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The completion of the human genome
project has brought us one step closer to understanding the molecular
basis of health and disease. The removal of this bottleneck from the drug
discovery process shifts the emphasis toward translating this knowledge
into clinical practice, specifically, validating specific genes as determinants
for disease.
However, for many important diseases, genetic determinants
were identified many years ago. Unfortunately, knowledge of the genetic
mutation has not led to effective drugs in many cases. While the standard
approach to most diseases is to formulate a drug that can modulate the
symptoms or to supplement missing proteins, gene therapy represents a
more intuitive approach of directly correcting or replacing aberrant or
missing genes. This approach carries the hope of a permanent cure. While
progress in gene therapy has been difficult, its potential benefits are
so overwhelming that many companies are working in this area and have
gene therapy drugs in trials.
Diseases Targeted
Over 50% of gene therapy trials are for the treatments
of various cancers such as head and neck, ovarian, or metastatic cancers.
In these cases the idea is to deliver a toxic gene to the cancer cells
or to deliver an immunogen to stimulate specific immune responses that
destroy the cells. Most of the remaining trials address traditional single
gene genetic disorders such as ADA deficiency, hemophilias, cystic fibrosis,
and hemoglobin disorders.
How is the gene delivered?
While the idea of gene therapy seems relatively
simple, the actual delivery of the gene to the diseased area has proved
quite troublesome. The delivery vehicle for the gene must have several
characteristics: it must be safe, it must efficiently deliver the gene
to a high percentage of diseased cells, it must be targeted specifically
to the appropriate cells, and it should be able to regulate levels of
expression of the therapeutic gene (e.g. turning the gene on or off).
Interestingly, viruses possess many of these characteristics, with different
viruses having different advantages. Thus, viral "vectors" have
been the most popular choice to deliver therapeutic genes. Obviously,
however, their one drawback is safety and scientists are still working
to limit the inflammation and immune response caused by giving high doses
of viral vector to patients.
Viral Vectors:
Turning a virus into a gene therapy
delivery vehicle requires altering the virus both to prevent it from causing
disease and to add the therapeutic gene. This universally involves making
replication-defective viruses that have been engineered so they can’t
reproduce in the body once delivered. The genes required for viral reproduction
have been removed and are only present in helper cell lines that are used
to grow the viruses initially. Other modifications to the virus are made
so that it cannot acquire these needed genes from viruses they might encounter
in the patient. While this prevents the virus from spreading in the patient,
and limits the amount of inflammation caused in the body, it makes the
virus less efficient at getting into cells and delivering the therapeutic
gene. Thus, very high doses of virus vector are required to treat a patient,
which are harder to produce and can lead to safety issues.
Around thirty five percent of gene
therapy trials have therapies using retroviruses, making them a very common
gene therapy vehicle. These curious pathogens are single-stranded RNA
viruses that produce double-stranded DNA upon infecting cells. The defining
advantage of these viruses is that the can integrate, meaning that the
double-stranded DNA they produce can incorporate in and become part of
our genome. The consequence of this is that when the infected cells divide,
all of the progeny will receive copies of the incorporated gene. This
allows the therapeutic gene to persist in the host cells for a very long
time, perhaps permanently. The major limitation of these viruses is that
most will only infect dividing cells, which limits the diseases that it
can be used to treat. Most of the vectors are derived from Moloney murine
leukemia virus which can infect mouse and human cells. Of new interest
are the lentiviruses, which are a subset of retroviruses. The most infamous
of these viruses is HIV, but it has great promise as a vector. This is
because lentiviruses can infect non-dividing cells as well as dividing
cells, and thus can deliver therapeutic genes to almost any diseased cell
type. Additionally, HIV is very good at evading our immune response, perhaps
allowing high dosages and multiple treatments.
