might overcome some of these limitations, but further
research will be needed to determine whether
embryonic stem cells are better suited to meet the
needs of gene therapy applications than are adult
stem cells.
One important feature of the optimal cell for delivering
a therapeutic transgene would be its ability to
retain the therapeutic transgene even as it proliferates
or differentiates into specialized cells. Most of the
cell-based gene therapies attempted so far have
used viral vehicles to introduce the transgene into the
hematopoietic stem cell. One way to accomplish this
is to insert the therapeutic transgene into the one of
the chromosomes of the stem cell. Retroviruses are
able to do this, and for this reason, they are often
used as the vehicle for infecting the stem cell and
introducing the therapeutic transgene into the
chromosomal DNA. However, mouse retroviruses are
only efficient at infecting cells that are actively dividing.
Unfortunately, hematopoietic stem cells are quiescent
and seldom divide. The percentage of stem
cells that actually receive the therapeutic transgene
has usually been too low to attain a therapeutic
effect. Because of this problem, investigators have
been exploring the use of viral vehicles that can
infect nondividing cells, such as lentiviruses (e.g., HIV)
or adeno-associated viruses. This approach has not
been entirely successful, however, because of
problems relating to the fact that the cells themselves
are not in an active state [13, 19].
One approach to improving the introduction of transgenes
into hematopoietic stem cells has been to
stimulate the cells to divide so that the viral vehicles
can infect them and insert the therapeutic transgene.
Inder Verma of the Salk Institute has noted, however,
that this manipulation can change other important
properties of the hematopoietic stem cells, such as
plasticity, self-renewal, and the ability to survive and
grow when introduced into the patient [23]. This possibility
might be overcome with the use of embryonic
stem cells if they require less manipulation. And in
fact, some preliminary data suggest that retroviral
vectors may work more efficiently with embryonic
stem cells than with the more mature adult stem
cells. For example, researchers have noted that retroviral
vectors introduce transgenes into human fetal
cord blood stem cells more efficiently than into cord
blood stem cells from newborns, and that the fetal
cord blood stem cells also had a higher proliferative
capacity (i.e., they underwent more subsequent cell
divisions). This suggests that fetal cord blood stem cells
might be useful in cell-based in utero gene therapy
to correct hematopoietic disorders before birth [15, 21].
In some cases
—such as a treatment of a chronic
disease
—achieving continued production of the therapeutic
transgene over the life of the patient will be
very important. Generally, however, gene therapies
using hematopoietic stem cells have encountered a
phenomenon known as
“gene silencing,” where, over
time, the therapeutic transgene gets
“turned off” due
to cellular mechanisms that alter the structure of the
area of the chromosome where the therapeutic
gene has been inserted [6, 7, 11, 22, 24]. Whether
the use of embryonic stem cells in gene therapy
could overcome this problem is unknown, although
preliminary evidence suggests that this phenomenon
may occur in these cells as well [8, 18].
Persistence of the cell containing the therapeutic
transgene is equally important for ensuring continued
availability of the therapeutic agent. Verma noted
that the optimal cells for cell-mediated gene transfer
would be cells that will persist for
“the rest of the
patient
’s life; they can proliferate and they would
make the missing protein constantly and forever
”
[23].
Persistence, or longevity, of the cells can come about
in two ways: a long life span for an individual cell, or
a self-renewal process whereby a short-lived cell
undergoes successive cell divisions while maintaining
the therapeutic transgene. Ideally, then, the genetically
modified cell for use in cell-based gene therapy
should be able to self-renew (in a controlled manner
so tumors are not formed) so that the therapeutic
agent is available on a long-term basis. This is one of
the reasons why stem cells are used, but adult stem
cells seem to be much more limited in the number
of times they can divide compared with embryonic
stem cells. The difference between the ability of adult
and embryonic stem cells to self-renew has been
documented in the mouse, where embryonic stems
cells were shown to have a much higher proliferative
capacity than do adult hematopoietic stem cells [25].
Researchers are beginning to understand the biological
basis of the difference in proliferative capacity
between adult and embryonic stem cells. Persistence
of cells and the ability to undergo successive cell
divisions are in part, at least, a function of the length
of structures at the tips of chromosomes called
telomeres. Telomere length is, in turn, maintained by
an enzyme known as telomerase. Low levels of
telomerase activity result in short telomeres and, thus,
fewer rounds of cell division
—in other words, shorter
longevity. Higher levels of telomerase activity result in
longer telomeres, more possible cell divisions, and
overall longer persistence. Mouse embryonic stem
cells have been found to have longer telomeres and
higher levels of telomerase activity compared with
adult stem cells and other more specialized cells in
the body. As mouse embryonic stem cells give rise to
hematopoietic stem cells, telomerase activity levels
drop, suggesting a decrease in the self-renewing
potential of the hematopoietic stem cells [3, 4]. (For
more detailed information regarding telomeres and
telomerase, see Figure C.2. Telomeres and
Telomerase.)
