Biology Reference
In-Depth Information
stem cells.
116
By transferring mutant MGMT genes into
murine hematopoietic stem cells with gamma-retro-
virus, we observed that transduced HSCs were effec-
tively protected against BG and BCNU or BG and
TMZ treatments.
117,118
The strategy is quite simple. After
viral transduction, heterogeneous population of HSCs,
containing mutant MGMT-expressing and untrans-
duced HSCs, were infused back into the recipients. By
giving treatments of BG, which inactivate endogenous
MGMT protein, plus methylating or chloroethylating
agents, untransduced HSCs will accumulate DNA
damage and die. Mutant MGMT-expressing HSCs are
protected from the treatment, survive, and expand
(
Figure 2.4
). The published results clearly showed
MGMT-P140K lentiviral transduced human HSCs were
also resistant to BG and BCNU treatments, and
in vivo
selection of human HSCs can enhance the repopulation
of human HSCs in non-myeloablated NOD/SCID recip-
ients.
119,120
In small animal imaging study, we have also
observed persistent hematopoietic cell clusters
throughout the animals only after
in vivo
MGMT-
P140K mediated selection.
121
However, those stem cell-
derived cells were not readily detected by peripheral
blood analysis, indicating that blood sampling may
underrepresent the extent of gene transfer and selection
of persistently expanding stem cell populations. Mutant
MGMT
in vivo
selection in larger animals, such as dogs
and primates, shows stable engraftment after drug
treatment.
122
e
124
In these studies, genotoxicity associ-
ated with MGMT after
in vivo
selection has not been
observed. However, it has been argued that only in large
Mutants of MGMT that were resistant to BG, but still
able to repair DNA, were discovered.
112
With further
development by our lab and other labs, MGMT-P140K
and MGMT-G156A were the two mutants that were
most widely used in studies. While both mutants were
highly resistant to BG-mediated degradation,
112,113
MGMT-G156A was less able to transfer alkyl adduct
in
vitro
, and MGMT-G156A protein was also less stable in
cells.
114
Therefore, MGMT-P140K has been preferred in
both preclinical and clinical studies today. By using
MGMT-P140K as the drug resistance gene, initial BG
treatment will not affect transduced cells and further
potentiate the effectiveness of methylating and chloroe-
thylating agents because the transduced cells not only
express MGMT but are not sensitive to MGMT inhibi-
tion by BG. Of note, methylating and chlorotheylating
agents cause DNA damage in both nuclear and mito-
chondria. Mitochondria directed MGMT-P140K
provided better BCNU resistance than nuclear MGMT-
P140K in K562 cells and human primary CD34
cells.
115
An important application of MGMT gene therapy is
in vivo
selection. Low transduction efficiency and low
cell numbers have always been hurdles for successful
gene therapy.
Ex vivo
expansion of viral transduced
HSCs has been difficult due to their tendency to differ-
entiate
in vitro
. Our lab and others have developed an
effective i
n vivo
selection strategy to sustain and enrich
gene-modified stem and progenitor cells with mutant
MGMT genes.
MGMT-P140K has been shown to be the most effec-
tive and promising for
in vivo
selection of hematopoietic
รพ
BG + alkylating
agents or methylating
Agents treatments
Gene-modified HSCs
BG +
BCNU or TMZ
MGMT-P140K &
therapeutic gene
transfer vector
WT MGMT
MGMT-P140K
Therapeutic protein
FIGURE 2.4
MGMT-P140K mediated
in vivo
selection. After infusion of transduced heterogeneous population of HSCs, transduced HSCs
containing MGMT-P140K can be enriched
in vivo
by treating patient with BG and BCNU or TMZ. Untransduced cells will die due to DNA
damage caused by BG and BCNU or TMZ treatment, and transduced cells with MGMT-P140K are protected and will survive. A therapeutic gene
can also be inserted together with MGMT-P140K and it can express a therapeutic protein with MGMT-P140K. (
Please refer to color plate section
).
