ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: Abstract 165FN2 CB and KB contributed equally and should be considered aequo loco. Wiskott-Aldrich-Syndrome (WAS) is a rare and life-threatening immune-disorder characterized by autoimmunity, microthrombocytopenia, immunodeficiency and susceptibility to lymphoma. WAS is caused by mutations in the WAS gene which encodes WASP, a key regulator of actin polymerization exclusively expressed in hematopoietic cells. WASP deficiency causes defects in lymphocytes, myeloid cells, and platelets. We here report a comprehensive analysis of ten patients treated by hematopoietic stem cell gene therapy between 2006 and 2009 (median follow up time 29.6 months, range 15 to 58 months). Patients were mobilized with G-CSF alone (3/10) or G-CSF combined with anti-CXCR4/AMD3100 (7/10), conditioned with busulfan (8mg/kg body weight) and received between 2.8×106 and 24.9×106 cells/kg bw, with a median transduction efficacy of 52%. Upon transplantation of retrovirus-transduced WASP-expressing progenitor cells, the proportion of corrected platelets and lymphocytes increased steadily over time reaching 85–90% and 80–85%, respectively. Interestingly, also myeloid cells showed a continuously increasing percentage of WASP-expressing fractions (20 to 70%). Due to transplantation of insufficient numbers of WASP-transduced HSC, one patient failed to engraft. He had no evidence of corrected myeloid/hematopoietic progenitor cells and continued to suffer from life-threatening infections and autoimmunity. He was successfully treated by haploidentical HSCT. All other patients had marked improvement of their clinical status. Bleeding diatheses, susceptibility to infections, and autoimmunity resolved. Patients had evidence of significant and sustained increase in platelet counts (p=0.01) together with a reconstituted WASP expression and normalization of thrombocyte size (p

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: Lymphoid enhancer-binding factor 1 (LEF-1) belongs to the canonical Wnt signaling pathway acting in transcriptional complexes with β-catenin. LEF-1 can also act independent of β-catenin (i.e. in the TGF-β or Notch pathway). Additionally, recent studies described LEF-1 dominant negative isoform (dnLEF-1), which lack the β-catenin binding domain and functions as either a transcriptional repressor or activator. To date, analysis of the role of LEF-1 in hematopoiesis has been restricted to the lymphoid compartment. Previously we described the crucial role of lymphoid enhancer-binding factor 1 (LEF-1) in granulopoiesis. We found that LEF-1 mediates proliferation, survival, and differentiation of granulocyte progenitor cells. Moreover, abrogated LEF-1 expression is one of the pathomechanism of severe congenital neutropenia CN (Skokowa et al., Nature Medicine, in press). Based on these findings, we aimed to characterize the molecular mechanisms of LEF-1 in the regulation of granulocytic differentiation. C/EBPα is well known as a key transcription factor in granulopoiesis and we found it to be a target gene directly regulated by LEF-1. A screen of the known 566 bp upstream promoter of C/EBPα gene revealed a putative LEF-1 binding site (− 559 bp to − 538 bp). We confirmed LEF-1 binding to C/EBPα promoter in nuclear extracts from CD34+ and CD33+ cells in the transcription factor binding NoShift and ChIP assays. Interestingly, LEF-1 binds to the C/EBPα promoter more efficiently in CD33+ myeloid progenitors than in CD34+ cells. The direct regulation of C/EBPα by LEF-1 was further confirmed in functional studies. We found that in line with down-regulation of LEF-1, expression of C/EBPα was also significantly reduced in CD33+ myeloid progenitors of CN patients. Moreover, LEF-1 rescue of these cells resulted in a marked up-regulation of C/EBPα mRNA expression and in vitro restoration of defective granulocytic differentiation. Remarkably, transduction of CN CD33+ cells with dnLEF-1 isoform, which lacks the ß-catenin-binding domain, resulted in up-regulation of C/EBPα to a similar degree as it was observed with full-length LEF-1. A direct regulatory link between LEF-1 and C/EBPα was additionally confirmed in LEF-1 inhibition experiments. C/EBPα expression was significantly down-regulated in CD34+ cells of healthy individuals, transduced with LEF-1 shRNA. Therefore, we propose that LEF-1 is a key regulator of myeloid differentiation acting in a β-catenin-independent manner, similar as it is known for LEF-1 regulation of T-lymphocyte development.

