stem cell gene therapy have been used for adenosine deaminase deficiency before being applied in other more frequent diseases. In the present paper, the development of bone marrow transplantation and stem cell gene therapy, as well as treatment with purified enzyme, for adenosine deaminase deficiency are discussed.

The Level of Serum Adenosine Deaminase Activity in Leprosy and Tuberculosis

To the Editor: The aim of this study was to investigate whether there could be a resemblance of the expected values of serum adenosine deaminase activity in tuberculosis and leprosy. The study was carried out with 85 tuberculosis patients and 46 leprosy patients within the age range of 15-65 years, and the results were compared with those obtained from 76 healthy control group subjects. The specific activity of adenosine deaminase is high in the human spleen, duodenum and T lymphocytes. In recent years, clinical interest in this enzyme has been focused on immunodeficiency. The deficiency of this enzyme can be observed in severe combined immune deficiency disease. 1,2 Leprosy and tuberculosis are caused by the same group of bacteria, Mycobacterium tuberculosis and Mycobacterium leprae. In these diseases, the cellular immunity was stimulated. Blood samples were obtained from 85 patients (32 female, 53 male) between the ages of 15 and 66 years in Elazig Tuberculosis Dispensary, Turkey. Sixty-five of these patients were diagnosed with active tuberculosis and 20 had inactive tuberculosis. The diagnosis was supported by clinical, bacteriological and x-ray radiography. The group termed as inactive tuberculosis was the clinically recovered group followed without medication, but with calcification findings in x-ray radiography. Blood samples were obtained from 46 leprosy patients (15 female, 31 male) aged between 18 and 65 years in Elazig Leprosy Hospital. All patients were LL (lepromatous leprosy) type without clinical evidence of reaction, and three of these patients were bacilli-positive under multidrug therapy, and the remainder were bacilli-negative. The control group was composed of 77 subjects (25 female, 52 male) in the same age group who applied to the blood bank as donors. Human blood serum was kept at –20°C until the analysis. The serum adenosine deaminase activity was determined by Giusti’s spectophotometric method—adenosine hemisulfate 12 mmol/L and phosphate buffer 50 mmol/L (pH: 6.5). 3

Interference with thymocyte differentiation by an inhibitor of S-adenosylhomocysteine hydrolase.

Adenosine deaminase (ADA) deficiency causes severe combined immune-deficiency disease in humans. It is believed that the accumulation of the ADA substrate deoxyadenosine affects T cell development through interference with deoxynucleotide metabolism and/or S-adenosylmethionine-mediated methylation processes. In this study, we used a specific inhibitor of methylation, (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (FddA), to study the effect of inhibition of methylation on intrathymic T cell development in a murine model for ADA deficiency. FddA causes inhibition of thymocyte differentiation specifically at the CD8low and CD4CD8 double-positive stages. This inhibition is not due to induction of apoptosis, rather it is a result of specific inhibition of regulation of the levels of the mRNAs coding for TCR and CD4 and CD8 co-receptor molecules that normally occurs at these stages of thymocyte differentiation. We hypothesize that the transcription of these T cell-specific molecules is regulated by a common developmental stimulus involving a S-adenosylmethionine-mediated methylation step. Identification of this step will help in understanding the role of methylation in intrathymic differentiation and will also provide a molecular explanation for the tissue and developmental stage specificity observed in severe combined-immune-deficient patients with ADA deficiency.

Identification of astrovirus serotypes from children treated at the Hospitals for Sick Children, London 1981–93

An enzyme immunoassay (EIA) for astrovirus type 1 together with immune electronmicroscopy (IEM) was used to type a collection of 162 astroviruses obtained from 1981-93 from children with diarrhoea. The EIA was found to be specific for astrovirus type 1. Astrovirus types 2-4 were typed by IEM. Astrovirus type 1 was the prevalent serotype 107/125 (86%), followed by type 3 (8%), type 4 (6%) and type 2 (1%). Six samples containing astrovirus could not be typed or detected by EIA because they were coated with coproantibodies; 11 others were not identified. Virus particles could no longer be detected in 15/162 (9%) samples following storage for > or = 2 years. Selected samples containing astrovirus types 1-4 were passaged in CaCO2 cells and their identity confirmed by one or both assays. One sample was shown to have remained viable for 10 years when stored as an aqueous suspension at -20 degrees C. Two patients with severe combined immune deficiency disease (SCID) were shown to be excreting astrovirus type 1 for 32 and 102 days respectively. One child was simultaneously shedding rotavirus and the other child was excreting adenovirus.

