Retroviral Integrations in Gene Therapy Trials

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis

Analyzing the Number of Common Integration Sites of Viral Vectors – New Methods and Computer Programs

Vectors based on γ-retroviruses or lentiviruses have been shown to stably express therapeutical transgenes and effectively cure different hematological diseases. Molecular follow up of the insertional repertoire of gene corrected cells in patients and preclinical animal models revealed different integration preferences in the host genome including clusters of integrations in small genomic areas (CIS; common integrations sites). In the majority, these CIS were found in or near genes, with the potential to influence the clonal fate of the affected cell. To determine whether the observed degree of clustering is statistically compatible with an assumed standard model of spatial distribution of integrants, we have developed various methods and computer programs for γ-retroviral and lentiviral integration site distribution. In particular, we have devised and implemented mathematical and statistical approaches for comparing two experimental samples with different numbers of integration sites with respect to the propensity to form CIS as well as for the analysis of coincidences of integration sites obtained from different blood compartments. The programs and statistical tools described here are available as workspaces in R code and allow the fast detection of excessive clustering of integration sites from any retrovirally transduced sample and thus contribute to the assessment of potential treatment-related risks in preclinical and clinical retroviral gene therapy studies

The genetics and kinetics of BCL2 driven lymphoid malignancies

Introduction: Non-Hodgkin Lymphoma (NHL) is rising in incidence. Treatment of this genetically heterogeneous disease has toxic side effects and significant numbers of relapsers / non-responders. BCL2, an anti-apoptotic protein, is commonly overexpressed in NHL as a result of the t(14;18) translocation. A number of BCL2 inhibitors have shown success in clinical trials but variable efficacy has meant that none have been licenced for use. Methods: Retroviral insertional mutagenesis (RIM), using Moloney Murine Leukaemia Virus (MoMuLV) in transgenic mice overexpressing BCL2, was used to identify putative target genes deregulated alongside BCL2 in lymphomagenesis. This project aimed to update MoMuLV integration site identification and sequencing, allowing quantification of integration site clonal abundance. Cohorts of mice were sacrificed at time points prior to disease onset in order to interrogate integration site kinetics. To test the oncogenic potential of candidate genes, C57BL/6 Vav-BCL2 p53+/- mouse B cells were retrovirally transduced with genes of interest and transplanted into mice to study the speed of lymphoma onset. Results & Conclusions: A novel, high throughput, quantitative library preparation and sequencing protocol compatible with an Illumina platform was validated. RIM screening in BCL2 transgenic and wild-type mice identified different insertion sites profiles, detecting known oncogenes and tumour suppressor genes as well as novel candidate genes involved in pathways of lymphoid organ development, B-cell activation and differentiation. Study of insertion kinetics over time showed three patterns of clonal abundance and also allowed the study of specific gene deregulation prior to disease onset. Overexpression of Cd86 slowed disease onset whilst Ildr1 expedited disease onset suggesting the former is a tumour suppressor gene and the latter an oncogene. Discovering genes mutated with BCL2 in lymphoma may help to explain the lack of efficacy of BCL2 inhibitors and also identify novel therapeutic targets.Open Acces

Dissecting genetic determinants of transcription factor activity

Understanding how phenotype relates to genotype, in terms of the myriad molecular processes that govern the behavior of cells and organisms, has been one of the central goals of biology for a long time. Transcription factors (TFs) play a mediating role connecting genotype with gene expression, which provides high-dimensional information about end phenotype. In particular, gene expression levels depend on their cis-regulatory sequence bound by TFs and condition-specific regulatory activity of TFs determined by its modulators through interaction with cofactors or signaling molecules. This thesis consists of two parts that related to the overall goal of dissecting upstream modulators of transcription factor activity. The first study is to dissect genetic determinants of transcription factor activity in a segregating population. We exploit prior knowledge about the in vitro DNA-binding specificity of a TF in order to map the loci (`aQTLs') whose inheritance modulates its protein-level regulatory activity. The second study is to identify regulatory mechanisms underlying tumorigenesis in mice by using genotyping and gene expression data across a set of 97 splenic tumors induced by retroviral insertional mutagenesis. We identify several instances of sequence-specific TFs whose activities are specifically affected by insertions mutations. Our results underscore the value of explicitly modeling TF activity and a strategy for finding upstream modulators of TF activity

Genome-wide analysis of transposon and retroviral insertions reveals preferential integrations in regions of DNA flexibility

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germline transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish, with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence integration of heterologous DNA in genomes, and have implications for targeted genome engineering

A Novel murine model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.Work in the Huntly laboratory is funded by CRUK, The European Research Council (ERC), Leukaemia Lymphoma Research, the Kay Kendall Leukaemia Fund, Wellcome Trust, the Medical Research Council (UK), the Leukemia Lymphoma Society America and the Cambridge NIHR Biomedical Research centre. David Adams is funded by Cancer Research UK and Wellcome Trust. Steffen Koschmieder has received funding from Deutsche José Carreras Leukämie-Stiftung (DJCLS; grant 10/23).This is the final published version. It first appeared at http://dx.doi.org/10.1084/jem.2014166

BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in approximately 80% of BRAFV600-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of BrafV618E (analogous to the human BRAFV600E mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of BrafV618E transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes