Biology Reference
In-Depth Information
DNA and accepted alkyl adduct, the formation of
Cys145-S-methyl or Cys145-S-chloroethyl is very stable
and unidirectional and leads to the inactivation and
degradation of MGMT protein (
Figure 2.2
). Thus vast
amounts of methyl or alkyl adducts on O
6
-guanine or
its analog could potentially deplete the MGMT reservoir
inside cells.
One of the limitations of current cancer chemo-
therapy is the dose-limiting effect of chemo-drugs
because of the severe myelosuppression from treatment,
resulting in neutropenia. This is particularly the case for
the nitrosoureas and methylating agents that appear to
be stem cell toxins. Introducing drug resistance genes
into non-cancer cells to protect them against chemother-
apeutic drugs has been explored using a number of
transgenes based on their properties protecting cells
from the cell death induced by chemotherapy. However,
for successful gene therapy targeting hematopoietic
stem cell populations, the efficiency of gene transfer is
crucial.
In order to protect and rescue bone marrow cells
against cancer chemotherapeutic agents, the ideal tar-
geting cells would be hematopoietic stem cells (HSCs)
because they are multipotent stem cells, can self-renew,
and can differentiate into all lineages of hematopoietic
cells over extended periods of time. To achieve long-
term MGMT expression in HSCs and their progeny
would be a goal to protect these cells from the toxic
effects of nitrosoureas and temozolomide. To have clin-
ical impact, however, stable integration of the transgene
into the HSC genome is required. Currently, gamma-
retrovirus and lentivirus are frequently used as the
gene transferring vectors in MGMT gene therapy
studies because they can integrate transgene directly
into the cellular genome. In the past two decades,
gamma-retrovirus, murine moloney leukemia virus
(MMLV), was the most popular choice, and about 60%
of clinical trials in the field of gene therapy have used
gamma-retroviruses. However, while expected, delete-
rious events resulted from gamma-retroviral gene thera-
pies were reported in the early 2000s, ending in
a temporary hold of all retroviral gene therapy tri-
als.
96
e
98
For instance, 5 of 20 patients successfully given
gene therapy treatment to cure X-SCID eventually
developed T-cell leukemic disease by gamma-retro-
virus-related insertional mutagenesis after 23
lentiviral vector is safer in terms of insertional mutagen-
esis because gamma-retrovirus have a higher frequency
in inserting into the 5' promoter or enhancer region of
a gene while lentivirus inserts the transgene throughout
the gene and more often in the coding region.
102,103
To protect bone marrow cells with drug resistance
genes, HSCs from patients are removed from the body,
and viral gene transfers are done
ex vivo
, and then trans-
duced cells are infused back into the patients (
Figure 2.3
).
Several drug-resistance genes have been studied over
the years for their ability to protect against various
chemotherapeutic agents, such as multidrug-resistance
protein 1 (
MDR-1
),
104
cytidine deaminase (
CDA
),
105
and dihydrofolate reductase (
DHFR
).
106,107
However,
for methylating agents and chloroethylating agents,
MGMT is the most effective. The severe cytotoxicity in
bone marrow is partly due to low expression of
MGMT in hematopoietic cells.
6
Thus O
6
-alkylguanine,
caused by chemotherapeutic drugs, is a potent genome
toxin, and it cannot be repaired by hematopoietic cells
because of low levels of MGMT protein, and if the
adducts were not repaired, they activate the apoptotic
process. However, by introducing wild-type MGMT to
bone marrow cells, only slight protection in bone
marrow was observed because of the endogenous
MGMT expression in tumor cells.
108,109
Furthermore, to
maximize tumor cell killing, MGMT inhibitor, benzyl-
guanine (BG), has been used to inactivate endogenous
MGMT and sensitize tumor cells to chemotherapeutic
drugs, resulting in even more marrow toxicity.
110,111
HSCs
MGMT-P140K
transfer vector
Gene-modified HSCs
68 weeks,
most often into the growth promoting
LMO-2
gene.
99
Recent studies have focused on using lentiviral vector
as the gene transfer method. Lentiviral vectors can trans-
duce non-dividing and quiescent cells, which is particu-
larly useful when transferring genes to quiescent stem
cells.
100,101
There is always a risk of insertional mutagen-
esis associated with integrating viral gene transfer.
However, it is now accepted that the integration patterns
of gamma-retrovirus and lentivirus are different and
e
Endogenous HSC
MGMT-P140K HSC
FIGURE 2.3
MGMT-P140K gene therapy. Human HSCs are
mobilized in peripheral blood and collected. MGMT-P140K gamma-
retroviral or lentiviral vector will deliver MGMT-P140K gene into
HSCs
ex vivo
. Then transduced heterogeneous population of HSCs
will be infused back into the patient. After HSCs home to their
hematopoietic niches, MGMT-P140K is expressed in active HSCs.
(
Please refer to color plate section
).