Bone marrow transplantation for genetic disorders

In 1967, a congenital disorder, severe combined immune deficiency disease, (SCID), was the first condition to be successfully corrected by bone marrow transplantation (BMT) from a histocompatible matched sibling donor. 1 Since then the number of inherited disorders in which BMT has been used has been greatly extended. 2 In preface, it should be stressed that BMT represents only one aspect of the management of genetic disorders which includes first and foremost detection and prevention by antenatal screening. Enzyme replacement treatment and the development of genetic engineering techniques to correct the underlying fault are being actively explored. However, reliable screening programmes are only feasible in a minority of disorders, of which thalassaemia is an example. Enzyme replacement treatment has been largely unsuccessful, and despite considerable advances in the understanding of gene regulation, at present BMT represents the only practice capable of correcting genetic disorders and improving the quality of life of affected individuals.

Evidence that specific cellular immunity cannot account for death of Mus caroli embryos transferred to Mus musculus with severe combined immune deficiency disease.

Mus caroli blastocysts transferred to pseudopregnant Mus musculus implant but die about midgestation. Death of the M caroli embryos is associated with infiltration of the fetoplacental units by M musculus lymphocytes, including functional cytotoxic T lymphocytes (CTL). To determine if specific maternal antifetal immune responses are responsible for the initiation of M caroli failure, survival of M caroli embryos transferred to M musculus with severe combined immune deficiency disease (SCID) was studied. C.B.-17 scid mice, homozygous for the scid gene, are reported to be unresponsive to T cell or B cell mitogens and to alloantigens. We have evaluated responses of C.B.-17 scid mice to M caroli xenoantigens in vivo and in vitro. C.B.-17 scid fail to reject M caroli skin grafts, and their spleen cells do not proliferate when mixed with M caroli splenocytes in a 1-way mixed leukocyte culture. Further, neither primary nor secondary challenge of C.B.-17 scid footpads with M caroli splenocytes elicits a delayed-type hypersensitivity (DTH) response. These results suggest that C.B.-17 scid mice are unable to recognize or respond specifically to M caroli xenoantigens. M caroli embryos transferred to C.B.-17 scid mice implant but fail to survive while cotransferred M musculus embryos are normal. Histologic examination of resorbing embryos revealed granulocytes, macrophages, large granular lymphocytes, but very few small lymphocytes at the implantation sites. Thus, it appears that specific maternal antifetal immune responses are not responsible for initiation of M caroli embryo failure in the M musculus uterus. These studies also indicate that efficient resorption of embryos does not require the function of T cells or B cells.

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture.

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice.

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.

Natural cytotoxicity in immunodeficiency diseases: Preservation of natural killer activity and the in vivo appearance of radioresistant killing

We studied spontaneous natural killer (NK) cell activity and radiation-resistant NK mediated cytotoxicity in four patients with clinically documented severe combined immune deficiency disease (SCID), and in one subject each with intestinal lymphangiectasia and cartilage-hair hypoplasia. We observed the preservation of spontaneous NK activity in all patients despite the presence of profound B- and T-lymphocytopenia and clinical immunodeficiency. NK activity was associated with relatively normal circulating numbers of OKM1 + lymphocytes, a population known to contain NK effectors. Spontaneous NK activity resistant to 3000 rad was increased in all patients, indicating the presence of activated natural killer cells in vivo. The concept of a chronically activated immune system in these patients was further supported by the presence of increased Ia positive T cells in all subjects tested, suggesting that radioresistant NK activity may be a useful parameter to measure when assessing in vivo immune activation. Our data, as well as that of others, supports the hypothesis that at least one population of NK cells is a distinct lineage arising at the differentiation level of myeloid and lymphoid stem cells in the bone marrow.

