Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor γ (IL2RG) gene to CD34 + BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31-68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain-only 2 (LMO2) protooncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene, CCND2. Additional genetic abnormalities in the patients' blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.

The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas1,2. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability1,2. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent1,2. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become trans-fusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl–1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochasticor resultfrom a hithertobenign cellexpansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.

Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe β-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Sickle cell disease results from a homozygous missense mutation in the β-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling β-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling β-globin remained high (approximately 50% of β-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

The present study demonstrates that CD4 ؉ CD25 ؉ T cells, expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART), exhibit phenotypic, molecular, and functional characteristics of regulatory T cells. The majority of peripheral CD4 ؉ CD25 ؉ T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 ( IntroductionHIV infection is mainly associated with a progressive decrease in the number of CD4 ϩ T lymphocytes and defective CD4-specific and CD8-specific T-cell responses against HIV and other pathogens. It has been demonstrated that highly active antiretroviral therapy (HAART) results in reduced HIV-1 replication, increased CD4 cell counts in most treated patients, and progressive but incomplete recovery of CD4 ϩ T-cell functions. [1][2][3][4] Thus, treatment of chronic HIV infection does not result in most cases in a full recovery of HIV-specific helper and cytotoxic T lymphocyte (CTL) responses (reviewed in Sakaguchi et al 5 ).Recently, a CD4 ϩ T-cell subset with regulatory properties has been characterized in humans several years after their discovery in mice. [6][7][8][9][10] These cells, named "regulatory T" (Treg) cells express the ␣ chain of the interleukin 2 (IL-2) receptor (CD25) and can inhibit the proliferation of CD4 ϩ and CD8 ϩ T lymphocytes both in vitro and in vivo. 9,11 It has been demonstrated that transfer of such cells can protect neonatally thymectomized mice from autoimmune diseases, whereas their depletion results in the development of systemic autoimmune diseases. [12][13][14] Moreover, alloantigen-induced CD4 ϩ CD25 ϩ regulatory T cells were reported to prevent rejection initiated by CD4 ϩ cells in both organ and bone marrow transplantation. 15,16 Although CD25 is usually considered as an activation marker, it has been shown that CD4 ϩ CD25 ϩ lymphocytes do not proliferate in response to polyclonal, allogenic, or antigen-specific stimulation 8,9,17 but require activation through their T-cell receptor (TCR) to exert their suppressive function. Their anergic state could be partially reversed by IL-2 and IL-15. 9 Besides CD4 ϩ CD25 ϩ regulatory T cells, 2 other types of CD4 ϩ cells with suppressive function have been described: type 1 T regulatory (Tr1) cells 18 and T-helper 3 (Th3) cells. 19 Tr1 cells were reported to suppress the immune response via the secretion of the immunosuppressive cytokines IL-10 and transforming growth factor ␤ (TGF-␤). The mechanism by which CD4 ϩ CD25 ϩ T cells mediate suppression seems to require direct cell-cell contact. Levings et al 19 have recently demonstrated, at the clonal level, that Tr1 and CD4 ϩ CD25 ϩ T cells are distinct subsets of regulatory T cells and that the suppression mediated by CD4 ϩ CD25 ϩ T cells is partially dependent on TGF-␤.In healthy individuals, the CD4 ϩ CD25 ϩ T-cell subset represents 5% to 10% of peripheral CD4 ϩ T cells and is characterized by the constitutive expression of CD152 (CTL-associate...

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.)

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