• Use of genes to treat disease
• Several approaches:
– The fundamental idea is to administer a functional gene, so as to give targeted cells a
new protein-manufacturing capacity, one that would have been present but is missing
or defective, or sometimes never meant to be there.
• For correcting faulty genes
• A normal gene may be inserted into a nonspecific location within the
genome to replace a nonfunctional gene (most common).
• The abnormal gene could be repaired through selective reverse mutation,
which returns the gene to its normal function.
• The regulation of a particular gene could be altered.
• Some diseases can also be treated by interfering with the activity of the
• Cells normally switch genes on and off by attaching certain chemicals to
the gene. One therapeutic approach might be to exploit this process with a
drug that switches a harmful gene off.
• Insert some new DNA into the middle of the harmful gene.
• Insert a gene that is the exact chemical opposite of the sick gene. The
product of this "anti-sense" gene would neutralize the product of the sick
Why is gene therapy hard?
• has to be designed
• needs to be manufactured
• has to be directed to the right cells in the body.
• needs to be controlled, so the protein is produced only when it is needed.
Each of these problems has one or more solutions.
Together, however, they have so far prevented success in all but a few single-
A class of viruses that can create double-stranded
DNA copies of their RNA genomes. These copies of
its genome can be integrated into the genome of
host cells. Human immunodeficiency virus (HIV) is a
•efficient gene transfer into many cell types and can
stably integrate into the host cell genome
•have minimal risk because retroviruses have
evolved into relatively non-pathogenic parasites
(expection: e.g. HIV)
•accept up to about 8 kb of exogenous DNA.
•are not inactivated by human serum, and
transduce dividing cells.
•The DNA virus used most widely for in situ gene
• inserts up to 35 kb
• they are human viruses and are able to
transduce a large number of different human cell
types at very high efficiency
• can transduce non-dividing cells.
•non-pathogenic virus that is widespread in the
•Unfortunately appear to integrate in a nonspecific
•room for about 4.8 kb of added DNA
•hybrid systems have been reported where an
adenoviral vector is used to carry a retroviral
vector into a cell that is normally inaccessible to
Non viral vectors
•two factors suggest that non-viral gene delivery systems will be
the preferred choice in the future:
-ease of manufacturing
-more flexible with regard to size of the DNA being
-generally safer in vivo
-do not elicit a specific immno response and can therefor
be administered repeatedly
but less efficient en delivering DNA particularly when used in vivo
• low immunogenicity but toxicity of lipids and low transfection
• Polimer-based systems
– e.g. Using collagen, lactic or glycolic acid, polyanhdride or
polyethylene vinyl coacetate
– DNA encapsulation within the polymer can protect againgst
degradation until release
– release form the polymer and into the tissue can be designed to occur
rapidly or over extended period of time; thus the delivery system can
be tailored to a particular application.
– Intrinsic drawbacks with cationic carriers such as solubility,
cytotoxicity and low trasfection efficiendy, have limietd their use in
vivo. These vectors sometimes attract serum proteins resulting in
dynamic changes in their physicochemical properties.
•novel delivery sistems which can be administere
in novel ways (e.g. Aerosols) are being developed.
The smaller the size of the condensed DNA
particles, the better the in vivo diffusion towards
target cells and the trafficing within the cell.
•chemical biological hybrid composed of
oligonucleotide DNA, RNA or peptide covalently
attached to nanoparticle (TiO2 4.3 nm).
Could attach one molecule of DNA or pepetide
that could target it ot a particular cellular site, and
another peptide that can carry out an effective
What factors have kept gene therapy from
becoming an effective treatment for genetic
• Short-lived nature of gene therapy - Before gene therapy can become
a permanent cure for any condition, the therapeutic DNA introduced into
target cells must remain functional and the cells containing the therapeutic
DNA must be long-lived and stable. Problems with integrating therapeutic
DNA into the genome and the rapidly dividing nature of many cells prevent
gene therapy from achieving any long-term benefits.