Description: Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P 

Description: Abstract 3123 In gene therapy targeting hematopoietic cells, a quantitative assessment of the risk factors underlying insertional mutagenesis is required to assess the practical value of preventive actions. Emanating from an observation of the Copeland lab (Du et al., 2005) we developed an in vitro immortalization (IVIM) assay which determines the risk of transformation of murine bone marrow cells as a consequence of insertional upregulation of Evi1 or Prdm16. These functionally related genes encode master regulators of hematopoiesis which are involved in the pathogenesis of human leukemia and insertional transformation in human gene therapy. Using our standardized conditions, the assay can detect mutants arising with a low frequency (down to 1 in a million cells), based on their rescue and expansion upon replating. The genetic lesion associated with clonal transformation is easily identified, and we can quantify not only the incidence of mutants (number of cells required to form a mutant) but also their fitness (number of subclones obtained by replating). Using the IVIM assay, our published work has revealed the following: (1) relocating gammaretroviral enhancer-promoter sequences from the LTR to an internal position of a “self-inactivating” (SIN) vector reduces the fitness of mutants, as do mutations in transcription factor binding sites or insulators that reduce the enhancer activity; (2) cellular promoters located in SIN vectors, depending on their enhancer activity, may reduce the risk of transformation below the detection limit (〉3 logs compared to standard gammaretroviral vectors); (3) the post-transcriptional regulatory element of the woodchuck hepatitis virus does not affect insertional transformation; and (4) the lentiviral integration pattern reduces the risk of insertional transformation by a factor of ∼3 compared to gammaretroviral vectors. In the meantime, the assay has been used to assess the transforming potential of new vectors developed to treat a variety of hematopoietic disorders, most notably X-SCID, X-CGD, WAS and globinopathies. Reproducibly we found that vectors containing cellular promoters reduced the risk of insertional transformation when compared to retroviral promoters, although not all cellular promoters appeared to be free of risk. The assay has also revealed major functional differences of various insulator elements, including synthetic ones designed to block enhancer-crosstalk. Testing a battery of 8 insulators that we obtained from collaborators or designed ourselves, we found that only a subset was potent enough to significantly reduce the transforming potential of a strong retroviral enhancer-promoter. Furthermore, we assessed the transforming potential of our new alpharetroviral SIN vectors (Suerth et al., JV 2010), modified to remove a residual TATA box of the LTR. When containing a retroviral internal promoter, alpharetroviral SIN vectors were ∼9-times and 3-times, respectively, less likely than the corresponding gammaretroviral and lentiviral constructs to induce strongly replicating clones. Mutants obtained with alpharetroviral SIN vector insertions in Evi1 were not only less frequent but also had a greatly reduced fitness compared to those induced by similarly designed gammaretroviral vectors. Alpharetroviral SIN vectors containing the human elongation factor 1 alpha promoter did not immortalize cells in this assay, as previously shown for gammaretroviral SIN vectors. Finally, we performed experiments to explore the mechanistic basis of the IVIM assay. Our data suggest that its principle is the selection of mutants that resist the differentiation-inducing effect of a myeloid growth factor cocktail. Therefore, variations of the cell culture conditions have a significant impact on the sensitivity of the assay, and potentially also on the spectrum of mutants that can be isolated. The established conditions typically select for upregulation of Evi1, Prdm16, or, more rarely observed, Ras -related genes. In summary, the IVIM assay quantifies the risk of insertional mutagenesis in gene therapy, related to vector sequences and integration pattern. It is specifically useful to assess the risk of insertional upregulation of Evi1 and Prdm16 via enhancer-mediated mechanisms, in myeloid progenitor cells. It thus serves as an animal replacement assay to screen for safety-enhancing vector modifications. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.

