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

Gene Therapy and Molecular Biology Vol 1, page 31
(cell membrane and endosomal); destabilize lysosomal
membranes and promote release of plasmid in the cytoplasm.
After escaping serum components and immune cells,
crossing the cell membrane, released from endosomes to
the cytoplasm and transported through the nuclear pores to
the nucleus the transgene has to accomplish two additional
tasks: (i) to be efficiently transcribed and (ii) its
expression to last for long periods. These two very
important factors depend on the DNA regulatory elements
that drive the expression of the therapeutic gene. The use
of mammalian gene expression vectors has revolutionized
the field of direct gene delivery. The proper choice of
promoter and enhancer elements linked to the gene of
interest is decisive for the successful expression of the
gene in the desired tissue or cell type in gene therapy.
The majority of mammalian expression vectors make
use of promoter/enhancer elements from pathogenic
viruses including the immediately early promoter of the
human cytomegalovirus (CMV), the Rous sarcoma virus
(RSV) promoter, the enhancer/origin of replication of
SV40, the adenovirus type 2 major late promoter (Ad-
MLP), as well as promoters from the mouse mammary
tumor virus (MMTV), human immunodeficiency virus
(HIV), herpes simplex virus (HSV), Epstein-Barr virus
(EBV), and others.
Many studies have compared the strength of different
promoters in driving a therapeutic gene both in cell culture
and in vivo. I will mention a few sample studies here.
Recombinant adenoviruses carrying the HSV-tk gene
under control of the human cytomegalovirus (CMV)
immediate early gene promoter or the adenovirus type 2
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negatively charged serum proteins in vivo causing
transgene inactivation; gene expression is transient; i.v.
injection targets mainly the lung
Not taken up by tumor cells but remain in the
extracellular space.
Low transfection; not widely applicable method; naked
plasmid is cleared from blood rapidly.
Not broadly tested.
Stealth Non toxic, escape immune surveillance and concentrate into
liposomes solid tumors by extravasation.
Naked Suited for intramuscular injection and DNA vaccination; easy
plasmid DNA to use; no viral antigens.
Gene gun Easy to use (plasmid-coated gold particles are delivered to
tumor cells using helium pressure); rapid, suited for gene
transfer to tumor specimens from patients for immunotherapy.
major late promoter (Ad-MLP) were compared for their
XI. Promoters and enhancers for
killing efficiency in combination with GCV treatment; the
transgene expression
rat 9L model for brain tumor and leptomeningeal
metastases was used; the adenovirus containing the CMV
A. Viral promoters
promoter showed greater cell killing efficiency compared
to the Ad-MLP promoter; animals with brain tumors
showed significantly longer survival time and animals
with leptomeningeal metastases had symptom-free periods
(Vincent et al, 1997).
Figure 10. Effect of different introns (A)
and polyadenylation signals (B) on CAT
expression. ELM cells were co-transfected
with equimolar amounts of each plasmid using
DMRIE:DOPE and CAT protein levels in cell
lysates were assayed 48 h after transfection;
pCMVβ was used as an internal control. SVI is
the SV40 19S/16S intron; HI, hybrid intron,
SV40 pA, SV40 late polyadenylation signal;
BGH, bovine growth hormone polyadenylation
signal; β-Glo, rabbit β-globin polyadenylation
Doll et al (1996) have compared the efficiency of
expression of the β-galactosidase gene flanked by the
AAV ITRs in brain tumors and primary brain cell cultures
driven by four different promoters. The human CMV
immediate-early enhancer/promoter was always the
strongest, generally by at least one order of magnitude,
compared with the SV40 early enhancer/promoter, the JC
polymovirus promoter, and the chicken β-actin promoter
coupled to the CMV enhancer. High level of expression
was usually seen within 24 h of transgene delivery by
either transfection or infection, but dropped dramatically
within days; all four promoters showed the same decline
in sustaining gene expression of β-galactosidase with time
(Doll et al, 1996).
The type of regulatory elements on plasmid vectors,
including promoter, enhancer, intron, and polyadenylation
signals, were systematically evaluated by Yew et al (1997)
by constructing a series of plasmids. Figure 10 shows the
effect of different introns (panel A) and different poly(A)
signals (panel B) on CAT expression. A hybrid intron (HI)
appeared to be the most effective. There was a 4-fold
increase in CAT expression from the bovine growth
hormone (BGH) poly(A) signal vector compared to the
SV40 poly(A) signal vector.