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

proteins, an effect often associated with adenoviral and
retroviral gene delivery.
A number of strategies are being pursued to solve
these problems. Sustaining the expression of a transgene
into somatic cells for, lets say, 6 months would mean than
a gene therapy treatment would need to be repeated twice
a year, for example to a hemophilia patient or to a patient
who has undergone balloon treatment after coronary heart
disease and is being treated via arterial gene transfer.
An approach to sustain expression of the transgene is
via episomal replication of the plasmid carrying the
transgene for long periods of time, maintaining the
plasmid in high copy numbers, and in a form replicating in
synchrony with the cell cycle; even better a plasmid can be
replicated continuously independently of the cell cycle, an
approach to find application in the transfection of
nondividing cells by plasmids (which to date is a virtue of
adenoviruses, AAV, and HIV-1 vectors; see Table 1).
A way to sustain expression of the transgene could be
achieved via targeted integration into one or several
different chromosomal locations and the insulation of the
transgene from neighboring chromatin domains using
special classes of DNA sequences able to act as insulators
and maintain independent realms of gene activity (such as
matrix-attached regions, MARs). In this case flanking of
the foreign gene by two MAR sequences is expected to
insulate it against position effect variegation and prevent
inactivation of the gene at the chromatin level by
chromatin condensation or other mechanisms propagated
from the neighboring domains at the integration site
(Boulikas, 1995b).
Several studies have shown that linearization of
plasmids with restriction enzymes favor highly their
integration into the host's genome compared with
supercoiled, covalently-closed plasmid DNA. Free ends of
DNA are known to promote recombination and a number
of nuclear proteins including p53, poly(ADP-ribose)
polymerase, ligases I and II, Ku antigen, DNA-dependent
protein kinase are known to bind to free ends of DNA,
whereas other molecules such as helicases and
endonucleases are known to function during repair of
lesions in DNA inducing the appearance of strand breaks;
especially important in this aspect are members of the
RAD50-57 family of proteins involved in recombination
and in repair of double-strand breaks.
B. Episomal plasmids for gene transfer
Integration or replication of a foreign gene introduced
as a plasmid into mammalian cells is a very rare event;
plasmid DNA resides transiently in the nucleus as an
episomal, extrachromosomal element for short periods of
time after transfection of cells in culture (usually up to one
or very few days) during which transcription can take
place; after that the episomal DNA is degraded and lost
permanently from the cells.
Boulikas: An overview on gene therapy
40
Viral origins of replication have been introduced into
the same plasmid as the reporter gene and found to
increase the persistence of expression. A polyoma virusbased
plasmid containing the polyoma virus origin of
replication and the T antigen gene, as well as the neo R
gene was maintained extrachromosomally in mouse
embryonic stem (ES) cells at 10-30 copies per cell for at
least 74 cell generations in the presence of G418
(Gassmann et al, 1995).
Prolonged episomal persistence may be an advantage
for gene therapy of nondividing cells. A limited number of
studies in gene transfer have used plasmids able to
replicate episomally. Most of the plasmids used contain
viral origins of replication but also the gene of the
replication initiator protein that after its expression in the
host will interact with the origin of the plasmid to maintain
a relatively high copy number of plasmids which will
persist for some time. The advantage using episomal
replication of plasmids is enormous in somatic human
gene therapy as it can sustain expression of a transgene for
a few months after a single injection of the plasmid as
compared to the loss of expression after about 1-10 days
(maximum at day 2) following injection of nonepisomal
plasmids (Zhu et al, 1993). Thierry and coworkers (1995)
have succeeded in sustaining the expression of the
luciferase reporter gene in mice for up to 3 months after a
single intravenous injection of a plasmid including the
human papovavirus BKV early region and origin of
replication, the large tumor antigen (T antigen) as the
replication initiator protein, and the late viral capsid
proteins in the same construct harboring the luciferase
gene; this plasmid was shown to be replicated
extrachromosomally for 2 weeks in the lung.
Episomal replication of a hybrid HSV-1/EBV vector
was achieved when the latent oriP of EBV and the EBNA-
1 cDNA, which encodes for the replication initiator
protein of EBV, were included in the vector (Wang and
Vos, 1996).
Expression of viral replication initiator proteins (e.g. T
antigen) is oncogenic. Of special interest in human gene
therapy is to determine human DNA sequences able to
sustain the extrachromosomal replication of plasmids into
permissive human cells for longer periods. Such DNA
sequences known to act as origins of replication, although
poorly understood, have been found in human, monkey,
and other mammalian genomes and could be used to
sustain the replication of the plasmid thus increasing its
copy number in the cell and the time of its persistence (see
page 122-123).
To this end, a technology has been developed in our
laboratory that permits us to isolate human origins of
replication (ORIs) and to include selected ORIs together
with the cDNA of the replication initiator protein
responsible for activating this particular ORI, in plasmids
with therapeutic genes (Boulikas et al, in preparation).