reference
for picture
Another mainstay of the field is adenovirus,
which normally causes the common cold. Its advantage is that it will readily
infect non-dividing cells such as neurons and cells of the heart. The
genetic material will not integrate into the genome, so its length of
expression is limited to a few months. While this may seem like a bad
characteristic, it may not be. Integrating into the genome does allow
almost permanent expression, but retroviruses can integrate anywhere,
and could possibly integrate right in the middle of an essential gene
like a tumor suppressor. Thus, the actual application of a retrovirus
gene therapy might create an entirely new disease. While adenoviruses
do not have this danger, their limited temporal expression requires multiple
treatments over time. Also, our bodies mount an impressive immune response
to adenovirus, such that application of the therapy can cause high levels
of inflammation and will prohibit multiple doses of the vector.
reference
for Adenovirus picture
Adeno-associated viruses (AAV) are
the newest viral vector developed and reportedly cause little to no immune
response, making it perhaps the safest of the bunch. In fact, these viruses
have never been shown to cause any disease in people at all. These viruses
possess one other critical feature; they can integrate into our DNA for
long term expression. Even more importantly, they integrate at the same
spot every time and don’t disrupt any genes, thus eliminating the risk
of insertional mutagenesis discussed above. Fascinatingly, if an AAV infected
cell is then infected with a regular adenovirus, the integrated AAV will
un-incorporate itself from the genome. Thus, even if AAV happened to integrate
at a certain place that disrupted a tumor suppressor and caused cancer,
it could be released from this location by an adenovirus, and the normal
function of the gene should return. The final advantage is that the virus
will invade both dividing and non-dividing cells. Why then isn’t everyone
using AAV? The answer is simple, the virus is very small and many therapeutic
genes simply will not fit. See picture above for comparison of AAV particle
size to that of adenovirus.
Non Viral Delivery Tools
| While viruses have
many advantages as carriers for therapeutic genes, their main limitations
are safety and difficulty to produce them in concentrated amounts.
Thus, other methods of gene delivery have been investigated. The dominant
non-viral method of delivery is the plasmid/ liposome conjugate. Plasmids
are closed circles of DNA that are very stable and will reproduce
in bacteria, yeast, and human cells. The therapeutic gene is encoded
in the plasmid DNA with regulatory elements that can control its expression
levels. The plasmid is mixed with fatty-acid molecules, the same as
surround our cells. The fatty-acids create a shell around the DNA
called a liposome. Because liposomes are made of the same molecules
as the outside of our cells, they can mix and fuse with cells when
they touch, spilling their contents into the cell. |
reference
for picture |
Unfortunately, they are not very efficient and
there is no integration of the plasmid DNA. Therefore, the gene is not
stable and the procedure must be repeated every few months. The main advantages
are that plasmid/liposome complexes are really easy to manufacture, cause
no immune response, and can carry large genes. Additionally, the insertion
of proteins into the outside of the liposome may allow them to bind to
only certain cell types, making it possible to target them to diseased
cells and no others.
Companies
Targeted
Genetics (TGEN)
Targeted Genetics has several delivery systems
in development, realizing that no one system will suit all needs. They
were first movers with adeno-associated virus vectors and have modified
the virus in numerous ways to improve its safety. However, they have also
licensed a new type of non-viral lipid delivery system that shows promise.
This allows them to develop gene therapies for a wider variety of diseases
than if they were to focus on one method of delivery.
Current Gene Therapy Projects include cystic fibrosis,
hemophilia A, arthritis, head and neck cancer, ovarian cancer, and metastatic
cancers. Their most advanced product is a liposomal system called DC-Cholesterol
that delivers tumor-suppressing genes. They obtained exclusive licensing
of a gene called E1A that, when introduced into cancer cells, makes them
more sensitive to chemotherapy. The drug successfully completed phase
II results, but for phase III trials the company is planning to combine
the treatment with traditional forms of chemotherapy to increase efficacy.
Cell Genesys
(CEGE)
Cell Genesys’ gene therapy strategy is to first
develop ex vivo therapies, or therapies that are performed on cells
outside the body. This provides the distinct advantages that there is
no immune response against the therapy because it is done outside the
body, and it allows 100% of cells to gain the therapeutic gene before
putting them back into the patient. This is a simpler first step that
they believe will lead to direct therapy for cells in the body. They also
divide potential products into two categories: off the shelf and patient-specific.
