Hematopoietic stem and progenitor cells are important targets for somatic gene therapy, considering their availability for in vitro manipulation and their enormous biological capacity. The first clinical reports describing successful gene therapeutic correction of severe combined immunodeficiencies by retroviral-vector mediated gene transfer into hematopoietic cells have created new perspectives for the entire field. However, recently at least two cases of secondary leukemias have been reported in which insertional activation of a cellular oncogene by a retrovirally transduced transgene represented the initiating event. We have previously described a similar pathogenesis of leukemic complications in a mouse model of retroviral gene marking. To overcome the current uncertainty which developed in the scientific and regulatory community following these publications, systematic research needs to be conducted to address the frequency of insertional mutagenesis, the impact of vector design, and the role of further contributing factors. Our group has a well-documented expertise in the development of retroviral vectors, selection markers and preclinical assay systems with improved predictive value for hematopoietic gene therapy. On the basis of our findings in preclinical studies and in the literature, we recently developed a classification for side effects related to the genetic manipulation of hematopoietic cells and derived hypotheses addressing the potential for combinatorial interactions of side effects in gene therapy. Three of these hypotheses generate the basis for the present research proposal: (1) In contrast to insertional gene inactivation, insertional gene activation (IGA) may generate more dangerous dominant effects and is expected to be strongly related to the specific architecture and sequence of the inserted transgene. (2) Using retroviral (including lentiviral) delivery technologies, insertional oncogene activation (IOA, a subform of IGA) may be more frequent than previously anticipated. (3) To promote malignant progression of a cell clone with IOA, synergy is required with biological features mediated by transgene expression and systemic conditions of the recipient. The present research proposal addresses these hypotheses by developing novel experimental approaches in cell lines and mouse models of bone marrow transplantation (specific aim 1). Using these models, we will explore the mechanisms of transformation following IOA, by elucidating the impact of vector architecture and its specific regulatory sequences and a potential contributing role of the transgenes involved (specific aim 2). These insights will allow us to develop and evaluate transgene technologies in which the risk of insertional tumorigenesis is significantly reduced, by improving vector design and/or using selectable marker technologies (specific aim 3). We hope that the experimental systems established in this project will have a long-term impact for human gene therapy. Our experiments will also reveal new mechanisms in the genealogy of leukemias, which may have implications for human disease. Quantitative and mechanistic insights into vector safety may become a reality, and the experimental approaches should be applicable for any type of inserting transgene technology developed in the future.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA107492-04
Application #
7430267
Study Section
Developmental Therapeutics Study Section (DT)
Program Officer
Li, Jerry
Project Start
2005-08-01
Project End
2009-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
4
Fiscal Year
2008
Total Cost
$224,720
Indirect Cost
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Li, Zhixiong; Beutel, Gernot; Rhein, Mathias et al. (2009) High-affinity neurotrophin receptors and ligands promote leukemogenesis. Blood 113:2028-37
Arumugam, Paritha I; Higashimoto, Tomoyasu; Urbinati, Fabrizia et al. (2009) Genotoxic potential of lineage-specific lentivirus vectors carrying the beta-globin locus control region. Mol Ther 17:1929-37
Modlich, Ute; Baum, Christopher (2009) Preventing and exploiting the oncogenic potential of integrating gene vectors. J Clin Invest 119:755-8
Modlich, Ute; Navarro, Susana; Zychlinski, Daniela et al. (2009) Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther 17:1919-28
Zychlinski, Daniela; Schambach, Axel; Modlich, Ute et al. (2008) Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther 16:718-25
Schambach, Axel; Baum, Christopher (2008) Clinical application of lentiviral vectors - concepts and practice. Curr Gene Ther 8:474-82
Modlich, U; Schambach, A; Brugman, M H et al. (2008) Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16. Leukemia 22:1519-28
Kustikova, Olga S; Baum, Christopher; Fehse, Boris (2008) Retroviral integration site analysis in hematopoietic stem cells. Methods Mol Biol 430:255-67
Schambach, Axel; Baum, Christopher (2007) Vector design for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. DNA Repair (Amst) 6:1187-96
Meyer, J; Rhein, M; Schiedlmeier, B et al. (2007) Remarkable leukemogenic potency and quality of a constitutively active neurotrophin receptor, deltaTrkA. Leukemia 21:2171-80

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