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- Relander, Thomas
(författare)
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Retroviral gene transfer to repopulating hematopoietic cells
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hematopoietic stem cells (HSCs) have the capacity to maintain hematopoiesis throughout life. HSCs are ideal targets for permanent gene transfer, as the transgene will be expressed in all the progeny of the gene-modified stem cells. The aim of this thesis was to optimize conditions for retroviral gene transfer to human candidate HSCs and to study mechanisms interfering with efficient gene transfer. First, long-term bone marrow culture transduction of murine hematopoietic cells was investigated, showing inefficient gene transfer and loss of repopulating ability of the transduced cells. Thereafter, transduction of human CD34+ cells was studied using the MGIN vector with the GALV, amphotropic and VSV-G envelopes. Amphotropic, and, particularly, GALV vector containing medium (VCM) inhibited transduction on fibronectin preloaded with vector. GALV VCM inhibited transduction of NOD/SCID repopulating cells (SRCs) as well. Optimal transduction was seen with vector preloaded to fibronectin and cells added in medium without vector. To study the mechanisms underlying GALV vector mediated inhibition, the GLVR1 receptor was overexpressed using phorbol ester or a retroviral vector, showing complete abolishment the inhibitory effect from GALV VCM. Together our results show that low levels of GLVR1, in combination with non-infectious vector particles in VCM, limit transduction of hematopoietic progenitors. Finally, vectors with GALV, amphotropic and VSV-G envelopes were compared regarding their ability to transfer genes to SRCs. The different pseudotypes were equally efficient in the presence of serum with an average transduction efficiency of 25-33 % of SRCs, but the engraftment levels were reduced compared to fresh cells. Only the GALV vectors could be used under serum-free conditions showing high level transduction of SRCs (46 %) without loss of SRC frequency as analyzed by transplantation at limiting cell numbers. In conclusion, under optimal conditions, highly efficient gene transfer human hematopoietic cells with NOD/SCID repopulating ability could be accomplished. However, for clinical applications, safety issues, particularly regarding the risk of mutagenesis from retroviral vector insertion into the genome of repopulating cells, need to be adressed carefully.
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