• Problems with viral vectors - Viruses, present a variety of potential
problems to the patient --toxicity, immune and inflammatory responses, and
gene control and targeting issues. There is always the fear that the viral
vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders - Conditions or disorders that arise from mutations in
a single gene are the best candidates for gene therapy. Unfortunately, some
the most commonly occurring disorders, such as heart disease, high blood
pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the
combined effects of variations in many genes.
Gene therapy and cancer
• Researchers are studying several ways to treat cancer using gene therapy.
Some approaches target healthy cells to enhance their ability to fight cancer.
Other approaches target cancer cells, to destroy them or prevent their growth.
Some gene therapy techniques under study are described below.
• replace missing or altered genes with healthy genes.
• stimulate the body’s natural ability to attack cancer cells.
• In some studies, scientists inject cancer cells with genes that make
them more sensitive treatments.
• “suicide genes” are introduced into cancer cells. Later, a pro-drug (an
inactive form of a drug) is given to the patient. The pro-drug is
activated in cancer cells containing these “suicide genes,” which leads
to the destruction of those cancer cells.
• Other research is focused on the use of gene therapy to prevent cancer
cells from developing new blood vessels.
•Phase I trial:are the first step in testing a new approach in humans. In these studies,
researchers evaluate what dose is safe, how a new agent should be given (by mouth,
injected into a vein, or injected into the muscle), and how often. Researchers watch
closely for any harmful side effects. Phase I trials usually enroll a small number of
patients and take place at only a few locations.
•Phase II trial study the safety and effectiveness of an agent or intervention, and
evaluate how it affects the human body. Phase II studies usually focus on a particular
type of cancer, and include fewer than 100 patients.
•Phase III trial compare a new agent or intervention (or new use of a standard one)
with the currentstandard therapy. In most cases, studies move into phase III testing only
after they have shown promise in phases I and II. Phase III trials may include hundreds
of people across the country.
•Phase IV trial are conducted to further evaluate the long-term safety and
effectiveness of a treatment. They usually take place after the treatment has been
approved for standard use. Several hundred to several thousand people may take part in
a phase IV study. These studies are less common than phase I, II, or III trials.
• At present over 300 clinical protocols have been approved
• Only one phase III and most of the rest of the approved gene therapy protocols
are for smaller phase I/II trials.
• Genetic Therapy Inc./Novartis is carrying out the phase-III clinical trial. The
target disease is glioblastoma multiforma, a malignant brain tumour. The
rationale is to insert a gene capable of directing cell killing into the tumour while
protecting the normal brain cells.
– The retroviral vector used (G1TkSvNa) contains the neomycin-resistance gene as a
selective marker and the herpes simplex thymidine kinase (HSTK) gene. The actual
material injected into the tumour mass is a mouse producer cell line (PA317) which
generates retroviral particles carrying the G1TkSvNa vector. As the only dividing cells
in the area of a growing brain tumour are the tumour cells and cells of the vasculature
supplying blood to the tumour, and retroviral vectors only transduce dividing cells, the
only cells to receive the vector should be the cells of the tumour and its blood vessels.
– In theory, the tumour cells that have been transduced with the vector containing the
HSTK blocks the DNA synthesis machinery and kills the cells. In fact, at least four
distinct mechanisms contribute to tumour cell death in this protocol.
• Several phase II trials are underway testing gene-therapy vectors as 'vaccines',
either against cancer (48) or against AIDS (49).
W. French Anderson, who led the first gene therapy trial in
1990, thinks gene therapy will outgrow its problems just like
other technologies have. In the journal Science, he wrote:
The field of gene therapy has been criticized for promising too much
and providing too little during its first 10 years of existence. But gene
therapy, like every other major new technology, takes time to develop.
Antibiotics, monoclonal antibodies, organ transplants, to name just a
few areas of medicine, have taken many years to mature. Early hopes
are always frustrated by the many incremental steps necessary to
produce "success." Gene therapy will succeed with time. And it is
important that it does succeed, because no other area of medicine
holds as much promise for providing cures for the many devastating
diseases that now ravage humankind.