350. Ectopic Retroviral Expression of LMO2, but Not IL2Rγ, Blocks Human T-Cell Development from CD34+ Progenitor Cells: Implications for Gene Therapy

Abstract

The occurrence of leukemia in 3 out of 11 patients in a gene therapy trial for SCID-X1 emphasized insertional mutagenesis as an adverse effect. In two out of three patients retroviral integration occurred near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2). It has remained unclear why LMO2 was preferentially targeted. We show here that of classical T-ALL oncogenes, mainly LMO2 is transcribed in CD34+ progenitor cells derived from umbilical cord blood (UCB). Upon stimulation with growth factors typically used in gene therapy protocols, transcription of LMO2, LYL1 and TAL1 increased 2|[ndash]|3 fold in CD34+ UCB cells, whereas the other T-ALL oncogenes remained unaffected. Similarly, when mobilized peripheral blood CD34+ cells were stimulated according to transduction protocols used for clinical SCID-X1 gene therapy trials, the same pattern of T-ALL oncogene transcription was observed, suggesting that cytokine stimulation leads to upregulation of proto-oncogenes irrespective of the source of CD34+ progenitor cells used. Transcription of LMO2, LYL1 and TAL1 is particularly high and their loci could therefore be preferentially accessible for viral integration. Retroviral overexpression of LMO2 in CD34+ cells led to severe abnormalities in human T-cell development, but B-cell, NK cell and myeloid development remained unaffected. The interleukin-2 receptor gamma chain (IL2R|[gamma]|), causing SCID-X1 when mutated, has been proposed to act as an oncogene cooperative to LMO2. However, we found that overexpression of IL2R|[gamma]| had no effect on T-cell development. Moreover, IL2R|[gamma]| normally is expressed at high levels in CD34+ progenitor cells and at even higher levels in the thymus. Thus, transduction of IL2R|[gamma]| in CD34+ progenitor cells does not cause ectopic expression of this gene. In conclusion, our data provide an explanation why LMO2 was preferentially targeted over other known T-ALL oncogenes. Furthermore, IL2R|[gamma]| does not appear to act as an oncogene. Rather, the normal IL2R|[gamma]| chain restores IL7 receptor signalling in the progeny of transduced SCID-X1 cells developing in the thymus, allowing T-cell development to progress to stages where ectopic LMO2 expression hampers T-cell development in the thymus, creating a probable pre-leukemic condition by accumulation of immature cells under intense proliferative pressure.