Description: The variety of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. However, a number of patients that underwent gene therapy in different trials developed hematological malignancy caused by integration of the provirus in the vicinity of proto-oncogenes. These severe adverse advents prompted intense research efforts towards safer gene therapy, leading to the removal of the long terminal repeat enhancer elements and the use of internal promoters in retroviral vectors. Still, a bottleneck of transition from basic research to clinical application is the test for safety of integrating retro- and lentiviral vectors. Instead of laborious in vivo models with limited predictive value, in vitro assays to screen for insertional mutagenesis are strongly desirable. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of viral vectors, which has been widely used to complete preclinical safety documentation of newly developed integrating vector systems. Despite general acceptance in the field of hematopoietic gene therapy, bias for insertional mutants of the myeloid lineage, a low sensitivity and a long assay run time are clear limitations. We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. As input we used murine lineage-negative hematopoietic stem and progenitor cells (HSPC) that were cultured as described for the IVIM assay. The murine HSPC were transduced with a number of different gammaretro- and lentiviral vectors, including vectors that have been employed in clinical trials for X-SCID and Wiskott-Aldrich Syndrome. After 14 days, whole mRNA was isolated from transduced and non-transduced samples and analyzed by Agilent custom microarrays (n=86) and qPCR from nine independent SAGA assays. We applied several Machine Learning algorithms to derive a core set of genes which distinguishes transformed from non-transformed samples in each individual SAGA assay. This set of genes from the individual analysis was further analyzed to derive a core set of genes that is able to robustly separate transformed from non-transformed samples in all assays performed. In order to account for platform-specific effects we validated all microarray results by conventional qPCR-methodology. The SAGA gene set was then cross-validated in an independent validation cohort of SAGA-assays that were not part of the SAGA-training set from which the signature was derived from. The SAGA assay was used to quantify the mutagenic potential of several benchmark vectors. It correctly assigned a high mutagenic potential to vectors (MFG.yc and CMMP.WASP) which led to serious adverse events (SAEs) in clinical trials. Most importantly, the SAGA assay reliably scored high for mutagenic vectors, even when the vector did not transform in IVIM-assays conducted in parallel, demonstrating the higher sensitivity of the SAGA-principle. In contrast, SIN lentiviral vectors with weaker internal promoters (LV.EFS.yc and LV.EFS.ADA) showed no enrichment of the SAGA-core signature and hence scored much safer in the SAGA test. We present the results for these vectors side-by-side either using IVIM or SAGA. In summary, we generated an advanced version of the currently used in vitro insertional mutagenesis screening system by integrating a molecular read-out which enhances reproducibility, sensitivity and reduces assay duration, paving the way for a better preclinical risk assessment of gene therapy vectors. Disclosures No relevant conflicts of interest to declare.

Description: Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of “transgene toxicology,” in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.

Description: Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.

Description: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and an aggressive malignancy. Galectin-3 (gal-3), the only antiapoptotic member of the galectin family, is overexpressed in DLBCL. While gal-3 can localize to intracellular sites, gal-3 is secreted by DLBCL cells and binds back to the cell surface in a carbohydrate-dependent manner. The major counterreceptor for gal-3 on DLBCL cells was identified as the transmembrane tyrosine phosphatase CD45. Removal of cell-surface gal-3 from CD45 with the polyvalent glycan inhibitor GCS-100 rendered DLBCL cells susceptible to chemotherapeutic agents. Binding of gal-3 to CD45 modulated tyrosine phosphatase activity; removal of endogenous cell-surface gal-3 from CD45 with GCS-100 increased phosphatase activity, while addition of exogenous gal-3 reduced phosphatase activity. Moreover, the increased susceptibility of DLBCL cells to chemotherapeutic agents after removal of gal-3 by GCS-100 required CD45 phosphatase activity. Gal-3 binding to a subset of highly glycosylated CD45 glycoforms was regulated by the C2GnT-1 glycosyltransferase, indicating that specific glycosylation of CD45 is important for regulation of gal-3–mediated signaling. These data identify a novel role for cell-surface gal-3 and CD45 in DLBCL survival and suggest novel therapeutic targets to sensitize DLBCL cells to death.