We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

https://science.sciencemag.org/content/sci/302/5644/415.full.pdf

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

To the Editor: We recently reported (April 18 issue)1 the sustained correction of X-linked severe combined immunodeficiency disease by ex vivo, retrovirally mediated transfer of the γc gene into CD34+ cells in four of five patients with the disease. These results have since been confirmed in four additional patients with typical X-linked severe combined immunodeficiency. Of the first four successfully treated patients, three continue to do well up to 3.6 years after gene therapy, whereas a serious adverse event occurred in the fourth patient. At a routine checkup 30 months after gene therapy, lymphocytosis consisting of a monoclonal population . . .

A Serious Adverse Event after Successful Gene Therapy for X-Linked Severe Combined Immunodeficiency

Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor gamma (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) proto-oncogene. 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.

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Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1

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.

https://science.sciencemag.org/content/sci/326/5954/818.full.pdf

Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy

Factors contributing to retroviral integration have been intractable because past studies have not precisely located genomic sites of proviruses in sufficient numbers for significant analysis In this study, 903 murine leukemia virus (MLV) and 379 human immunodeficiency virus-1 (HIV-1) integrations in the human genome were mapped The data showed that MLV preferred integration near the start of transcriptional units (either upstream or downstream) whereas HIV-1 preferred integration anywhere in the transcriptional unit but not upstream of the transcriptional start Defining different integration site preferences for retroviruses will have important ramifications for gene therapy and may aid in our understanding of the factors directing the integration process

https://science.sciencemag.org/content/sci/300/5626/1749.full.pdf

Transcription Start Regions in the Human Genome Are Favored Targets for MLV Integration

Background X-linked severe combined immunodeficiency due to a mutation in the gene encoding the common γ (γc) chain is a lethal condition that can be cured by allogeneic stem-cell transplantation. We investigated whether infusion of autologous hematopoietic stem cells that had been transduced in vitro with the γc gene can restore the immune system in patients with severe combined immunodeficiency. Methods CD34+ bone marrow cells from five boys with X-linked severe combined immunodeficiency were transduced ex vivo with the use of a defective retroviral vector. Integration and expression of the γc transgene and development of lymphocyte subgroups and their functions were sequentially analyzed over a period of up to 2.5 years after gene transfer. Results No adverse effects resulted from the procedure. Transduced T cells and natural killer cells appeared in the blood of four of the five patients within four months. The numbers and phenotypes of T cells, the repertoire of T-cell receptors, and the in vitro pro...

https://www.nejm.org/doi/pdf/10.1056/NEJMoa012616

Sustained Correction of X-Linked Severe Combined Immunodeficiency by ex Vivo Gene Therapy

Erratum: Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy (New England Journal of Medicine (April 18, 2002) 346 (1185-1193))

Cécile Bouneaud

Papers

Sustained Correction of X-Linked Severe Combined Immunodeficiency by ex Vivo Gene Therapy

Erratum: Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy (New England Journal of Medicine (April 18, 2002) 346 (1185-1193))

Gene therapy of RAG-2−/− mice: sustained correction of the immunodeficiency