Aberrant Expression of LMO2, TAL1 (SCL) and T- RALDH2 in the T-Cell Clone of a Patient with T-ALL like Syndrome after Gene Therapy for X1-SCID.
Authors: Layh-Schmitt Gerlinde , Rosenstiel Antonia , Klanke Chuck , Anderson Kathleen , Hacein-Bey-Abina Salim , Fischer Alan , Cavazzano-Calvo Marina , von Kalle Christof 
Published: April 2020

Abstract
In a gene therapy trial with patients suffering from X-linked Severe Combined Immunodeficiency (X1-SCID) led by Fischer and Cavazzana-Calvo (Hacein-Bey-Abina et al., Science, 2003) full restoration of the immune function was observed after retrovirally mediated gamma c transfer. However, three years after treatment, two out of eleven patients developed a T cell lymphoproliferative disorder, which was associated with LMO2 activation as a result of integration of the retroviral vector into the LMO2 locus. LMO2 is required for normal hematopoiesis and is usually only expressed in erythroid cells and immature T-cells as a component of a multifactorial transcription regulation complex consisting of TAL1, LMO2 (mediating protein-protein interaction), GATA1/2, Ldb-1, and E2A. A number of studies with transgenic mice and observations in T-ALL patients suggest that in addition to aberrant LMO2 expression, secondary events, such as mutations in an oncogene like SCL (TAL1) or in tumor suppressor genes are responsible for the onset of malignancy. The goal of this project is to unravel on molecular level specific events which might occur after retroviral mediated gamma c transfer and to determine possible secondary independent events which finally lead to uncontrolled clonal T-cell proliferation. Experiments were initiated with the T-cell clone of one of the patients (patient 5) who developed leukemia-like symptoms after gene therapy. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2, TAL1 as well as RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes were transcribed. By immunoblot analyses and RT-PCR we were able to confirm over expression of LMO2 and TAL1 in the patient lymphoblasts as well as in Jurkat (T-ALL cell line) and K562 (erythroleukemia cell line) cells. In the patient T-cell clone and in the Jurkat cells, RALDH2 was found to be expressed as an N-terminal truncated form on both the mRNA and protein level, suggesting that the molecular mechanism leading to the T-ALL-like lymphoproliferation in patient 5 resembles the findings in T-ALL cell lines described by Ono et al., (Molecular and Cellular Biol., 1998). Ono et al. identified N-terminal truncated (T) RALDH2 as a target gene for a protein complex consisting of LMO2, TAL1, E47 and GATA3 in T-ALL cell lines, in which GATA 3 mediates DNA binding of the complex. Retinoic acid is known to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. We have cloned and overexpressed the T-RALDH variant by transfection and retrovirus transduction to test whether the truncated form of RALDH2 is still capable of converting retinal to retinoic acid and to establish siRNA procedures to examine whether depletion of T-RALDH2 in T-ALL cells changes cellular proliferation.