Pharmakokinetische Untersuchungen genkorrigierter Hämatopoese in klinischen retroviralen Studien

Die somatische Gentherapie mit retroviralen Vektoren konnte erfolgreich zur Behandlung monogenetischer Erbkrankheiten eingesetzt werden. Jedoch traten bereits in 3 klinischen Gentherapiestudien schwerwiegende vektorinduzierte Nebenwirkungen auf. Integrationsstellen (IS)-Analysen erlaubten die Detektion von Integrationen in oder in der Nähe von Protoonkogenen, die zu einer Überexpression der Gene und zur malignen Entartung der betroffenen Zellen führten, an deren Folgen 2 der 7 erkrankten Patienten verstarben. Umfassende Analysen der Vektor-IS und deren Einfluss auf zelluläre biologische Prozesse sind daher von größter Wichtigkeit und können dabei helfen, mögliche Risiken schon frühzeitig auf der molekularen Ebene zu erkennen. Die Bestimmung der IS erfolgte in den untersuchten klinischen Gentherapiestudien mithilfe der linearen amplifikationsmediierten PCR (LAM-PCR). Zur Verbesserung des zugänglichen Anteils der IS wurde ein mathematisches Modell entwickelt, das die genomische Zugänglichkeit von IS in Abhängigkeit der verwendeten Restriktionsenzyme a priori berechnet. Weiterer Bestandteil meiner Arbeit war die Etablierung einer nicht restriktiven (nr) LAM-PCR, die eine IS-Analyse ohne die Verwendung von Restriktionsenzymen ermöglicht. Die im Rahmen dieser Arbeit durchgeführte vergleichende IS-Analyse in Kombination mit der Sanger Sequenzierung von 5 klinischen (2547 IS) und 3 präklinischen (1316 IS) gammaretroviralen Studien zeigte beeindruckende Übereinstimmungen. So wurden über 70% aller IS in genkodierenden Bereichen detektiert mit einer Anhäufung um die Transkriptionsstartstelle (TSS, 23%-39%). Weiterhin konnten wir die Protoonkogene MDS1-EVI1 (108 IS), PRDM16 (37 IS), LMO2 (13 IS), CCND2 (12 IS) und SETBP1 (10 IS) als gemeinsame bevorzugte Integrationsorte beobachten. Ein weiterer Fokus stellten die Klonalitäts- und pharmakokinetischen Analysen der Proben von insgesamt 9 Patienten aus zwei X-CGD, einer ADA-SCID und einer WAS gammaretroviralen Gentherapiestudie dar, die über einen Zeitraum von bis zu 5 Jahren nach Therapie erfolgten. In allen untersuchten Studien zeigte die (nr) LAM-PCR Analyse gefolgt von der Pyrosequenzierung (GS FLX, > 20000 IS) ebenfalls eine nicht zufällige Verteilung mit einer bevorzugten Integration in genkodierenden Bereichen (47%-72%) und einer Anhäufung der IS um die TSS (8%-16%). Weiterhin wurden ebenfalls bevorzugte Integrationsorte in oder in der Nähe der Protoonkogene MDS1-EVI1 (289 IS), PRDM16 (104 IS), LMO2 (52 IS), SETBP1 (34 IS) und CCND2 (33 IS) detektiert. Mittels LAM-PCR „Sequence Count“ Analysen konnte eine vektorinduzierte in vivo Selektion einzelner MDS1-Klone in 3 der insgesamt 4 X-CGD Patienten beobachtet werden. Reverse Transkriptase (RT)-PCR Untersuchungen zur Genexpression in einem X-CGD Patienten zeigten eine Überexpression der von der Integration betroffenen Gene MDS1-EVI1 und STAT3. In den weiteren Patienten der untersuchten Gentherapiestudien verlief die hämatopoetische Repopulation bis zum letzten analysierten Zeitpunkt polyklonal. Allerdings konnten wir auch in der WAS Gentherapiestudie eine in vivo Selektion eines CCND2- und eines MDS1-Klons über „Sequence Count“ Analyse und qPCR nachweisen. Die Klonalitätsanalysen mittels LAM-PCR/nrLAM-PCR und Hochdurchsatzsequenzierung (Pyrosequenzierung, GS FLX) führten zu einer einzigartigen Datensammlung von insgesamt > 20000 IS aus klinischem Patientenmaterial. Unsere Untersuchungen zeigten, dass herkömmliche retrovirale Vektoren mit aktivem LTR einen sehr signifikanten Einfluss auf die zelluläre Genexpression ausüben. Es bleibt längerfristigen Untersuchungen vorbehalten, zu klären, welche biologischen Auswirkungen die retro- und lentiviralen Vektorsysteme mit selbstinaktivierendem (SIN) LTR auf das Schicksal der transduzierten Zelle haben

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis

This article is available open access through the publisher’s website at the link below. Copyright @ 2013 The American Society of Gene & Cell Therapy.Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.Imperial College London, the Wellcome Trust, and Brunel University

VISMapper: ultra-fast exhaustive cartography of viral insertion sites for gene therapy

Background -- The possibility of integrating viral vectors to become a persistent part of the host genome makes them a crucial element of clinical gene therapy. However, viral integration has associated risks, such as the unintentional activation of oncogenes that can result in cancer. Therefore, the analysis of integration sites of retroviral vectors is a crucial step in developing safer vectors for therapeutic use. Results -- Here we present VISMapper, a vector integration site analysis web server, to analyze next-generation sequencing data for retroviral vector integration sites. VISMapper can be found at: http://vismapper.babelomics.org. Conclusions -- Because it uses novel mapping algorithms VISMapper is remarkably faster than previous available programs. It also provides a useful graphical interface to analyze the integration sites found in the genomic context

A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource

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

Intérêt du traitement par itraconazole dans l' endocartite à candida ( à propos d' une observation)

NANCY1-SCD Medecine (545472101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

The balance between the intronic miR-342 and its host gene Evl determines hematopoietic cell fate decision

Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Lin- Sca1+ cKit+ cells drives lymphopoiesis whereas miR-342 OE increases myeloid colony formation in vitro and in vivo, going along with a profound upregulation of canonical pathways essential for B-cell development or myelopoietic functions upon Evl or miR-342 OE, respectively. Strikingly, miR-342 counteracts its host gene by targeting lymphoid signaling pathways, resulting in reduced pre-B-cell output. Moreover, EVL overexpression is associated with lymphoid leukemia in patients. In summary, our data show that one common gene locus regulates distinct hematopoietic differentiation programs depending on the gene product expressed, and that the balance between both may determine hematopoietic cell fate decision

Cell Cycle Status of CD34+Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes

Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that similar to 10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis