Paterson Institute for Cancer Research SCIENTIFIC REPORT 2005

GENE THERAPY
ued our studies utililising the drug-resistance genes
encoding either the inactivator-insensitive P 140K
mutant version of methylguanine-DNA-methlytransferase
(MGMT), or multi-factoral chemoprotection
by the co-expression of MGMT with either
MDR1 or MRP1, both in vitro and in vivo. Lorna
Woolford has been investigating the potential of
MGMT P 140K resistance to inactivation by PaTrin2.
In this study we have tested the potential of
MGMT P 140K to confer inactivation-insensitive
protection to HSCs. She has shown in vitro that
mutant MGMT confers significant resistance to the
combination of PaTrin2 and temozolamide when
compared with controls and subsequently in vivo in
cells transduced with MGMT P 140K-2a-eGFP used
to reconstitute haematopoiesis in an in vivo model.
In this model she has shown protection of the
gene–modified cells following cytotoxic challenge
and an in vivo selective advantage. Secondary transplantation
experiments indicated that this selection
occurred at the level of the stem cell. To further
these studies Barbara Verbeek has developed
inducible version of MGMT that will add increased
safety and efficacy to the gene therapy selection
strategies that we have been developing.
We have also initiated a study into the potential of
enhancing DNA repair as a way of protecting the
HSCs against other types of chemotherapeutics
such as topoisomerase I inhibitors. Tom Southgate
and Dorothy Gagen have over-expressed XRCC1
within HSCs and demonstrated protection against a
class of chemotherapeutic agents which induce
high levels of collateral toxicity. This is now being
tested in vivo in order to assess whether XRCC1 may
also be used to aid selection. Along with Rebecca
Schofield, Tom has also developed gene therapy
strategies that confer radioprotection. Previously
Tom had demonstrated that SOD2 conferred significant
protection of the HSCs against ionising
radiation. They are now working on enhancing this
protection though the co-expression of Catalase
which will aid in the removal of reactive oxygen
species and their downstream consequences within
the cell. Furthermore, Tom has examined the ability
of XRCC1 to confer enhanced DNA repair after
ionising radiation damage. He has shown that overexpression
of XRCC1 mediates the greatest level of
radiation protection yet conferred to HSCs and that
the mechanism of action is likely to be through a
non-classical action of the XCRR1 protein. This
investigation is still ongoing but suggests that
XRCC1 may be a useful tool in both chemo - and
radioprotection of tissues.
Modifying Macrophages to Infiltrate and Attack
Tumours
Previous work by our group has demonstrated that
chimeric immune receptors targeted against tumour
antigens can specifically direct the Antibody-
Dependent Cellular Cytotoxicity (ADCC) pathway
of macrophages against tumour cells expressing
these antigens. Michael Lie-A-Ling has continued
this work by investigating the use of lentiviral vectors
in delivering chimeric immune receptors to
bone marrow cells. The goal of this is not only to
incorporate the chimeric receptors in a wider range
of effector cells but also to incorporate these receptors
in stem and progenitor cells ensuring long term
production of gene modified effector cells in vivo.
We are aiming to use the lentiviral technology to
express chimeric immune receptors in the myeloid
compartment of the immune system.
P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 5
(A) Illustrates comparisons
made between
HOXB4ERT arrays at two
timepoints. Equivalent
comparisons were also
made for ERT-control
arrays and fold changes
calculated for each probeset.
(B) Illustrates scatterplots
for each of these
c o m p a r i s o n s
(HOXB4ERT time 0 v 48h
time point shown).
Colours identify probesets
with fold changes of ≥
|1.5|; red in all 9 comparisons;
blue in 8; green in 7;
and purple in 6.
Publications listed
on page 59
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