Stuart Adams

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Hematopoietic Stem Cell Gene Therapy for Adenosine Deaminase–Deficient Severe Combined Immunodeficiency Leads to Long-Term Immunological Recovery and Metabolic Correction

Gene therapy can restore immune and metabolic function in patients with adenosine deaminase immunodeficiency. Out of the Bubble As part of a normal day, most people will flush a toilet, open a door, or drink from a water fountain without even thinking about it—or about the lurking pathogens poised to infect us. We are afforded this luxury because of our immune system, which responds rapidly and specifically to just about anything thrown at it. Yet, for people with severe combined immunodeficiency (SCID), who carry a mutation that thwarts adaptive immunity, everyday activities can be deadly. Like the famous “bubble boy,” some people with SCID choose to live in a germ-free environment. Yet, matched hematopoietic stem cell (HSC) transplantation, which can replace the patient’s ailing immune system with functional cells from a related donor, can offer these patients a normal life. Sometimes, however, donor relatives aren’t available. Now, two new studies provide clinical support for treatment options that may allow SCID patients without matched donors to live relatively normal lives as well. One such treatment option is gene therapy. Removing HSCs from SCID patients, repairing the underlying genetic defect in these cells, and returning the repaired cells to the original host can replace the faulty immune system in SCID patients without the graft rejection or graft-versus-host disease that follows transplantation of cells from unrelated donors. Gaspar et al. do just that for two types of SCID: X-linked SCID (SCID-X1) and adenosine deaminase–deficient SCID (ADA-SCID). The authors repaired the underlying genetic defect in 10 of 10 patients with SCID-X1 and in 4 of 6 patients with ADA-SCID, resulting in the development of a functional polyclonal T cell repertoire that persisted for at least 9 years after therapy. The procedure produced minimal side effects and permitted all patients to attend typical schools. One patient in the SCID-X1 cohort developed a blood cancer, acute lymphoblastic leukemia (ALL), a complication observed in previous SCID-X1 gene therapy studies, but this patient is currently in remission. No cases of ALL developed in the ADA-SCID cohort. The promising results of these and similar studies, albeit with an increased risk of ALL in SCID-X1 patients, support the development of new safer and more efficient vectors for this and other kinds of gene therapy. Long-term follow-up of patient participants in early gene-therapy trials such as the ones described here is critical for scientists to decipher the parameters of success and failure for gene therapy in general—and for SCID-specific treatments to bubble over into the clinic. Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen–matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.

Long-Term Persistence of a Polyclonal T Cell Repertoire After Gene Therapy for X-Linked Severe Combined Immunodeficiency

Gene therapy results in long-term persistence of T cells in immunodeficient patients. Out of the Bubble As part of a normal day, most people will flush a toilet, open a door, or drink from a water fountain without even thinking about it—or about the lurking pathogens poised to infect us. We are afforded this luxury because of our immune system, which responds rapidly and specifically to just about anything thrown at it. Yet, for people with severe combined immunodeficiency (SCID), who carry a mutation that thwarts adaptive immunity, everyday activities can be deadly. Like the famous “bubble boy,” some people with SCID choose to live in a germ-free environment. Yet, matched hematopoietic stem cell (HSC) transplantation, which can replace the patient’s ailing immune system with functional cells from a related donor, can offer these patients a normal life. Sometimes, however, donor relatives aren’t available. Now, two new studies provide clinical support for treatment options that may allow SCID patients without matched donors to live relatively normal lives as well. One such treatment option is gene therapy. Removing HSCs from SCID patients, repairing the underlying genetic defect in these cells, and returning the repaired cells to the original host can replace the faulty immune system in SCID patients without the graft rejection or graft-versus-host disease that follows transplantation of cells from unrelated donors. Gaspar et al. do just that for two types of SCID: X-linked SCID (SCID-X1) and adenosine deaminase–deficient SCID (ADA-SCID). The authors repaired the underlying genetic defect in 10 of 10 patients with SCID-X1 and in 4 of 6 patients with ADA-SCID, resulting in the development of a functional polyclonal T cell repertoire that persisted for at least 9 years after therapy. The procedure produced minimal side effects and permitted all patients to attend typical schools. One patient in the SCID-X1 cohort developed a blood cancer, acute lymphoblastic leukemia (ALL), a complication observed in previous SCID-X1 gene therapy studies, but this patient is currently in remission. No cases of ALL developed in the ADA-SCID cohort. The promising results of these and similar studies, albeit with an increased risk of ALL in SCID-X1 patients, support the development of new safer and more efficient vectors for this and other kinds of gene therapy. Long-term follow-up of patient participants in early gene-therapy trials such as the ones described here is critical for scientists to decipher the parameters of success and failure for gene therapy in general—and for SCID-specific treatments to bubble over into the clinic. X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor γ chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)–matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34+ hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukemia because of up-regulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.

Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells

Universal gene-edited CAR19 T cells eliminate infant leukemia. CAR sharing Chimeric antigen receptor (CAR) T cells can be very effective in treating acute lymphocytic leukemia. Unfortunately, these therapeutic cells have to be custom-made for each patient, and this is not always feasible, especially for patients who do not have sufficient healthy T cells. Qasim et al. demonstrate that there may be another option for these patients. By using gene editing to simultaneously introduce the CAR and disrupt TCR and CD52 in T cells, the authors generated functional CAR T cells that could evade host immunity for use in unmatched recipients. These “off-the-shelf” CAR T cells were then used to treat two infants with relapsed refractory acute lymphocytic leukemia and bridge them to allogeneic stem cell transplantation. Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non–human leukocyte antigen–matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)–mediated gene editing of T cell receptor α chain and CD52 gene loci. Two infants with relapsed refractory CD19+ B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. This bridge-to-transplantation strategy demonstrates the therapeutic potential of gene-editing technology.

Omission of in vivo T‐cell depletion promotes rapid expansion of naïve CD4+ cord blood lymphocytes and restores adaptive immunity within 2 months after unrelated cord blood transplant

Umbilical cord blood transplant (UCBT) is associated with impaired early immune reconstitution. This might be explained by a lower T‐cell dose infused, the naivety of cord blood T‐cells and the use of in vivo T‐cell depletion. We studied the pattern of early immune reconstitution and the clinical outcome of children undergoing unrelated UCBT when in vivo T‐cell depletion was omitted. Thirty children affected by malignancies (46%) or immunodeficiencies (54%) underwent an unrelated UCBT. Prospective assessment of immune reconstitution and clinical outcome was performed. We observed an unprecedented CD4+ T‐cell reconstitution, with a median cell count at 30 and 60 d post UCBT of 0·3 × 109/l and 0·56 × 109/l, respectively. Early T‐cell expansion was thymic‐independent, with a rapid shift from naïve to central memory phenotype and early regulatory T‐cell recovery. Viral infections were frequent (63%) but resolved rapidly in most cases and virus‐specific T‐lymphocytes were detected within 2 months post‐UCBT. Acute graft‐versus‐host disease (GvHD) was frequent (grade II = 34%, grade III–IV = 16%) but steroid responsive, and the incidence of chronic GvHD was low (14%). The omission of in vivo T‐cell depletion promotes a unique thymic‐independent CD4+ T‐cell reconstitution after unrelated UCBT in children. We postulate that this relates to the specific immunological and ontological qualities of fetal‐derived lymphocytes.

Immune Reconstitution and Recovery of FOXP3 (Forkhead Box P3)-Expressing T Cells After Transplantation for IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked) Syndrome

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is a rare X-linked disorder that usually presents in early childhood with immune enteropathy, diabetes mellitus, and other autoimmune complications. The disease is caused by mutations in the forkhead box P3 gene, a transcription factor that is essential for the development and function of regulatory T cells. This population of cells plays an essential role in controlling immune responses and preventing autoimmunity. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is often initially treated with immunosuppressive drugs, but only allogeneic hematopoietic stem cell transplantation has offered the possibility of cure. We recently performed an unrelated donor transplant in a child with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome by using a reduced-intensity conditioning regimen. This transplant provided a rare opportunity to gain valuable insight into the regeneration of the immune system after transplantation. Clinical recovery was associated with the emergence of regulatory T cell populations, the majority of which expressed memory phenotype markers and raised important questions about the origin and longevity of the FOXP3+ regulatory T cell pool.

