A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID.

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.

The Antioxidant TEMPOL Protects Human Hematopoietic Stem Cells From Culture-Mediated Loss of Functions.

In a steady state, hematopoietic stem cells (HSC) exhibit very low levels of reactive oxygen species (ROS). Upon stress, HSC get activated and enter into proliferation and differentiation process to ensure blood cell regeneration. Once activated, their levels of ROS increase, as messengers to mediate their proliferation and differentiation programs. However, at the end of the stress episode, ROS levels need to return to normal to avoid HSC exhaustion. It was shown that antioxidants can prevent loss of HSC self-renewal potential in several contexts such as aging or after exposure to low doses of irradiation suggesting that antioxidants can be used to maintain HSC functional properties upon culture-induced stress. Indeed, in humans, HSC are increasingly used for cell and gene therapy approaches, requiring them to be cultured for several days. As expected, we show that a short culture period leads to drastic defects in HSC functional properties. Moreover, a switch of HSC transcriptional program from stemness to differentiation was evidenced in cultured HSC. Interestingly, cultured-HSC treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO or Tempol) exhibited a higher clonogenic potential in secondary colony forming unit cell (CFU-C) assay and higher reconstitution potential in xenograft model, compared to untreated cultured-HSC. By transcriptomic analyses combined with serial CFU-C assays, we show that Tempol, which mimics superoxide dismutase, protects HSC from culture-induced stress partly through VEGFα signaling. Thus, we demonstrate that adding Tempol leads to the protection of HSC functional properties during ex vivo culture.

Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients.

BACKGROUND: Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE: We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS: We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS: Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS: Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy.

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPß expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Human kidney-derived hematopoietic stem cells can support long-term multilineage hematopoiesis.

Long-term multilineage hematopoietic donor chimerism occurs sporadically in patients who receive a transplanted solid organ enriched in lymphoid tissues such as the intestine or liver. There is currently no evidence for the presence of kidney-resident hematopoietic stem cells in any mammal species. Graft-versus-host-reactive donor T cells promote engraftment of graft-derived hematopoietic stem cells by making space in the bone marrow. Here, we report full (over 99%) multilineage, donor-derived hematopoietic chimerism in a pediatric kidney transplant recipient with syndromic combined immune deficiency that leads to transplant tolerance. Interestingly, we found that the human kidney-derived hematopoietic stem cells took up long-term residence in the recipient's bone marrow and gradually replaced their host counterparts, leading to blood type conversion and full donor chimerism of both lymphoid and myeloid lineages. Thus, our findings highlight the existence of human kidney-derived hematopoietic stem cells with a self-renewal ability able to support multilineage hematopoiesis.

Transient mTOR inhibition rescues 4-1BB CAR-Tregs from tonic signal-induced dysfunction.

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition.

Retrieval of vector integration sites from cell-free DNA.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors.

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

Adenylate kinase 2 expression and addiction in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) represents the malignant expansion of immature T cells blocked in their differentiation. T-ALL is still associated with a poor prognosis, mainly related to occurrence of relapse or refractory disease. A critical medical need therefore exists for new therapies to improve the disease prognosis. Adenylate kinase 2 (AK2) is a mitochondrial kinase involved in adenine nucleotide homeostasis recently reported as essential in normal T-cell development, as defective AK2 signaling pathway results in a severe combined immunodeficiency with a complete absence of T-cell differentiation. In this study, we show that AK2 is constitutively expressed in T-ALL to varying levels, irrespective of the stage of maturation arrest or the underlying oncogenetic features. T-ALL cell lines and patient T-ALL-derived xenografts present addiction to AK2, whereas B-cell precursor ALL cells do not. Indeed, AK2 knockdown leads to early and massive apoptosis of T-ALL cells that could not be rescued by the cytosolic isoform AK1. Mechanistically, AK2 depletion results in mitochondrial dysfunction marked by early mitochondrial depolarization and reactive oxygen species production, together with the depletion of antiapoptotic molecules (BCL-2 and BCL-XL). Finally, T-ALL exposure to a BCL-2 inhibitor (ABT-199 [venetoclax]) significantly enhances the cytotoxic effects of AK2 depletion. We also show that AK2 depletion disrupts the oxidative phosphorylation pathway. Combined with pharmaceutical inhibition of glycolysis, AK2 silencing prevents T-ALL metabolic adaptation, resulting in dramatic apoptosis. Altogether, we pinpoint AK2 as a genuine and promising therapeutic target in T-ALL.

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or ß hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Extensive multilineage analysis in patients with mixed chimerism after allogeneic transplantation for sickle cell disease: insight into hematopoiesis and engraftment thresholds for gene therapy.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges.

Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation in the treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertional mutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation of chimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies.

A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells.

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.

T cell dynamics and response of the microbiota after gene therapy to treat X-linked severe combined immunodeficiency.

BACKGROUND: Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction. METHODS: Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples. RESULTS: Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure. CONCLUSIONS: This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.

INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions.

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand?

More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome.

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.

A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).