folding, routing and/or stabilization of
P-glycoprotein but does not seem to play a role in the drug efflux activity of
P-glycoprotein (see (Gottesman et al., 1995; Schinkel et al., 1993)). Although sequence analysis revealed more than 40 consensus PKC
and/or PKA phosphorylation sites distributed throughout the primary structure
of the human P-glycoprotein, only very few (~4) are thought to be
phosphorylated. Phosphorylation of these sites does not seem to play an
essential role in drug transport (Germann
et al., 1996).
Unlike other members of the ABC superfamily of
transporters, the multidrug transporter can detect and extrude a wide spectrum
of structurally and functionally diverse compounds (Table 2). These include various anticancer drugs like the
vinca alkaloids, anthracyclines and epipodophyllotoxins as well as other
cytotoxic agents. It is also capable of mediating transport of non-cytotoxic
clinically useful pharmacological agents like calcium channel blockers (e.g.
verapamil), immunosuppressive drugs (e.g. cyclosporine A), antihistamines, etc.
These drugs or their analogues, also known as reversing agents or
chemosensitizers, can serve as competitive inhibitors to block and sensitize
the multidrug transporter to the anticancer drugs and reverse the multidrug
resistance phenotype. An examination of the types of compounds that serve as MDR1 substrates reveal that they are generally
hydrophobic and are usually positively charged at physiological pH (see (Gottesman
and Pastan, 1993)). Both photoaffinity labelling and mutational analyses suggest that
the transmembrane domains in the two halves of P-glycoprotein are important in
determining the substrate specificity of the drug transport function. The fifth
and sixth transmembrane domains including the extracytoplasmic loop between
them in the N-terminal half as well as the corresponding 11th and 12th
transmembrane domains and their intervening loop at the C-terminal have been
shown to be the major drug binding sites (see (Gottesman
et al., 1995)).
III. Distribution and role of the multidrug
transporter in normal tissues
An appreciation of the normal distribution and
physiological role of MDR1 is
important for the design and implementation of any intervention strategy. The
multidrug transporter is expressed in a cell and tissue specific manner
providing some hints as to the physiological role of this transporter. Some of
these roles were recently confirmed when homologous genes in mice were
inactivated using gene knockout technology (see (Borst
and Schinkel, 1996)). Three major roles can be deduced from the major tissue distribution
patterns of the multidrug transporter.
1. Role in steroid transport.
The plasma membrane of the adrenal cortex, site for
steroid secretion, contains the highest concentration of MDR1. Another tissue involved in steroid secretion, the
endometrium of gravid uterus and placenta also expresses MDR1. This suggests that the multidrug transporter may be
involved either in the secretion process itself or protecting the membranes of
secreting cells from the toxic effects of high concentration of steroids.
Interestingly, mdr1(a/b) knockout
mice are fully viable and have normal litters.
2. Role in transepithelial transport of endogenous
metabolites and xenobiotics.
High concentrations of MDR1 can also be found in the luminal surfaces of
epithelial tissues like the brush border of proximal renal tubules; mucosal
surfaces of the large and small intestines as well as the apical surfaces of
pancreatic ductules and biliary canalicular surface of hepatocytes. The
localization of MDR1 in these
tissues implies that the transporter may also be involved in the normal
excretion of various endogenous metabolites or exogenous xenobiotics. This role
is further suggested by the phenotype of the mdr1 knockout mice. These mice exhibited delayed
clearance kinetics of the drug, vinblastine, which is consistent with a defect
in either liver or kidney excretion of the drug (Schinkel
et al., 1997; Schinkel et al., 1994; Schinkel et al., 1995).
3. Role in blood-brain, blood-germ cell/fetus
barrier.
Expression of the MDR1 in the capillary endothelial cells of the brain, testis and placenta
is suggestive that the multidrug transporter may be involved in keeping
cytotoxic products out of the brain, germ cells and fetus. This role is
supported by the observation that transgenic knockout mice lacking the mouse mdr1b gene accumulate toxic levels of the anticancer
drug, vinblastine and the antihelminthic agent, ivermectin, in their brains (Schinkel
et al., 1994; Schinkel et al., 1996)
Some expression of MDR1 can be found in human hematopoietic progenitor cells
(CD34+) (Chaudhary and Roninson, 1991) and peripheral blood including certain subpopulation of T cells (Chaudhary
et al., 1992; Drach et al., 1992). Mature bone marrow cells expressed even lower levels of MDR1 suggesting a downregulation of this gene during
maturation of the hematopoietic stem cells. Possible roles of the multidrug
transporter in the hematopoietic stem cells include the export of a regulatory
molecule to modulate their differentiation and proliferation and/or the protection
of the stem cells against toxic insults.
