X-linked Severe Combined Immunodeficiency Disease Research Articles

Immune Reconstitution After Gene Therapy Approaches in Patients With X-Linked Severe Combined Immunodeficiency Disease.

X-linked severe immunodeficiency disease (SCID-X1) is an inherited, rare, and life-threating disease. The genetic origin is a defect in the interleukin 2 receptor γ chain (IL2RG) gene and patients are classically characterized by absence of T and NK cells, as well as presence of partially-functional B cells. Without any treatment the disease is usually lethal during the first year of life. The treatment of choice for these patients is hematopoietic stem cell transplantation, with an excellent survival rate (>90%) if an HLA-matched sibling donor is available. However, when alternative donors are used, the success and survival rates are often lower. Gene therapy has been developed as an alternative treatment initially using γ-retroviral vectors to correct the defective γ chain in the absence of pre-conditioning treatment. The results were highly promising in SCID-X1 infants, showing long-term T-cell recovery and clinical benefit, although NK and B cell recovery was less robust. However, some infants developed T-cell acute lymphoblastic leukemia after the gene therapy, due to vector-mediated insertional mutagenesis. Consequently, considerable efforts have been made to develop safer vectors. The most recent clinical trials using lentiviral vectors together with a low-dose pre-conditioning regimen have demonstrated excellent sustained T cell recovery, but also B and NK cells, in both children and adults. This review provides an overview about the different gene therapy approaches used over the last 20 years to treat SCID-X1 patients, particularly focusing on lymphoid immune reconstitution, as well as the developments that have improved the process and outcomes.

Review: Oncogenic Insertional Mutagenesis as a Consequence of Retroviral Gene Therapy for X-Linked Severe Combined Immunodeficiency Disease.

For X-linked severe combined immunodeficiency (SCID-X1), the practice of gene therapy has revealed an unusual effect: insertional mutagenesis that can lead to leukemia. Even though incorporation of the retrovirus close to the oncogene for T-cell acute lymphoblastic leukemia (T-ALL), LIM-only protein 2 (LMO2) is observed frequently, but it is not clear why LMO2 expression is affected. It was demonstrated that in all the typical T-ALL oncogenes, there is mainly transcription of LMO2 in CD34+ progenitor cells. LYL1, TAN1, and TAL1 are very important intensification factors that are classically used in the gene therapy for copying LMO2 when they are stimulated. For this reason, oncogenes are susceptible to amalgamation with viruses. The IL-2R-gamma (IL-2 receptor γ chain) was found to be a supporting oncogene to LMO2. Nevertheless it was illustrated that excessive expression of IL-2R-gamma did not affect T-cell growth. In comparison to it, the excessive expression of LMO2 in CD34+ cells can cause ongoing increases in the development of T cells. Conversely, there is no effect on the development of B cells and myeloid cells. This information helps explain why LMO2 is mostly affected by various identified T-ALL oncogenes. In addition, throughout the process of T-cell development, expression of IL2R-gamma mediated by retrovirus may not always be oncogenic. As an alternative, replacement of signals of common IL-7 receptors may increase development of T cells wherever LMO2 was expressed and caused abnormal thymocyte development.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy

AbstractHematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

Rapid Expansion of Gene-Marked Lymphocytes in X-SCID Dogs after AMD3100+G-CSF-Based Hematopoietic Stem/Progenitor Cell Mobilization and Intravenous Injection of a Common γ-Chain Expressing Foamy Viral Vector

