01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

A bicistronic retroviral vector (HaMID) containing a
modified human MDR-1 cDNA and a mutant human
dihydrofolate reductase cDNA bearing a leucine to
tyrosine substitution at codon 22 was constructed and used
to transduce the human CEM T lymphoblastic cell line as
well as primary murine myeloid progenitors; HaMIDtransduced
cells were highly resistant in the presence of 25
nM taxol and 100 nM trimetrexate simultaneously while
control cells were entirely growth inhibited (Figures 27,
28; Galipeau et al, 1997).
Gene Therapy and Molecular Biology Vol 1, page 79
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Several human clinical trials, approved by RAC and
FDA, are under way with the long-term goal of
transferring the MDR1 gene into bone marrow cells of
advanced cancer patients using retroviral infection. A
human gene therapy protocol (#100) for chemoprotection
of patients treated for testicular cancer with high doses of
carboplatin and etoposide proposes to use transplantation
of these patients with autologous peripheral blood stem
cells (drawn, purified and cryopreserved prior to
chemotherapy treatment) and transduced with the MDR1
Figure 27. Structure of the retroviral vectors used to deliver the MDR1 and DHFR genes. The vectors are based on the Harvey
murine sarcoma virus. A single transcript (arrow) is initiated in the retroviral 5’ LTR promoter. HaMDR1sc (top) contains the MDR1sc
cDNA and HaDHFR(L22Y) (middle) contains a mutant DHFR cDNA. The bicistronic (two-gene) vector HaMID (bottom) contains both
MDR1 and DHFR genes. From Galipeau J, Benaim E, Spencer HT, Blakley RL, Sorrentino BP (1997) A bicistronic retroviral vector for
protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs. Hum Gene Ther 8, 1773-1783. Reproduced with
kind permission from the authors and Mary Ann Liebert, Inc.
Figure 28. Growth inhibition
assays comparing the effect of 25
nM taxol, 100 nM trimetrexate
(TMTX) alone and in combination
on CEM cells transduced with
HaDHFR(L22Y), HaMDR1sc, or
HaMID. Drug-selected CEM cells
were washed and seeded at 1x10 5
cells/ml in 2 ml of media containing
the indicated concentrations of
drugs. After 72 hr, the percentage of
growth was calculated by dividing
the number of cells at each drug
concentration by the number of cells
present in control medium (100%
growth). Quadruplicate experiments
are shown. a Cells preselected in 100
nM trimetrexate. b Cells preselected
in 25 nM taxol. From Galipeau J,
Benaim E, Spencer HT, Blakley RL,
Sorrentino BP (1997) A bicistronic
retroviral vector for protecting