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

C. Considerations of chromatin structure
of plasmids during gene delivery
Almost all supercoiled plasmids used in gene transfer,
as produced in bacteria, are under negative supercoiling.
Immediately after their import into nuclei plasmids are
packaged into nucleosomes that absorb and constrain part
or most likely all of the negative supercoils. This is true
assuming that no cuts on the DNA are introduced during
its passage through the cell membrane barrier to
cytoplasmic lysosomes before entering nuclei; if DNA is
cut the supercoils on the plasmid will be relaxed. Nicked
DNA might be repaired and ligated in nuclei by DNA
ligases and be subject to the same constrains as
chromosomal DNA. Use of linear plasmids is expected to
stimulate recombination during repair of double strand
breaks (also would increase degradation of the plasmid in
the nucleus and loss of the transgene) ultimately resulting
in plasmid integration at variable chromosomal loci,
determined to some extend by the nature of the free ends
of DNA and the short terminal sequence of the DNA at the
ends as well as the type of recombinase molecules in the
cell type used.
Treatment of cell cultures with sodium butyrate
inducing hyperacetylation of core histones would reverse
in part the relieving of the negative torsional strain by the
wrapping of the plasmid around histone octamers and will
provide DNA in a negatively superhelical of underwound
form able to sustain transcription of the template (Schlake
et al, 1994).
D. Overcoming the influences of
chromosomal surroundings at plasmid
integration sites
Use of two MARs each flanking the reporter gene on
either side is expected to form a minidomain after
integration of the foreign gene into a chromosomal site.
MARs potentiate the effect of promoters and enhancers
when two MAR elements are placed one upstream and the
other downstream from control elements but not between
them. MARs will (i) shield reporter genes from the
influences of chromosomal surroundings that most often
cause inactivation of foreign genes. This effect of
chromatin structure on neighboring sequences is known as
position effect variegation. Indeed about 85% of the
chromosomal sites are transcriptionally inactive assuming
that 15% of the genomic DNA is transcribed; however,
even integration of a foreign gene into an active chromatin
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locus may not warrantee its transcriptional activation as
other parameters, such as proximity of the integration site
to the natural promoter and enhancer elements of the
active chromatin domain, or orientation of the integrated
gene with respect to the active gene in the chromosomal
DNA may determine its level of expression. (ii) MARs
will maintain a supercoiled DNA topology within the
domain thus increasing the negative supercoiling at local
promoter and enhancer sites, a prerequisite for efficient
transcription (see Boulikas, 1995b).
XIV. Transfer of reporter genes
A. Transfer of the β-galactosidase (lacZ)
reporter gene
Before a gene therapy preclinical study or even gene
transfer to cells in culture begins it is essential to test the
variables and pinpoint the conditions leading to the
success of the operation using reporter gene transfer.
LacZ, encoding the β-galactosidase (β-Gal) from E. coli is
one of the most commonly used reporter genes. A staining
procedure for this enzymatic activity can result in the
generation of blue color using X-Gal as a substrate leading
to the direct visualization of its activity, for example, in
thin sections through animal tissues.
Transfer of the reporter β-galactosidase gene to human
liver tumors in nude mice was performed by Wang and
Vos (1996) using a hybrid HSV-1/EBV vector which
replicates episomally when the latent oriP of EBV and the
EBNA-1 cDNA were included.
Many mammalian tissues, especially intestine, kidney,
epididymis, and lung contain endogenous β-Gal, a
lysosomal enzyme participating biochemically in
glycolipid digestion. Weiss et al (1997) were able to detect
mammalian β-Gal activity on histochemical preparations
of mouse, rat and baboon lung tissue (Figure 16) and also
to distinguish between the endogenous and bacterial β-Gal
activity in airway epithelial cells in the transgenic Rosa-26
mouse in based upon the differences in pH optima
between the mammalian and bacterial enzymes (Figure
17). Time and temperature of exposure to X-Gal could not
be used to distinguish between endogenous and exogenous
β-Gal activity; thus, exposure of tissue preparation to pH
8.0-8.5, which minimized detection of the endogenous
activity allowed unambiguous discrimination and was the
method of choice to detect reporter β-Gal activity.