In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to <1500 cells/uL with <5% gene marking in unmobilized dogs treated with EF1a-gammaC-FV and to <1000 cells/uL with <50% gene marking in unmobilized dogs treated with PGK-gammaC-FV at all time points post-therapy. The expansion of CD3+ T-cells at 6 weeks post injection for the mobilized dogs was about 2700 cells/uL, as compared to <380 cells/uL in the PGK-gammaC-FV and <210 cells/uL in the EF1a-gammaC-FV unmobilized dogs. Notably, in human clinical trials, CD3 T cell counts were <250 cells/uL following transplantation with autologous CD34+ cells modified with EF1a-gammaC-SIN gamma-retrovirus (Hacein-Bey-Abina, NEJM, 2014). In conclusion, mobilization with cG-CSF and AMD3100 prior to in vivo injection of PGK-gammaC-FV substantially improved the lymphocyte expansion and immune reconstitution in X-SCID dogs and resulted in a higher level of gene marking in myeloid cells (about 1%) at one-month post injection than seen in our previous studies in unmobilized dogs. These results suggest remarkable potential for an accessible and portable approach for treatment of human X-SCID clinical trials using combination of hematopoietic stem/progenitor cells mobilization and in vivo foamy viral vector delivery. [Display omitted] [Display omitted] [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Perspectives on stem cell gene therapy for genetic disorders

Gene transfer into haematopoietic stem cells (HSCs) has now come to the stage where it can be recognized as a form of established therapy for patients with a certain range of genetic diseases. Primary immunodeficiency disease (PID) represents an ideal pathological target for this type of genetic medicine, that is HSC gene therapy, as exemplified well by the successful cases with adenosine‐deaminase (ADA) deficiency. Other diseases including X‐linked severe combined immunodeficiency disease (X‐SCID), chronic granulomatous disease (CGD) and Wiskott–Aldrich syndrome (WAS) have also been the targets of gene therapy, but the efficacy reportedly varies considerably. In addition, all those diseases except ADA deficiency have been demonstrated to be subject to an inherent risk of insertional leukaemogenesis as long as gene transfer accompanies genomic insertion. Researchers therefore should continue to aim for further improvement of HSC gene therapy to maximize both safety and efficacy. To achieve this aim, an ideal model system is desired, by which faithful recapitulation of the disease‐related pathophysiology is feasible. Recently, a new model system possibly constituting such an ideal platform has come to reality; that is, patient‐/disease‐specific induced pluripotent stem cells (iPSCs). iPSCs should particularly be useful for modelling genetic disorders, because of their excellent potential to differentiate into any types of somatic cells while retaining the patient‐specific genetic mutations. These model cells should allow testing various kinds of genetic manipulation and also detailed analysis of the target cells before/after differentiation, thus providing a disease‐specific platform for preclinical studies to test safety and efficacy of each gene‐modification procedure.

Viral Vectors for Gene Therapy of Genetic Diseases: Challenges and Prospects

Viruses naturally possess specialized molecular mechanisms to efficiently transfer their genetic materials into the infected cells. Viral vectors can be used to modify specific cell type or tissue and can be directed to express therapeutic genes. These unique abilities made them desirable for engineering viral vectors to deliver the therapeutic genes. The efficient delivery of therapeutic genes and correct gene expression are important for clinically relevant gene therapy. The most commonly used viral vectors are derived from retroviruses, adenoviruses, and adeno associated viruses (AAV). Gene therapy has a turbulent history because of failure of the first clinical trial. Jesse Gelsinger [1] who was suffering from a partial deficiency of ornithine trans carbamyalse (OTC), took part in a gene therapy clinical trial at the University of Pennsylvania, Philadelphia. Due to immune responses caused by a high dose of adenoviral vector containing OTC, Gelsinger died four days later because of multi-organ failure [1]. In another clinical trial conducted in France and UK, among twenty X-linked severe combined immunodeficiency disease (X-SCID) patients, 19 were successfully cured their immunodeficiency by retroviral vector containing IL-2RG (γc). Four of the treated patients developed T cell leukemia because the gamma retrovirus integrated in/near the proto-oncogenes that turned the gene on. But the patients were successfully treated with chemotherapy and in complete remission [2].