Host natural killer immunity is a key indicator of permissiveness for donor cell engraftment in patients with severe combined immunodeficiency

Background Severe combined immunodeficiency (SCID) can be cured by using allogeneic hematopoietic stem cell transplantation, and the absence of host immunity often obviates the need for preconditioning. Depending on the underlying genetic defect and when blocks in differentiation occur during lymphocyte ontogeny, infants with SCID have absent or greatly reduced numbers of functional T cells. Natural killer (NK) cell populations are usually absent in the SCID-X1 and Janus kinase 3 forms of SCID and greatly reduced in adenosine deaminase deficiency SCID but often present in other forms of the disorder. Objective To determine if SCID phenotypes indicate host permissiveness to donor cell engraftment. Methods A retrospective data analysis considered whether host NK cells influenced donor T-cell engraftment, immune reconstitution, and long-term outcomes in children who had undergone nonconditioned allogeneic stem cell transplantation between 1990 and 2011 in the United Kingdom. Detailed analysis of T- and B-cell immune reconstitution and donor chimerism was compared between the NK+ (n = 24) and NK− (n = 53) forms of SCID. Results Overall, 77 children underwent transplantation, with survival of 90% in matched sibling donor/matched family donor transplants compared with 60% when alternative donors were used. Infants with NK−SCID were more likely to survive than NK+ recipients (87% vs 62%, P < .01) and had high-level donor T-cell chimerism with superior long-term recovery of CD4 T-cell immunity. Notably, 33% of children with NK+SCID required additional transplantation procedures compared with only 8% of children with NK−SCID (P < .005). Conclusions NK−SCID disorders are highly permissive for donor T-cell engraftment without preconditioning, whereas the presence of NK cells is a strong indicator that preparative conditioning is required for engraftment of T-cell precursors capable of supporting robust T-cell reconstitution.

Dysregulation of the HIF pathway due to VHL mutation causing severe erythrocytosis and pulmonary arterial hypertension.

To the editor: Hereditary erythrocytosis can be caused by mutations in genes involved in the hypoxia-inducible factor (HIF) pathway.[1][1]–[3][2] For example, Chuvash polycythemia is caused by an R200W substitution in the von Hippel–Lindau protein (VHL).[1][1] There is increasing evidence

Polymorphism in the PAI-1 (SERPINE1) gene and the risk of osteonecrosis in children with acute lymphoblastic leukemia.

To the editor: The success of modern therapy for childhood acute lymphoblastic leukemia (ALL) has led to an increased focus on avoidance of treatment-related morbidity in long-term survivors. It is clear that the prognosis for older patients in particular has been improved by protocols based on

Production and First-in-Man Use of T Cells Engineered to Express a HSVTK-CD34 Sort-Suicide Gene

Suicide gene modified donor T cells can improve immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), but can be eliminated in the event of graft versus host disease (GVHD) through the administration of prodrug. Here we report the production and first-in-man use of mismatched donor T cells modified with a gamma-retroviral vector expressing a herpes simplex thymidine kinase (HSVTK):truncated CD34 (tCD34) suicide gene/magnetic selection marker protein. A stable packaging cell line was established to produce clinical grade vector pseudotyped with the Gibbon Ape Leukaemia Virus (GALV). T cells were transduced in a closed bag system following activation with anti-CD3/CD28 beads, and enriched on the basis of CD34 expression. Engineered cells were administered in two escalating doses to three children receiving T-depleted, CD34 stem cell selected, mismatched allogeneic grafts. All patients had pre-existing viral infections and received chemotherapy conditioning without serotherapy. In all three subjects cell therapy was tolerated without acute toxicity or the development of acute GVHD. Circulating gene modified T cells were detectable by flow cytometry and by molecular tracking in all three subjects. There was resolution of virus infections, concordant with detectable antigen-specific T cell responses and gene modified cells persisted for over 12 months. These findings highlight the suitability of tCD34 as a GMP compliant selection marker and demonstrate the feasibility, safety and immunological potential of HSVTK-tCD34 suicide gene modified donor T cells. Trial Registration ClinicalTrials.gov NCT01204502