IV. Protecting bone marrow cells from the toxic
effects of chemotherapy
As mentioned earlier, MDR1-based multidrug resistance during chemotherapy can
be circumvented either by the use of reversing agents, which may cause other
side effects or by increasing the dose of the chemotherapeutic agent. However,
high doses of chemotherapy causes myelotoxicity resulting in leukopenia,
thrombocytopenia and anemia in the patient since the hematopoietic system is
highly sensitive to anti-cancer drugs. One possible solution would be to
introduce the MDR1 gene into the
sensitive hematopoietic cells to protect these cells from anticancer agents.
This has been demonstrated to be feasible in animal studies. A transgenic mouse
model has been made in which the MDR1 cDNA driven by the chicken b-actin promoter was expressed in bone marrow cells (Galski
et al., 1989). Normal bone marrow function was observed in these transgenic mice and
they tolerated higher doses various anti-cancer drugs (Galski
et al., 1989; Mickisch et al., 1992; Mickisch et al., 1991; Mickisch et al.,
1991). Furthermore, bone marrow from these transgenic mice was successfully
engrafted into lethally irradiated sensitive mice confering a multidrug
resistant phenotype on the receipient mice (Mickisch
et al., 1991). Retroviral transduction of the MDR1 cDNA in mouse bone marrow cells and subsequent
introduction of these cells into irradiated sensitive mice also render the bone
marrow of recipient mice drug resistant (Hanania
and Deisseroth, 1994; Licht et al., 1995; Podda et al., 1992; Sorrentino et
al., 1992). Several observations in these mice suggest that the MDR1 gene was in fact delivered into stem cells. These
include long term expression of the multidrug transporter in different
hematopoietic lineages, a single proviral integration site in different cells
and persistence of gene expression following serial transplantation of bone
marrow from resistant to sensitive mice. Greater drug tolerance was also
observed in simian models transduced with the MDR1 gene (Boesen
et al., 1995). The multidrug transporter gene has also been introducted into human
hematopoietic cells via retroviral transduction (Hegewisch-Becker
et al., 1995). Besides bone marrow, various other sources of hematopoietic stem
cells can be used to introduce the MDR1, including cord blood (Bertolini
et al., 1994; Williams and Moritz, 1994) and mobilized peripheral blood progenitors (Chen
et al., 1995; Prosper et al., 1997; Scott et al., 1997; Sekhsaria et al., 1993;
Sutherland et al., 1995). Several clinical trials are being performed to evaluate the
feasibility of dose escalation in advanced cancer patients receiving bone
marrow that has been transduced with MDR1 retroviruses (Deisseroth et al., 1994; Hesdorffer et al., 1994;
O'Shaughnessy et al., 1994; Rosenberg et al., 1996).
V. Retroviral vs non-viral delivery
Successful gene transfer is dependent on two important
steps, namely efficient delivery of the transgene to the appropriate cells and
its subsequent maintenance and expression. Delivery modalities can be viral or
non-viral. The most exploited system for gene transfer is via murine retroviral
vectors (Miller, 1992; Miller et al., 1993; Miller and Rosman,
1989). These vectors have thus far been the main route of MDR1 gene transfer into hematopoietic cells. They are
replication-defective with their “non-essential genes” deleted and
replaced with the gene of interest. The “non-essential genes” are
then supplied in trans since they encode proteins that are important for viral
functions incuding replication and packaging. Gene transfer is effected by the
binding of the vector to receptors on the target cells. The advantages of the
retroviral system are that they are relatively safe and efficient, their host
range can be manipulated, and they can stably integrate into the host chromosome
for persistent expression. Disadvantages of these vectors include the
requirement of murine retroviruses for active DNA replication/cell division and
their propensity to integrate randomly into the host genome increasing the risk
for insertional mutagenesis. Another safety concern is the generation of
unpredictable replication competent viruses via recombination of the viral
vector sequences with endogenous or exogenous helper viruses. Hence, non-viral
modalities of gene delivery as a plausible alternative has been given
increasing attention.