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Current status and future prospects of stem cell gene therapy for primary immunodeficiency

Patients affected by primary immunodeficiency (PID) can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). In the absence of HLA-matched donors, however, incidence of HSCT-related complications is observed. Therefore, gene therapy has been developed as a highly desirable alternative treatment for patients lacking suitable donors. Retrovirus-based gene therapy was begun in 1990 for the patients of adenosine deaminase deficiency, followed by X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome and chronic granulomatous disease. Although treated patients have had excellent immune reconstitution and resolution of ongoing infections, complications such as a lymphoproliferative syndrome and a disappearance of gene-modified cells were observed in some clinical trials. To overcome these, ongoing and upcoming clinical trials use some new strategies. The use of preconditioning chemotherapy makes space in the bone marrow for the gene-treated stem cells and allows engraftment of multi lineage stem/progenitor cells. Self-inactivating vectors in which strong enhancers of long terminal repeat are eliminated may reduce the risk of insertional activation of proto-oncogene resulting in leukemia. These modifications will surely increase the safety and efficacy of stem cell gene therapy for PID.

Phoenix rising: gene therapy makes a comeback

Despite the first application of gene therapy in 1990, gene therapy has until recently failed to meet the huge expectations set forth by researchers, clinicians, and patients, thus dampening enthusiasm for an imminent cure for many life-threatening genetic diseases. Nonetheless, in recent years we have witnessed a strong comeback for gene therapy, with clinical successes in young and adult subjects suffering from inherited forms of blindness or from X-linked severe combined immunodeficiency disease. In this review, various gene therapy vectors progressing into clinical development and pivotal advances in gene therapy trials will be discussed.

Site-specific Gene Insertion Mediated by a Cre-loxP-carrying Lentiviral Vector

Retroviral vectors have been used to treat patients with the X-linked severe combined immunodeficiency disease and chronic granulomatous disease. In both cases, success has been undermined by clonal expansion of transduced cells in some patients due to insertional mutagenesis induced by random vector integration. This outcome underscores the importance of designing vectors for site-specific gene insertion to avoid unanticipated gene disruption or gene activation. In the present study, we incorporated the sequence-specific Cre protein into lentiviral virions. We demonstrated that the virion-associated Cre protein remained enzymatically active and was capable of directing site-specific insertion of a gene in the vector into a defined loxP site in the host genome. As there are loxP-like sequences throughout human genome that can be recognized by either wild-type Cre or Cre variants, our study demonstrates a new strategy of designing lentiviral-based vector for gene targeting.

Megadose CD34+ Cell Grafts Improve Recovery of T Cell Engraftment but not B Cell Immunity in Patients with Severe Combined Immunodeficiency Disease Undergoing Haplocompatible Nonmyeloablative Transplantation

To determine whether T cell engraftment and recovery of B cell immunity could be improved, we prospectively treated 15 children with severe combined immunodeficiency disease (SCID) with megadoses of haplocompatible CD34+ cells and a fixed number of CD3+ cells without previous myeloablative chemotherapy. Evidence of T cell engraftment was seen in 73% of patients (95% confidence interval [CI] = 48%-90%). Engraftment was more likely in patients with X-linked SCID and in those with evidence of maternal engraftment at the time of diagnosis. In patients with T cell engraftment, the median time to development of a CD4 count > 200 cells/mm3 and a phytohemagglutinin response > 50% of control was 1.2 and 4.9 months, respectively. Clearance of preexisting infections occurred after a median of 2.8 months. B cell function developed in 33% of engrafted patients (95% CI = 14%-61%). The 1-year event-free survival (EFS) rate was 60% (95% CI = 36%-80%), and the overall survival (OS) rate was 87% (95% CI = 61%-98%), with a median follow-up of 39 months. The use of megadoses of CD34+ cells with a fixed number of CD3+ cells in nonmyeloablative hematopoietic stem cell transplantation (HSCT) in patients with SCID is associated with excellent engraftment, T cell recovery, and OS; however, B cell function does not recover in most patients.

Reducing the genotoxic potential of retroviral vectors.

