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

Boulikas: An overview on gene therapy
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Li et al, 1994; Zhao et al, 1997
Glutathione transferase DNA alkylating agents reviewed by Maze et al, 1997
O 6 -methyl guanine transferase Nitrosoureas Allay et al, 1995
Cytidine deaminase Cytosine arabinoside (Ara-C) Momparler et al, 1996
Aldehyde dehydrogenase Cyclophosphamide reviewed by Koc et al, 1996
cell lines accompanied by an increased expression of the
4,500 to 5,000-nt in size mRNA for P-glycoprotein (Chen
et al, 1986).
Rates of drug influx for lipid-soluble drugs are
proportional to drug concentrations in the medium; Pglycoprotein
alone or in conjunction with other cellular
components seems to transport drugs to the exterior of the
cell, a mechanism pronounced in drug-resistant cell lines.
Consistent with the presence of a membrane-bound,
exchangeable pool of drug and a cytoplasmic, non
exchangeable pool, P-glycoprotein was proposed to
directly interact via its hydrophobic transmembrane
domains with the membrane-associated drug molecules
(anthracyclins, vinca alcaloids) to mediate their efflux to
the extracellular milieu (Gros et al, 1986). Doxorubicin, an
inhibitor of topoisomerase II which is a major nuclear
matrix component, has been shown to interact with
hydrophobic regions in calmodulin; calmodulin is also a
nuclear matrix protein. Photoaffinity-labeled analogs of
vinblastine showed direct binding of this drug to Pglycoprotein
(Safa et al, 1986).
Expression of P-glycoprotein is consistently low in
bone marrow cells rendering them particularly sensitive to
certain MDR-type of anticancer drugs; chemotherapy with
these drugs largely depletes or wipes off bone marrow
pluripotent stem cells from patients (myelosuppression).
One approach to this problem has been removal and deepfreezing
of bone marrow samples from cancer patients
prior to chemotherapy; in a second phase CD34 + cells are
isolated from the frozen bone marrow specimen using
negative selection on soybean agglutinin plates followed
by a positive selection on plates coated with anti-CD34 +
antibody (Ward et al, 1994) which are then reimplanted to
the patient or are simply injected intravenously and find
their way to the bone marrow where they implant; this is a
costly undertaking.
Gene therapy approaches are being aimed at
transferring the MDR1 gene under the control of a strong
promoter/enhancer into bone marrow stem cells;
transfected stem cells, from which all B and T cells are
derived, would be rendered resistant to chemotherapeutic
drugs used to treat cancer patients and allow
administration of higher doses of these drugs.
Furthermore, even if a small percentage of cells are
successfully transfected, these cells could be expanded by
selection with MDR-drug. The same approach could be
used to express a nonselectable gene such as the β-globin
gene to treat sickle cell anemias and thalassemias inserted
in the same construct with the MDR1 gene as has been
suggested by Ward and coworkers (1994).
C. Transfer of the MDR1 gene into bone
marrow cells
The purpose of this approach is to overexpress the
MDR1 gene in bone marrow cells in ex vivo or in vivo
protocols in order to render stem cells resistant to cancer
chemotherapy; this will prevent destruction of the bone
marrow stem cells during treatment of cancer patients with
antineoplastic drugs for killing tumor cells. Transfer of the
MDR1 cDNA into primary human hematopoietic
progenitor cells of cancer patients undergoing high-dose
chemotherapy will protect the bone marrow from the doselimiting
cytotoxicity of cytostatic agents.
Transgenic mice expressing the human MDR cDNA in
their bone marrow cells were resistant to doxorubicin
(Galski et al, 1989; Mickisch et al, 1991). Retroviral
transfer of MDR1 resulted in high level expression of both
RNA and P-glycoprotein; taxol-treatment of mouse bone
marrow cells killed those that had not been transfected and
resulted in an enrichment of the cells containing the
human gene (Sorrentino et al, 1992; Podda et al, 1992).
Transfer of the MDR1 gene via a retrovirus into human
CD34 + cells, isolated from bone marrow and stimulated
with IL-3, IL-6, and stem cell factor, showed that 20-70%
of the CFU-GM or BFU-E cells contained the transferred
MDR1 gene by PCR analysis (Ward et al, 1994).
AAV and cationic liposomes have been used for the
transfer of the human MDR1 cDNA to NIH-3T3 cells
followed by selection of successfully transfected cells
based on the drug-resistant phenotype conferred by the Pglycoprotein
efflux pump; a single intravenous injection of
the bicistronic vector complexed to cationic liposomes
into recipient mice, achieved delivery of MDR1 and
human glucocerebrosidase cDNAs in all the organs tested
(Baudard et al, 1996).
Eckert et al (1996) have designed novel retroviral
vectors termed SF-MDR and MP-MDR which
significantly elevated survival of transduced primary
human hematopoietic progenitor cells under moderate
doses of colchicine and paclitaxel in vitro when compared
with a conventional MoMuLV-based vector; the novel
vectors were based on the spleen focus-forming virus or
the myeloproliferative sarcoma virus for the enhancer
DNA sequence and the murine embryonic stem cell virus
for the leader.