Insertional mutagenesis following gamma-retroviral gene transfer in mature T lymphocytes

  • Retroviral vectors are powerful tools in clinical gene therapy as they integrate permanently into the target cell genome and thus guarantee long-term expression of transgenes. Therefore, they belong to the most frequently used application platforms in clinical gene therapy involving a broad range of different target cells and tissues. However, stable genomic integration of retroviral vectors can be oncogenic, as reported in several animal models and in clinical trials. In particular, γ-retroviral vectors, which derive from naturally mutagenic γ-retroviruses, integrate semirandomly into the host genome with regard to the target sequence, but have a preference for regions of active transcription and regulatory elements of transcriptionally active genes. The integration can result in overexpression of adjacent genes or disruption of ‘target’ gene expression. Moreover, γ-retroviral integration can cause modified transcripts and proteins through alternative or aberrant splicing or through premature termination of transcription. Initially, the event of insertional mutagenesis and subsequent induction of leukemia by the genotoxicity of a γ-retroviral vector was described in a mouse model after genetic modification of hematopoietic stem cells (HSCs). Vector-related activation and overexpression of the oncogene ecotropic viral integration site-1 (Evi1) fostered clonal outgrowth and leukemogenesis. Additional genotoxic events of γ-retroviral vectors were observed in clinical HSC gene therapy trials for X-linked severe combined immune deficiency (SCID-X1), chronic granulomatous disease (X-CGD), and Wiskott-Aldrich Syndrome (WAS). But, genotoxicity induced by γ-retroviral vectors has never been described in clinical gene therapy trials involving adoptive transfer of genetically modified mature T lymphocytes. This fact is surprising, since T cells are long-lived and have a high capacity of self-renewal. In a previous study, the susceptibility towards oncogenic transformation of mature T cells and HSCs after genetic modification was compared. It could be demonstrated that T-cell receptor (TCR)-polyclonal mature T cells are far less prone to transformation after γ-retroviral transfer of (proto-)oncogenes in vivo than HSCs. Additional experiments revealed that TCR-oligoclonal (OT-I and P14) mature T cells are transformable in the same setting and give rise to mature T-cell lymphomas (MTCLs). In the present thesis, the susceptibility of mature T cells towards insertional mutagenesis was investigated. Within the first part of the thesis, retroviral integration sites (RISs) from 33 murine MTCLs were retrieved and subsequently analyzed in terms of integration pattern, detection of common integration sites (CIS) and gene ontology (GO). As these bioinformatic results demonstrated that insertional mutagenesis most likely contributed to mature T-cell lymphomagenesis, the susceptibility of mature T cells was directly assessed in a mouse model. Therefore, murine TCR-oligoclonal OT-I T cells were transduced with an enhanced green fluorescent protein (EGFP) encoding γ-retroviral vector and gene-modified T cells were transplanted into RAG1-/- mice. After 16 months, including one round of serial transplantation, a case of MTCL emerged. Tumor cells were characterized by CD3, CD8, TCR and ICOS expression. Integration site analysis via ligation-mediated polymerase chain reaction (LM-PCR) revealed a proviral insertion in the Janus kinase 1 (Jak1) gene. Subsequent overexpression of Jak1 could be demonstrated on transcriptional and protein level. Furthermore, T-cell lymphoma cells were characterized by an activated Jak/STAT-pathway as signal transducer and activator of transcription 3 (STAT3) was highly phosphorylated. The overexpression of Jak1 was causally implicated in tumor growth promotion as specific pharmacological inhibition of Jak1 using Ruxolitinib significantly prolonged survival of mice transplanted with these Jak1-activated tumor cells. A concluding systematic metaanalysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis. This was the first reported case of an insertional mutagenesis event in mature T cells in vivo. Thus, the results obtained in this thesis underline the importance of long-term monitoring of genetically modified T cells in vivo and the evaluation of vector toxicology and safety in T-cell based gene therapies. In particular, the transduction of T cells with a recombinant TCR or CAR (chimeric antigen receptor) bears a risk enhancement, as normal T-cell homeostasis is perturbed besides the general risk of insertional mutagenesis.
  • Retrovirale Vektoren sind vielversprechende Werkzeuge für den Gentransfer in klinischen Applikationen/Studien, da sie stabil ins Genom der Zielzellen integrieren und folglich eine langfristige Transgen-Expression ermöglichen. Jedoch können bei Vektoren, deren Cis-Elemente identisch mit denen von Wildtyp Retroviren sind, schwere Nebenwirkungen auftreten. Die insertionsbedingte Aktivierung von flankierenden Proto-Onkogenen durch die retroviralen LTRs hat sowohl in präklinischen als auch in klinischen Studien zur Transformation von hämatopoetischen Stammzellen nach γ-retroviralem Gentransfer geführt. So konnten Patienten, die an der Immunschwächekrankheit SCID-X1 (X-linked severe combined immunodeficiency) litten, zunächst erfolgreich durch die genetische Modifikation autologer Blutstammzellen mit dem Gen IL2RG (gemeinsame γ-Untereinheit der Interleukin-2 Rezeptor Familie) behandelt werden. Als schwere Nebenwirkung entwickelten jedoch bisher fünf der 19 behandelten Patienten eine reifzellige T-ZellLeukämie infolge der genetischen Stammzell-Modifikation mittels γ-retroviraler Vektoren. In vier Fällen konnte diese erfolgreich mit Chemotherapie behandelt werden. Die Tumorgenese war in den meisten Fällen auf eine Insertions-bedingte Aktivierung des Proto-Onkogens Lmo2 (LIM only domain 2) zurückzuführen. Weitere Fälle von klonaler Expansion und Insertionsmutagenese wurden in klinischen Gentherapie-Studien der septischen Granulomatose (X-CGD), des Wiskott-Aldrich Syndroms (WAS) und der β-Thalassämie beobachtet. ...

Download full text files

  • PhD_TH_Final.pdf

Export metadata

Additional Services

Share in Twitter Search Google Scholar
Author:Tim Heinrich
Referee:Bernd LudwigGND, Dorothee von LaerORCiDGND
Advisor:Dorothee von Laer
Document Type:Doctoral Thesis
Year of Completion:2013
Year of first Publication:2013
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Release Date:2019/03/28
Page Number:158
Diese Dissertation steht außerhalb der Universitätsbibliothek leider (aus urheberrechtlichen Gründen) nicht im Volltext zur Verfügung, die CD-ROM kann (auch über Fernleihe) bei der UB Frankfurt am Main ausgeliehen werden.
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Sammlung Biologie / Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
Licence (German):License LogoArchivex. zur Lesesaalplatznutzung § 52b UrhG