The recent development of leukemia in gene therapy patients with X-linked severe combined immunodeficiency disease because of retroviral vector insertional mutagenesis has prompted reassessment of the genotoxic potential of integrating vector systems. In this chapter, various strategies are described to reduce the associated risks of retroviral genomic integration. These include deletion of strong transcriptional enhancer-promoter elements in the retroviral long terminal repeats, flanking the retroviral transcriptional unit with enhancer blocking sequences and designing vectors with improved RNA 3' end processing. Protocols are provided to evaluate the relative biosafety of the modified vectors based on their ability to immortalize hematopoietic progenitor cells and propensity to trigger clonal hematopoiesis or leukemogenesis following hematopoietic stem cell transplantation.

Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

Integrating Ideas on Insertional Mutagenesis by Gene Transfer Vectors

The 3rd Stem Cell Clonality and Genotoxicity Retreat was held 12–13 December 2006, directly following the 48th annual meeting of the American Hematological Society held in Orlando, Florida. This series of retreats was organized by David Williams, Chris Baum, and Christof von Kalle of Cincinnati Children's Hospital. The impetus was the incidence of insertional leukemogenesis arising in the French gene therapy trial treating X-linked severe combined immunodeficiency disease (X-SCID) in 2002. Prior to 2002, the risk of insertional mutagenesis from gene transfer vectors was a theoretical one that had not been followed up with rigorous proactive studies. Of course, this all changed after the adverse events in the French trial and a case of insertional mutagenesis discovered in an animal study earlier the same year by Chris Baum's group. In the following years, evidence was found for clonal dominance in hematopoietic stem cells transduced with retroviral vectors in both mice and macaques, and in patients with chronic granulomatous disease (CGD) treated with gene-corrected hematopoietic cells.

Epstein-Barr–associated Leiomyomatosis and T-cell Chimerism After Haploidentical Bone Marrow Transplantation for Severe Combined Immunodeficiency Disease

The clinical course of Epstein-Barr virus (EBV)-associated smooth muscle tumors is variable and there are no reports in patients with mixed T-cell chimerism after bone marrow transplantation (BMT). A child with X-linked severe combined immunodeficiency disease developed multiple renal and pulmonary leiomyomata 8 years after haploidentical BMT. Epstein-Barr viral DNA was detectable in the blood and in situ hybridization for EBV-encoded RNAs was positive in the tumor. The tumors have been radiographically stable, chimerism remains mixed, and plasma EBV DNA has been repeatedly negative for over 2 years after donor lymphocyte infusion. EBV-associated smooth muscle tumors may occur in patients who are partially reconstituted after BMT for severe combined immunodeficiency and may not require surgery or chemotherapy.

CD34+ Selected T-Cell Depleted Peripheral Blood Stem Cell Transplantation from HLA-Haplocompatible Donors for the Treatment of Severe Combined Immunodeficiency Disease.

We report the results of haplocompatible peripheral blood stem cell transplantation (PBSCT) utilizing CD34+ selection and T-cell depletion for 17 patients with severe combined immunodeficiency disease (SCID). Of these patients, 11 had T−B−NK+, 1 had T−B+NK+, and 5 had T−B+NK− phenotype. A total of 15 cell preparations were processed immediately after collection, in which two were shared between two twin siblings. Total viable nucleated cells (TVNC) in the original cell collections were between 5.9 and 9.13×1010 (median 7.0×1010) with 0.47–2.39% (median 0.9%) CD34+ cells. After Isolex 300i (Baxter Inc., n=14) or CliniMACS System (Miltenyi Biotec Inc., n=1) processing, a median number of 379×106 (89–970×106) TVNC were recovered, with a median viability of 98% (83–100%) and median purity of 96% (89–100%) CD34+ cells. All Isolex processed (n=14) cells were further T-cell depleted with OKT3 monoclonal antibody, yielding a median of 0.09% CD3+ cells (0.008–0.4%). One preparation that utilized the CliniMACS System yielded only 0.06% CD3+ cells, hence did not receive further T-cell depletion. Recovery of CD34+ cells after complete processing was from 13.3% to 60.2% (median 50.1%). Twelve patients (70.6%) are alive 2 months to 8.7 years post transplant. A total of five patients died from infections or transplant-related complications. Four patients suffered from autoimmune hemolytic anemia, which resulted in one death. Fourteen patients engrafted. One of three patients who did not engraft subsequently received a boost from the same donor but eventually died without engraftment, and 2 received a matched unrelated BMT with myeloablative conditioning and recovered T- and B-cell function. At last follow-up the median time for the recovery of T- and B-cell function was 8.5 months and 1 year, respectively. The dose of CD3 did not show any influence on T- or B-cell function recovery (p=0.48 and 0.09, respectively). And the dose of CD34+ cells did not influence T-cell function recovery (p=0.1), but did influence B-cell function recovery, which was statistically significant (p=0.02). The B− SCID phenotype is associated with a poorer outcome compared to the B+ SCID phenotype, with 50% and 100% survival rates, respectively. However, this result was not statistically significant (p=0.07). Of the 9 surviving patients followed for more than 2 years, most are in good general health. The body height growth curve is within the 5th and 10–25th percentiles in 3 and 4 patients, respectively. For body weight, the growth curve is within 10–25th and 50–75th percentiles in 5 and 2 patients, respectively. Five have achieved successful recovery of both T- and B-cell immunity and require no medication at last visit; however, 2 of these had graft failure following their initial haplocompatible transplant, and received a second BMT from a matched unrelated donor with conditioning. Three patients with X-linked SCID (3/5) didn't achieve B-cell reconstitution and still require IVIG replacement therapy. Based on these results, we conclude that for SCID patients who lack an HLA-matched related donor, CD34+ selected T-cell depleted haplocompatible PBSCT is an effective treatment.

