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

A closely related family of ubiquitous DNA binding
proteins, called MDBP, binds with high affinity to two 14
base pair (bp) sites within the human cytomegalovirus
immediate early gene 1 (CMV IE1) enhancer; these
MDBP sites did not require cytosine methylation for
optimal binding; mutation of one of the enhancer MDBP
sites to prevent MDBP recognition modestly increased the
function of a neighboring CREB binding site in a transient
transfection assay (Zhang et al, 1995). Furthermore, the
CMV promoter competed with the Egr1 promoter for
transcription factors or co-factors which might be required
for activation by WT1; WT1 was converted from an
activator to a repressor by co-transfection of an excess of
the parental CMV-based vector (Reddy et al, 1995).
C. Tissue-specific promoters in gene
therapy
A number of studies have used tissue-specific
promoters and enhancers from mammalian genes in order
to attain a cell type-specific expression of the transgene.
The discovery of genes which are expressed at high levels
in specific tumor cell types has prompted the idea of the
use of their promoter or enhancer DNA sequences to
express in this particular cancer cell type therapeutically
important genes (Venkatesh et al, 1990; Brady et al, 1994;
Dimaio et al, 1994; Osaki et al, 1994; Pang et al, 1995).
Examples include the expression of the suicidal CD
gene under control of the regulatory regions of the tumor
marker gene carcinoembryonic antigen (Richards et al,
1995), the expression of HSV-tk gene, under control of αfetoprotein
enhancer and albumin promoter, into adult
liver cells in transgenic animals (Su et al, 1996), the
expression of β-galactosidase in tyrosine hydroxylaseexpressing
neurons in the substantia nigra midbrain of
adult rats using the tyrosine hydroxylase promoter (Song
et al, 1997), and the expression of the lacZ marker gene
under control of the murine pancreatic amylase promoter
in the pancreas in neonatal and adult mice (Dematteo et al,
1997). Transduction of the human LDL-R cDNA under
the transcriptional control of the liver-type pyruvate kinase
promoter allowed high and tissue specific expression of
the gene in primary hepatocytes (Pages et al, 1996b).
Fibroblasts, infected with recombinant retroviruses and
selected with G418 for the expression of the vector
carrying the therapeutic gene, have been used for the ex
vivo treatment in animal models; when the therapeutic
gene was either under control of the viral LTR or an
heterologous internal promoter, expression of the
transgene from the integrated retrovirus was shut off
(Scharfmann et al, 1991). The use of the dihydrofolate
reductase housekeeping gene promoter which is expressed
in all cell types, led to sustained expression, albeit at very
low levels (Scharfmann et al, 1991); it appears that the
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combination of a suitable enhancer and promoter for a
particular cell type and the method of introduction of the
transgene is crucial for sustained expression.
Combination of the mouse muscle creatine kinase
enhancer with the human cytomegalovirus promoter to
drive the expression of the canine factor IX gene in ex vivo
infected mouse primary myoblasts led to the expression of
factor IX and its secretion in the blood of mice
transplanted with these myoblasts for over 6 months;
however, the levels of factor IX protein secreted into the
plasma (10 ng/ml for 10 7 injected cells) were not sufficient
to be of therapeutic value (Dai et al, 1992).
Joki and coworkers (1995) have used the promoter of
the early growth response gene 1 (EGR-1, also known as
Zif/268, TIS-8, NFGI-A, or Krox-24) to confer selective
expression of the luciferase gene in glioma cell lines
exposed to ionizing radiation; a 10-fold higher activity in
luciferase activity was found after irradiation of the cells
which was detectable at 1-3 h after stimulation with 20 Gy
(stereotactic radiosurgery during treatment of isolated
brain metastases, arteriovenous malformations,
meningiomas, craniopharyngiomas, and glioblastomas
uses a single dose of 20-30 Gy). Transfection of the HSVtk
gene under control of the EGF-1 promoter rendered
irradiated, but not nonirradiated, cells sensitive to GCV.
Irradiation induces DNA repair, cell cycle arrest, and
reinitiation of DNA synthesis in surviving cells; γradiation
also induces higher levels of a number of
proteins including p53, AP-1, NF-κB, TNF, IL-1, and
EGF-1. Therefore, use of the EGF-1 promoter can activate
gene expression selectively in radiation fields and could be
used to drive the expression of cytotoxic genes (HSV-tk)
for the killing of cancer cells.
Peptides containing the three zinc fingers of Zif268
could efficiently repress activated transcription from
promoter constructs prepared with Zif268 binding sites
inserted at various positions with respect to the TATA box
(Kim and Pabo, 1997); such strategies could find
important applications in gene therapy leading to
construction of artificial promoters able to activate or
repress transcription of transferred genes. A potent hybrid
CAG promoter was used to drive the HSV-tk gene and
showed effective eradication of pancreatic tumors in
animal xenografts (Aoki et al, 1997).
D. Molecular switch systems
The ability to regulate gene expression via exogenous
stimuli will facilitate the study of gene functions in
mammalian cells. Molecular switch systems have been
devised (Wang et al, 1994) allowing the researcher to turn
on or off individual genes; the switch used by Delort and
Capecchi (1996) is composed of three elements: (i) the
inducible UAS promoter, a synthetic promoter containing