A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.

Myeloid-Derived Growth Factor Protects Against Pressure Overload-Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca2+-ATPase Expression in Cardiomyocytes.

BACKGROUND: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. METHODS: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf-deficient (Mydgf-/-), and Mydgf bone marrow-chimeric or bone marrow-conditional transgenic mice with pressure overload-induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography-mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. RESULTS: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf-/- mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow-derived inflammatory cells attenuated pressure overload-induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein-coupled receptor agonist-induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf-/- mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf-/- mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload-induced hypertrophy and dysfunction. Pressure-overloaded Mydgf-/- mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. CONCLUSIONS: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Multimodal Lentiviral Vectors for Pharmacologically Controlled Switching Between Constitutive Single Gene Expression and Tetracycline-Regulated Multiple Gene Collaboration.

Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied. Doxycycline-dependent switching can be tracked via fluorescent markers coupled to constitutive and tetracycline-regulated GOIs. This article describes transduction of murine primary HPCs with different doses of multimodal LVs, distinct cytokine conditions, and their influence on the number and viability of cells co-expressing both collaborating GOIs upon doxycycline induction. A 2-week protocol is provided for multimodal LV production, titer determination, and evaluation of tetracycline responsive promoter background activity in a murine fibroblast cell line. The power of this model to assess the dose/time/order-controlled contribution of single and multiple genes into hematopoietic networks opens new routes in reprogramming, stem cell, and leukemia biology.

Comparison of Tetracycline-regulated Promoters in Lentiviral-based Vectors in Murine Transplantation Studies.

Tetracycline-regulated systems with efficient temporal and dose regulation of transgene expression are useful for development of new physiologic/pathophysiologic experimental models and gene therapy approaches. Lentiviral vectors with improved tetracycline-regulated promoters help to overcome the existing limitations such as basal activity in the drug absence, poor inducibility or unstable transgene expression. To compare conventional and improved tetracycline-regulated promoters in lentiviral based vectors in vivo, we investigated doxycycline-regulated gene transfer/expression levels in a long-term murine transplantation model and demonstrated that the lentiviral vector with the improved T11 promoter exhibited more efficient inducibility and higher gene transfer level. The time required to reverse transgene expression after doxycycline removal was increased for animals with higher gene expression levels and vector copy numbers. Examination of peripheral blood leukocytes and splenocytes revealed similar cell lineage distributions for transgene positive and negative cell populations from experimental and control mice, but increased variability in the percentages of myeloid and lymphoid cells was detected in transgene positive bone marrow cells. However, no indication of lineage bias in total bone marrow cells and no signs of hematopoietic disease were observed seven months after transplantation. Our results showed that the T11 tetracycline-regulated promoter enabled improved transgene expression in a murine transplantation model. The established system allows further development of tetracycline-regulated experimental models to investigate normal and malignant hematopoiesis.

Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors.

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin(-)), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells.

Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.

Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.

Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells.

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells and provide the opportunity for time- and dose-controlled reversible transgene expression in hematopoietic stem cells in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetPs) in the absence of induction or vector integration in the vicinity of proto-oncogenes can diminish the advantages of the system. Here we investigated the effect of lentiviral transduction rate on tetP background activity, vector copy number (VCN), and clonal variability as a consequence of vector integration. We found an exponential relationship between VCN and gene transfer/expression level, accompanied by a linear relationship between VCN and tetP background activity. Long-term murine transplantation studies demonstrated stable and reversible transgene expression in serial recipients. Although analysis of associated clonal composition revealed development of clonal dominance in the presence and absence of induction, no indications of disease presented during the observation period. The majority of tetracycline-regulated vector integration sites were identified in intron/exons of metabolic/housekeeping and signaling genes or in noncoding/repeat regions of the genome. Furthermore, we demonstrated that the nature of the selected transgene might affect tetP background activity and inducibility in vivo. Limiting tetP-regulated gene transfer may avoid generation of clones with high VCN and enhanced tetP background activity. Our data help to establish physiologic and pathophysiologic systems to study dose-dependent mechanisms triggered by different levels of transgene expression in the context of basic HSC biology and cellular transformation models.

Polyclonal fluctuation of lentiviral vector-transduced and expanded murine hematopoietic stem cells.

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in "expanded" groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.

Cell-intrinsic and vector-related properties cooperate to determine the incidence and consequences of insertional mutagenesis.

In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient gamma-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of approximately 10(-4). Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10(-6)). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity.

Retroviral insertion site analysis in dominant haematopoietic clones.

Identification of retroviral vector insertion sites in single, dominating cell clones has become an important tool for the investigation of cellular signalling pathways involved in clonal expansion and malignant transformation. Also, recent severe adverse events in clinical trials resulting from retroviral vector-mediated insertional mutagenesis underline the need of well-designed safety studies including integration site analyses to estimate cost/benefit ratios in gene therapy. We have recently described a modified ligation-mediated PCR (LM PCR) method allowing preferential retrieval of insertion sites causally linked to clonal dominance of an affected clone. In the first part of the given work we focus on particularities of the LM PCR procedure to be taken into account when working with self-inactivating as compared to 'classical' retrovectors. In the following sections we focus on data acquisition, processing, organisation, and analysis. Thus the protocol presented here should be helpful in establishing and utilising databases of retroviral integration sites.

Retroviral integration site analysis in hematopoietic stem cells.

Stable transgene insertion into a host genome irrevocably and unambiguously marks individual cells and all their descendants, i.e., the respective cell clone. Based thereon, retroviral gene marking has become an important tool for investigating the in vivo fate of different cell types, both in animal models and in clinical gene transfer. Moreover, identification of (vector) insertion sites in malignant clones transformed because of insertional activation of proto-oncogenes after experimental as well as therapeutic retroviral gene transfer has resulted in new insights into oncogenic transformation of hematopoietic stem cells (HSCs). However, because of the high sensitivity of the PCR-based methods for insertion site detection, researchers are often confronted with large numbers of different insertion sites/cell clones whose contribution to the given state is hard to judge. A relatively simple ligation-mediated polymerase chain reaction (LM-PCR) method allows the preferential analysis of insertion sites in those cell clones that significantly contributed to the cell pool analyzed. In murine bone marrow transplantation models, we have shown that this method is very useful to analyze the impact of retroviral insertion sites on both malignant and benign clonal dominance of individual repopulating HSC.

Insertional mutagenesis by replication-deficient retroviral vectors encoding the large T oncogene.

Insertion sites of replication-deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor-suppressor proteins p53 and Rb by virtue of a chaperone-like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg-induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics ( approximately 8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D-derived monocytic leukemias showed hits in Pim2 and Max proto-oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector-mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.

Retroviral vector insertion sites associated with dominant hematopoietic clones mark "stemness" pathways.

Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major "stemness" pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.

Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule.

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.

Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population.

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.