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

a modified tTA transactivator gene engineered with the
ligand-binding domain of the estrogen receptor to the
carboxy terminus of the tTA transactivator; a single
retroviral vector could transduce both the transactivator
gene and the VSV-G protein gene controlled by the tTAinducible
promoter into mammalian cells (Iida et al,
1996). The tetracycline-inducible system was modified by
fusing the ligand binding domain of the estrogen receptor
to the carboxy terminus of a tetracycline-regulated
transactivator to regulate VSV-G expression in a
tetracycline-dependent manner that could be modulated by
β-estradiol in stable packaging cell lines (Chen et al,
1996).
D. Limitations and advancements using
retroviral vectors
Before the in vivo gene therapy with retroviruses
becomes a successful reality a number of problems must
be overcome. Despite the extensive use of retroviral
vectors in gene therapy, there are still problems to be
solved and there is an ultimate need for the development
of new, improved retroviral vectors and packaging
systems to fuel further advances in the field of human
gene therapy. The principle limitation of retroviruses has
been poor gene expression in vivo which has been
overcome through the use of tissue-specific promoters.
Use of internal ribosome entry sites from picornaviruses in
retroviral vectors has provided stable expression of
multiple gene enhancers (reviewed by Naviaux and
Verma, 1992; Boris-Lawrie and Temin, 1993).
Little is known about the factors that influence the
efficiency of retroviral infection in vivo. Many commonly
used experimental animal strains, such as mice, harbor
endogenous C-type proviruses, some of which are
expressed and have circulating antibodies against the viral
envelope glycoproteins that cross-react with the Mo-MLV;
the efficiency of retrovirus-mediated transfection in vivo
using a variety of mouse strains was affected by humoral
immune competence and interference between
endogenous MLVs and exogenous recombinant Mo-MLV
(Fassati et al, 1995).
One of the drawbacks of retroviruses for their
exploitation in gene therapy has been the low viral titers
obtained, too low to achieve therapeutic levels of gene
expression; methods for the efficient concentration from
large volumes of supernatant and purification of
amphotropic retrovirus particles have been developed in
several laboratories. For example, Bowles et al (1996)
have used concentration and further purification of virus
particles by sucrose banding ultracentrifugation; animal
studies have shown that viral transduction increased
proportionally with titer of the retrovirus.
Transduced cells producing retrovirus are tissueincompatible
and are, therefore, expected to be attacked by
Boulikas: An overview on gene therapy
6
the immune system; this will lead to the elimination of
therapeutic cells from the body, a phenomenon markedly
associated also with adenoviral gene transfer. A privileged
exception are brain tumor cells expressing recombinant
retrovirus which persist without immunologic rejection
(Culver et al, 1992).
Sodium butyrate treatment of murine retrovirus
packaging cells producing a CFTR vector increased the
production of the retrovirus vector between 40- and 1,000fold
(Olsen and Sechelski, 1995).
The Cre/LoxP recombinase strategy (see below) has
been used to generate retroviral vectors that have the
ability to excise themselves after inserting a gene into the
genome, thereby avoiding problems encountered with
conventional retrovirus vectors, such as recombination
with helper viruses or transcriptional repression of
transduced genes (Russ et al, 1996). Retroviral vectors
with the Cre/LoxP technology have also been used to
deliver the GM-CSF gene to K562 cell culture (Fernex et
al, 1997), for the development of retroviral suicide vectors
for gene therapy using the HSV-tk gene (Bergemann et al,
1995), and for the production of a high-titer producer cell
line containing a single LoxP site flanked by the viral
LTRs (Vanin et al, 1997).
Because retrovirus vectors are integrated into the
genome, transcriptional repression of transduced genes
will often take place from position effects exerted from
neighboring chromatin domains; two matrix-attached
regions (MARs), one at either flank of the transgene, are
proposed here to insulating the gene in the retrovirus
vector from chromatin effects at the integration site by
creating an independent realm of chromatin structure
harboring the transgene. MAR insulators have been used
and can enhance up to 2,000-fold the expression of genes
in transgenic animals and plants (McKnight et al, 1992;
Breyne et al, 1992; Allen et al, 1993; Brooks et al, 1994;
Thompson et al, 1994; Forrester et al, 1994).
E. Targeting of retrovirus to specific cell
types
A number of approaches have been directed to develop
retroviral vectors that are able to target particular cell
types; also efforts focus toward retroviral vectors that
incorporate nonretroviral features and are tailored to
desired needs for specific uses (reviewed by Vile and
Russell, 1995; Gunzburg and Salmons, 1996).
Ideally, therapeutic genes should be delivered only to
the relevant cell type and/or expressed in this cell type.
Viral and nonviral vectors can be targeted through ligandreceptor
interactions. Retroviral targeting through
protease-substrate interactions has also been described;
epidermal growth factor (EGF) was fused to a retroviral
envelope glycoprotein via a cleavable linker comprising a
factor Xa protease recognition signal. Vector particles