MISMATCHED BONE MARROW TRANSPLANTATION (BMT) USING SOYBEAN AGGLUTININ (SBA) NEGATIVE MARROW CELLS

The limitation of mismatched BMT is fatal graft vs. host disease (GVHD). We processed haplotype mismatched parental marrow with SBA to enrich for stem cells and reduce GVHD and successfully treated 6 patients with severe combined immune deficiency disease. HLA chimerism was found within 3 weeks post BMT. All the patients demonstrate T cell and 2 have evidence of B cell engraftment. T cell numbers and responses to phytohemagglutinin (PHA) and alloantigen pre and post BMT are: In #2,5,6 who required pre BMT immunosuppression for maternal GVHD, neutrophil, RBC, and platelet engraftment occurred by days 11,26,32 respectively. No immunosuppression was given post BMT. 2 patients had no GVHD, 3 had transient rash/fever, and only 1 developed persistent rash. Patients are now 3 to 17mo. post BMT. These results demonstrate that treated mismatched BMT can result in engraftment without significant GVHD and can be used for patients who otherwise have limited hope for survival.

917 SEVERE COMBINED IMMUNE DEFICIENCY DISEASE – A T-CELL DISORDER

Severe combined immune deficiency disease (SCID) represents a spectrum of disorders characterized by the inability to manifest normal cell-mediated and humoral immunity. Recognition and grouping of these variants of SCID are important in order to provide guidelines for investigation and to evaluate and select appropriate therapeutic modalities. In contrast to earlier suggestions that these patients manifest abnormalities of lymphoid stem cells, our studies have demonstrated T and B-cell precursor cells in the majority of SCID patients. We have used in vitro assays for monitoring the induction of T-cell differentiation and have characterized the responsiveness of patient precursor T-cells to specific induction regimens including contact with thymic epithelium, epithelium conditioned medium, thymosin and theophylline. In this way, we have identified different groups of patients with an arrest of T-cell differentiation at different stages of maturation. In most of the patients with normal adenosine deaminase, the combined immune deficiency could be shown to reflect the failure of normal T-cell differentiation and consequent failure of T-cell dependent maturation of B lymphocytes to an antibody-secreting stage, rather than intrinsic abnormalities of the B cells themselves. These data emphasize the thymic dependence of specific B-cell differentiation in man and demonstrate the functional integrity of B lymphocytes in many patients with SCID.

Severe Combined Immune-Deficiency Disease

Severe Combined Immune-Deficiency Disease

Germ-free delivery. The initiation of management of infants with a high probability of congenital immune deficiency states

Germ-free delivery of an infant with a high probability of congenital severe combined immune deficiency disease was accomplished by cesarean section without major modification of routine medical and nursing procedures. The male sex of the infant had been determined by karyotype on amniotic fluid cells, thus the risk of being affected by an X-linked disease was established. Intensification of ordinary methods for preparation of operating area and personnel resulted in successful germ-free delivery. Highly technical equipment and specially trained people with attendant high cost were not required. The infant was placed in a sterile plastic flexible film isolator where he has been maintained in a gnotobiotic state for twelve months (to date) free of infection in spite of continued immune incompetence. With the likelihood that lesser forms of this disease may be more common than heretofore expected and with the advent of new obstetric and immunologic diagnostic techniques, sterile delivery may assume more importance in the future.

Serum IgE in clinical immunology and allergy

Serum IgE was measured with the use of the Rowe modification of the Mancini technique. Serum IgE was significantly elevated in atopic and questionably atopic patients and in patients with filariasis within 2 years of diagnosis. Children with thymic hypoplasia and with cystic fibrosis had higher IgE levels than did control clinic patients. Children with variable immunodeficiency and X-linked agammaglobulinemia had significantly less IgE than did control patients, and patients with severe combined immune deficiency disease when not treated with plasma had no detectable IgE. The value of IgE determinations in groups of patients was demonstrated.