Human embryonic stem cells have also been shown
to maintain pluripotency (the ability to give rise to
other, more specialized cell types) and the ability to
proliferate for long periods in cell culture in the laboratory
[2]. Adult stem cells appear capable of only a
limited number of cell divisions, which would prevent
long-term expression of the therapeutic gene needed
to correct chronic diseases.
“Embryonic stem cells
can be maintained in culture, whereas that is nearly
impossible with cord blood stem cells,
”
says Robert
Hawley of the American Red Cross Jerome H. Holland
Laboratory for Biomedical Sciences, who is developing
gene therapy vectors for insertion into human
hematopoietic cells [12].
“So with embryonic stem
cells, you have the possibility of long-term maintenance
and expansion of cell lines, which has not
been possible with hematopoietic stem cells.
”
The patient
’s immune system response can be
another significant challenge in gene therapy. Most
cells have specific proteins on their surface that allow
the immune system to recognize them as either
“self”
or
“nonself.” These proteins are known as major histocompatibility
proteins, or MHC proteins. If adult stem
cells for use in gene therapy cannot be isolated from
the patient, donor cells can be used. But because of
the differences in MHC proteins among individuals,
the donor stem cells may be recognized as nonself
by the patient
’s immune system and be rejected.
John Gearhart of Johns Hopkins University and Peter
Rathjen at the University of Adelaide speculate that
embryonic stem cells may be useful for avoiding
such immune reactions [10, 20]. For instance, it may
be possible to establish an extensive
“bank” of
embryonic stem cell lines, each with a different set
of MHC genes. Then, an embryonic stem cell that is
immunologically compatible for a patient could be
selected, genetically modified, and triggered to
develop into the appropriate type of adult stem cell
that could be administered to the patient. By genetically
modifying the MHC genes of an embryonic
stem cell, it may also be possible to create a
“universal”
cell that would be compatible with all patients.
Another approach might be to
“customize” embryonic
stem cells such that cells derived from them
have a patient
’s specific MHC proteins on their
surface and then to genetically modify them for use
in gene therapy. Such approaches are hypothetical
at this point, however, and research is needed to
assess their feasibility.
Ironically, the very qualities that make embryonic
stem cells potential candidates for gene therapy (i.e.,
pluripotency and unlimited proliferative capacity) also
raise safety concerns. In particular, undifferentiated
embryonic stem cells can give rise to teratomas,
tumors composed of a number of different tissue
types (see Chapter 10. Assessing Human Stem Cell
Safety). It may thus be preferable to use a differentiated
derivative of genetically modified embryonic
stem cells that can still give rise to a limited number
of cell types (akin to an adult stem cell). Cautions
Esmail Zanjani of the University of Nevada,
“We could
differentiate embryonic stem cells into, say, liver cells,
and then use them, but I don
’t see how we can take
embryonic stem cells per se and put genes into
them to use therapeutically
” [26].
Further research is needed to determine whether the
differentiated stem cells retain the advantages, such
as longer life span, of the embryonic stem cells from
which they were derived. Because of the difficulty in
isolating and purifying many of the types of adult
stem cells, embryonic stem cells may still be better
targets for gene transfer. The versatile embryonic
stem cell could be genetically modified, and then, in
theory, it could be induced to give rise to all varieties
of adult stem cells. Also, since the genetically modified
stem cells can be easily expanded, large, pure
populations of the differentiated cells could be produced
and saved. Even if the differentiated cells
were not as long-lived as the embryonic stem cells,
there would still be sufficient genetically modified
cells to give to the patient whenever the need
arises again.
Achieving clinical success with cell-based gene
therapy will require new knowledge and advances in
several key areas, including the design of viral and
nonviral vehicles for introducing transgenes into cells,
the ability to direct where in a cell the transgene is
introduced, the ability to direct the genetically modified
stem cells or the secreted therapeutic agent to
diseased tissues, optimization and regulation of the
production of the therapeutic agent within the stem
cell, and management of immune reactions to the
gene therapy process. The ability of embryonic stem
cells to generate a wide variety of specialized cell
types and being able to maintain them in the laboratory
would make embryonic stem cells a promising
model for exploring critical questions in many of
these areas.
“
There are possibilities of long-term
maintenance and
expansion of embryonic stem cells and of differentiation
along specific lineages that have not been
possible with hematopoietic stem cells,
”
Zanjani says.
“
And if they [embryonic stem cells]
could be used [in
the laboratory] as a model for differentiation, you
could evaluate
… vectors for gene delivery and get
an idea of how genes are translated in patients.
”
Cynthia Dunbar, a gene therapy researcher at the
National Institutes of Health, similarly notes that
embryonic stem cells could be useful not only in
screening new viral and nonviral vectors designed to
introduce therapeutic transgenes into cells, but especially
for testing levels of production of the therapeutic
agent after the embryonic stem cells differentiate
in culture [9]. Explains Dunbar,
“These behaviors are
hard to predict for human cells based on animal
studies
… so this would be a very useful laboratory
tool.
” Indeed, the major contribution of embryonic
stem cells to gene therapy may be to advance the
general scientific knowledge needed to overcome
many of the current technical hurdles to successful
therapeutic gene transfer.
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display extensive tropism for pathology in adult brain:
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