A popular non-viral approach to gene transfer is
liposome-mediated delivery. Entry of naked DNA into a cell is hindered by the
size of the DNA which is typically in the micron range as well as the similar
charge on the DNA and the cell membrane causing them to repel each other.
Liposomal delivery systems serve to compact the DNA and provide a
“double-sided sticky tape” to bind the anionic DNA to the anionic
cell membrane so as to favor membrane destabilization or endocytosis.
Basically, polycationic lipids are mixed with plasmid DNA to form liposomes
which will then fuse with the target cell and mediate gene transfer (Felgner
and Ringold, 1989). Different formulations of lipids have been developed. These usually
consist of mixtures of a neutral co-lipid exhibiting fusogenic properties with
a cationic lipid or cytofectin to form cationic liposomes which are then mixed
with DNA before being introduced into cells.
The formulation we have used was first described by
Behr (Behr, 1986) and refined by Thierry et al. (see (Thierry
et al., 1997)). These are lipopolyamines (DLS) consisting of
dioctadecylamidoglycylspermine (DOGS) (TransfectamTM; Promega) and
Dioleylphosphatidyl ethanolamine (DOPE) (see Figure 2) with a self-aggregating hydrocarbon tail linked to a
polycationic DNA-compacting headgroup. It has protonatable polyamines as lipid
head groups which can buffer the complex against endosomal degradation and
effectively condense DNA into a discrete DNA-lipopolyamine complex in the
nanomicron range and has been found to be relatively non-toxic.
One advantage of this gene delivery system is that
since liposomes are mainly made up of DNA and lipids and contain no proteins,
host response is minimized. Liposomes
can also potentially accomodate larger sized DNA. Furthermore, liposome-DNA
complexes are technically simpler to prepare, test and scale-up compared to
retroviral vectors. Due to their inherent modularity, they can be designed to
target different tissues. However, for this delivery system to be useful for
clinical gene therapy, a number of obstacles must be surmounted. Compared to
viral vectors, this method of introducing DNA into cells is relatively
inefficient (Baudard et al., 1996). Moreover, targeted gene delivery using conventional liposomes is
limited by the selective uptake of liposome-DNA complexes by cells of the
reticuloendothelial system (RES). This can be partially
Figure 2:
Structure of lipopolyamines, DLS. Top panel: Dioctadecylamidoglycylspermine or DOGS. Bottom
panel: Diolelylphosphatidyl
ethanolamine or DOPE.
circumvented by the use of long-circulating,
stearically stabilized or “stealth” liposomes which display
monosialoganglioside GM1 or polyethylene glycol (PEG) (Allen,
1994).
VI. Maintenance of the transgene within cells
Maintenance of the transgene can be achieved either
through the integration of the transferred DNA into the host genome or as an
autonomously replicating extrachro-mosomal element or episome. Retroviruses
have evolved effective ways to mediate integration of their reversed
transcribed DNA into the host genome. However, integration of a random
supercoiled DNA into the genome occurs only occasionally. Hence, the addition
of sequences that allow episomal replication may be advantageous. Vectors that
replicate episomally can be created by incorporating the origin of replication
and a gene product important for maintaining the episomal replication from either
the Epstein Barr Virus (EBV) virus (Margolskee,
1992; Sabbioni et al., 1995) or the BKV vectors (Sabbioni
et al., 1995; Thierry et al., 1995). These vectors can maintain high levels of expression via vector
amplification. EBV episomes have been found to replicate in lymphoid cells at
~10-50 copies per cell, while BK virus can replicate in diverse cell types into
~150 copies per cell. Maintaining the transgene episomally is attractive as it
reduces the risk of insertional mutagenesis as well as host cis-chromosomal
effects on the transgene expression.