EBV-associated leiomyomas following haploidentical transplantation for X-linked severe combined immunodeficiency disease

EBV-associated leiomyomas following haploidentical transplantation for X-linked severe combined immunodeficiency disease

A critical role for IL-21 in regulating immunoglobulin production.

The cytokine interleukin-21 (IL-21) is closely related to IL-2 and IL-15, and their receptors all share the common cytokine receptor gamma chain, gammac, which is mutated in humans with X-linked severe combined immunodeficiency disease (XSCID). We demonstrate that, although mice deficient in the receptor for IL-21 (IL-21R) have normal lymphoid development, after immunization, these animals have higher production of the immunoglobulin IgE, but lower IgG1, than wild-type animals. Mice lacking both IL-4 and IL-21R exhibited a significantly more pronounced phenotype, with dysgammaglobulinemia, characterized primarily by a severely impaired IgG response. Thus, IL-21 has a significant influence on the regulation of B cell function in vivo and cooperates with IL-4. This suggests that these gammac-dependent cytokines may be those whose inactivation is primarily responsible for the B cell defect in humans with XSCID.

Gene therapy in recovery phase

Last month's news that a team at London's Great Ormond Street Hospital used gene therapy to cure a child with X-linked severe combined immunodeficiency disease (SCID) suggests that a more conservative approach will not slow progress in the field.

Hematopoietic stem cell gene therapy for inherited bone marrow disorders: past accomplishments and continued challenges.

From the time that the genes encoding the defective proteins were cloned for a number of inherited diseases, it became a goal to correct those conditions by restoring the normal gene and thereby, its product. For the inherited disorders affecting the blood and its progenitor cells, the hematopoietic stem cells were the ideal target cells for gene transfer, because the normal gene would then be transferred to all of the progeny cells, theoretically for the lifetime of the recipient. However, the tasks of isolating the hematopoietic stem cells, introducing the new genes in such a manner as to preserve engraftment of the manipulated cells, and achieving long-term gene expression, have not been straightforward in the clinical trial setting, although there has been moderate success for cells in vitro, and in murine studies. With the report of clinical efficacy of gene transfer in children with X-linked severe combined immunodeficiency disease, the dream of clinical gene transfer to hematopoietic cells has become a reality. But there are still significant impediments remaining for a number of diseases. The innovations of introduction of synthetic receptors that confer growth advantage, the use of lentiviral vectors with increased stem cell transduction efficiency, and the addition of modified promoter/enhancer sequences to augment and preserve gene expression may bring wider success to gene therapy clinical trials for bone marrow disorders in the near future.