Off the shelf therapies are treatments that can be used on any patient
and could basically be packed in vials that a pharmacy could stock. Patient-specific
therapies would be packaged as kits that would be custom processed at
clinical labs on an individual basis to tailor the treatment for each
patient.
Current projects include the GVAX vaccine for several
types of cancers as well as gene therapy for hemophilias, restenosis,
and Parkinson’s disease. Their GVAX vaccine is in phase II trials. The
therapy is based on introducing GM-CSF into tumor cells from the patient.
When these cells are re-introduced to the patient, GM-CSF illicits a potent
immune response that may attack the remaining cancer cells.
Introgen
(INGN)
Introgen’s current flagship product, INGN 201,
is an adenovirus vector that delivers the p53 tumor suppressor gene. This
gene is believed to be lost due to mutation in about 50% of all cancers.
Moreover, presence of p53 has been postulated to confer sensitivity to
chemotherapy. They have used this therapy on about 400 patients so far
and are beginning a phase III trial for head and neck cancer, a phase
II trial for non-small cell lung cancer, and 6 phase I trials.
Vical (VICL)
Vical has focused on non-viral methods of delivery.
They use plasmid DNA enclosed in liposomes that may be infused or injected.
They believe this method will elicit a much more reduced immune response
than a virus-based system. Additionally, liposomes have several manufacturing
advantages in that they are far cheaper to produce and easier to store
than viruses. No cells are needed to produce them (unlike viruses) and
the purification of the product is therefore easier. Also, plasmid DNA
and liposomal reagents have long shelf lives. These features combined
may result in safer, cheaper therapy that could be given on an outpatient
basis. They have a drug in phase 3 trials for metastatic melanoma.
Allovectin is their leading product. It is in phase
three trials for metastatic melanoma, a type of skin cancer for which
there are about 30,000 cases per year, about half of which result in death.
This therapy is based on injecting liposomes carrying a gene for HLA-B7.
This is a highly immunogenic molecule that when taken up and expressed
by the cancer cells causes a strong and robust immune response. A generalized
immune response may then develop toward the cancer cells, attacking metastatic
tumors that haven’t received the drug.
Ribozyme (RZYM)
Ribozyme is engaged in the use of ribozymes to
degrade mRNA as an alternative to gene therapy. They therefore use fundamentally
different technology than most of the other companies here, but is included
because the goals are similar. However, they also have their own burden
of proof for safety and efficacy. Ribozymes are cheap to produce, but
as snippets of RNA, are fundamentally more difficult to store than DNA
is because RNA spontaneously degrades itself in time. The basic idea is
to deliver a ribozyme to a cell in which an incorrect protein is being
made and causes a disease. The ribozyme will chew up the specific mRNA
for that incorrect protein, so that the protein is never made. This type
of technology can only remove bad proteins, it cannot replace them with
the correct copy.
Their leading candidate, angiozyme, belongs to
the class of angiogenesis inhibitors. It is a catalytic RNA that binds
to and destroys the mRNA for vascular endothelial growth factor or VEGF.
VEGF is believed to be the signal that cancer cells emit to induce the
growth of blood vessels to nourish the tumor. RZYM plans to inject their
ribozyme into the tumor where it will shut off VEGF production and cut
off the blood supply to the tumor. The drug is in phase II trials.
Current Obstacles
Delivery remains the problem in the field. The
search continues for the perfect vector that will efficiently infect the
types of cells desired without causing overwhelming immune responses.
The entire field suffered a major setback from the death of an adolescent
in a trial in Pennsylvania. While every major clinical trial has some
patients who react adversely to the therapy, gene therapy almost became
a bad word among the public. The increased oversight and scrutiny brought
on by this event will ultimately be good for the public at large, but
it may take a while before the gene therapy field overcomes the stigma
created.
Many companies are working on next generation viral
systems such as adeno-associated virus that should prove safer. Improved
management of trials should guard against further setbacks in the field.
In addition, results from groups like Vical that employ non-viral methods
are also eagerly awaited. All in all, the promise of gene therapy still
lingers, and current research is getting us closer. However, the "magic-bullet"
quality that it originally had as a therapy will never again be attained.
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