The episomal vector that was utilized in our studies
is derived from EBV, a human lymphotropic herpesvirus (see (Margolskee,
1992)). Its genome is 172 kb long and encodes approximately 100 different
genes, most of which are responsible for viral production. The life cycle of
EBV comprises two phases, a lytic and a latent phase (Fig 3). More than 90% of the adult human population harbors
this virus in its latent phase asymptomatically. The virus exists latently as
an episome. The episomal phase of EBV is maintained by two elements interacting
to ensure that the viral genome is retained within the nucleus, efficiently
replicated and properly partitioned into daughter cells. Replication is
bidirectional occuring once per cell cycle in synchrony with the host. The
cis-acting element is the episomal origin of replication, known as OriP. The
OriP (Fig 4) maps to approximately
1.8 kb of the EBV genome and comprises two distinct sets of sequence motifs,
both of which are important for replication. They are the family of repeats
(FR) comprising 20 tandem repeats of 30 bp in length each and the dyad symmetry
(DS) component which is approximately 140 bp in length and contains a 65 bp
dyad symmetry. The FR is separated from the DS by about 960 bp of sequences.
Replication is initiated at the DS while the FR serves as a replication fork
barrier. The transacting element responsible for episomal maintenance of EBV is
the EBV nuclear antigen 1 (EBNA-1). EBNA-1 is a 65-80 kDa phosphoprotein that
is encoded by a 2 kb open reading frame (ORF) within a 3.7 kb transcript. This
protein comprises unique N and C-terminal domains joined by a central domain
that contains glycine-alanine (G-A) repeats. These G-A repeats were found not
to be essential for the transacting functions of EBNA-1 (Yates
et al., 1985) but were recently implicated in viral escape from the cytotoxic T
lymphocyte (CTL) surveillance (Levitskaya
et al., 1995). EBNA-1 plays an important role in replication as well as
transcriptional activation by binding to the FR and related sequences in DS.
Interaction of EBNA-1 with DS initiates bidirectional replication while binding
of EBNA-1 to FR enhances transcription from the episome and terminate DNA
replication. These two elements have been widely exploited to maintain other
genes of interest episomally.
To ensure long term expression of the transgene in
this episomal configuration in rapidly dividing cells, selective pressure has
to be applied. An episomally maintained MDR1 that is introduced into rapidly dividing
hematopoietic stem cells would be very useful to protect hematopoietic cells
human P-glycoprotein (Lee et al., unpublished data).
The resistant cells can be maintained in selective medium for several months
and extrachromosomal DNA could be recovered from HIRT supernatants (Hirt,
1967).
VIII. Other potential applications of liposome
delivered, episomally maintained mdr1.
Besides its utility in protecting sensitive bone
marrow cells from toxic chemotherapeutic drugs, such a vector can also be very
useful in various other gene therapy applications. For an episomal vector to be
clinically useful, an appropriate selectable marker is essential to maintain
selective pressure for stable expression transgene. Although neo-resistance due
to expression of a neomycin phosphotransferase DNA has been popularly used in
vitro, its applicability in vivo is limited because of the toxicity of G418 (Valera
et al., 1994). MDR1, in contrast, is a
clinically relevant in vivo
selectable marker. Surface expression of MDR1 allows for easy detection and sorting of cells
containing this transgene.
Mutations in different regions of the MDR1 gene change the relative resistance to different
drugs, making it possible to construct “designer” MDR1 gene that distinguish cells containing the transgene
from the endogenous gene (Gottesman et al., 1995). Empirically, MDR1 has
been successful as a dominant selectable marker for the coexpression of many
genes (see (Sugimoto et al., 1996)) including HSV-TK (Sugimoto
et al., 1994; Sugimoto et al., 1995), glucocerebrosidase (Aran
et al., 1996; Aran et al., 1994; Aran et al., 1996), a-galactosidase (Sugimoto
et al., 1995), a subunit of the phox flavocytochrome b558, gp91phox
(Sokolic et al., 1996) and ribozyme targetted to the U5 region of HIV1 (Lee
et al., 1997).
In summary, although retroviruses have been the
dominant method of gene transfer of MDR1 for chemoprotection, the usefulness of retroviral vectors may be
limited by safety concerns. This chapter discusses another alternative for the
transfer of MDR1 genes using
liposome delivery. Long-term expression is maintained episomally using the EBV
OriP/EBNA. Higher expression of the gene can be obtained using such a vector as
more copies of the gene are maintained within the cell. Such a vector system
combining liposome delivery, episomal maintenance, and MDR1 as clinically useful selectable marker, have
potential applications for gene therapy.
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