Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Leukemia in a Murine Bone Marrow Transplantation Model
Authors: Heckl Dirk , Schwarzer Adrian , Haemmerle Reinhard , Meyer Johann , Steinemann Doris , Rudolph Cornelia , Skawran Britta , Schlegelberger Brigitte , Baum Christopher , Modlich Ute 
Published: October 2019

Abstract
Abstract 671
Gene therapy with gammaretroviral and lentiviral vectors has shown its potential for the treatment of inherited hematopoietic disorders. However, the occurrence of severe adverse events, namely the induction of leukemias in clinical trails for the treatment of severe combined immunodeficiency (SCID)-X1, chronic granulomatous disease and Wiskott Aldrich Syndrome due to insertional activation of proto-oncogenes by the integrated vector raised safety concerns. Lentiviral vectors are assumed to be safer due to their integration bias to transcription units in comparison to gammaretroviral vectors that have an integration preference to promoter regions and CpG islands. However, the recent report on the development of clonal dominance in a clinical trial for b-thalassemia mediated by deregulated gene expression due to alternative splicing after lentiviral insertion into an intron of HMGA2 highlights potential risks of lentiviral gene therapy.
Here we report a case of B-cell lymphoblastic leukemia in a murine bone marrow (BM) transplantation model 199 days post transplantation of cells transduced by a lentiviral vector expressing eGFP from the human glycoprotein-I-b-alpha (GPIba) promoter (-286 to +309 relative to the transcription start site). The mouse developed leukocytosis (179×103/μl white blood cells counts) and leukemic infiltrations were found in the BM, spleen, liver and lung. The leukemia was clonal as determined by Southern blot analysis, derived from the donor cells as determined by the CD45.1 chimerism marker and reinitiated disease in secondary animals (n=7). Leukemic cells had the morphology of lymphoid blasts/progenitors, stained positive for the markers B220, CD43, and partially CD19 and were negative for IgM. We therefore concluded that the cells were arrested and the PrePro-B to Pro-B cell stage.
Two lentiviral integrations were identified in the leukemic clone of which one was mapped to intron 8 of the Early B-cell factor 1 (Ebf1) gene, a major regulator of B-cell development. The other insertion site was detected in the first intron of the Nance-Horan-Syndrome-gene that has no known function in hematopoiesis and we could not detect altered expression of this gene. No further genomic alterations were identified by spectral karyotyping and Array CGH analysis. Because the vector contained various splice sites in its backbone and the internal GPIba promoter, we next analyzed potential splice products and detected mRNAs formed from splice events between Ebf1 exons 8 and 9 to the integrated vector.
Some of these could result in early termination of the mRNA which would generate a truncated Ebf1 protein containing only the DNA-binding domain. However, the overall contribution of this early stop mRNAs was low (5% of total Ebf1 message) and we could not detect a truncated Ebf1 protein by Western blot. Overexpression of the truncated Ebf1 protein in a B-cell differentiation assay on OP9 cells in vitro did not interfere with B-cell development.
In contrast, we found the Ebf1 full length mRNA to be ∼4-fold downregulated and the EBf1 protein in leukemic cells to be reduced compared to stage matched BM B-cell progenitors (CD19+CD43+). Genome wide expression analysis of leukemic cells compared to B-cell progenitors revealed downregulation of Ebf1 target genes and definite signs of transdifferentiation to the myeloid lineage as it was described upon loss of Ebf1 or its downstream target Pax5 in other studies. In addition, Gene Set Enrichment and Gene Ontology analyses suggested upregulation of genes implicated in JAK-STAT signalling. Indeed, we observed strong upregulation of Flt3 and high levels of STAT5 activation, in line with recent reports of B-cell leukemia development in Ebf1+/− mice overexpressing constitutively active STAT5 (Heltemes-Harris et al., J. Exp. Med 2011). STAT5 activation is also found in a large subset of human B-ALL and the occurrence of mono- or bi-allelic EBF1 deletions in human B-ALL is known (Mullighan et al., Nature, 2007). As the lentiviral integration was an early event in the leukemia development in our experiments, haploinsufficiency of Ebf1 will have been most likely the leukemia initiating event.
Taken together our results highlight the risk of intragenic lentiviral vector integration of vectors containing splice sites that can induce alternative splicing, which, as shown here, may lead to inactivation of haploinsufficient tumour suppressor genes.

Disclosures:
Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.