Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome
Targeted killing of specific cells. This general approach is popular in cancer gene therapies. Genes are directed to the target cells and then expressed so as to cause cell killing. Direct cell killing is possible if the inserted genes are expressed to produce a lethal toxin (suicide genes), or a gene encoding a prodrug is inserted, conferring susceptibility to killing by a subsequently administered drug. Alternatively, selectively lytic viruses can be used. Indirect cell killing uses immune-stimulatory genes to provoke or enhance an immune response against the target cell.
Because of practical difficulties, this approach has yet to be applied but, in principle, it can be done at different levels: at the gene level (e.g. by gene targeting methods based on homologous recombination); or at the RNA transcript level (e.g. by using particular types of therapeutic ribozymes or therapeutic RNA editing).
Targeted inhibition of gene expression. If disease cells display a novel gene product or inappropriate expression of a gene (as in the case of many cancers, infectious diseases, etc.), a variety of different systems can be used specifically to block the expression of a single gene at the DNA, RNA or protein levels. Allele-specific inhibition of expression may be possible in some cases, permitting therapies for some disorders resulting from dominant negative effects. (The example shows correction of a mutation in a mutant gene by homologous recombination, but mutation correction may also be possible at the RNA level. ODN, oligodeoxynucleotide; TFO, triplex-forming oligonucleotide.)
Severe Combined Immune Deficiency (SCID) was one of the first genetic disorders to be treated successfully with gene therapy, proving that the approach could work. However, the first clinical trials ended when the viral vector triggered leukemia (a type of blood cancer) in some patients. Since then, researchers have begun trials with new, safer viral vectors that are much less likely to cause cancer.
Adenosine deaminase (ADA) deficiency is another inherited immune disorder that has been successfully treated with gene therapy. In multiple small trials, patients' blood stem cells were removed, treated with a retroviral vector to deliver a functional copy of the ADA gene, and then returned to the patients. For the majority of patients in these trials, immune function improved to the point that they no longer needed injections of ADA enzyme. Importantly, none of them developed leukemia.
Encouraging results from animal models (especially mouse, rat, and dog) show that gene therapy has the potential to slow or even reverse vision loss. The eye turns out to be a convenient compartment for gene therapy. The retina, on the inside of the eye, is both easy to access and partially protected from the immune system. And viruses can't move from the eye to other places in the body. Most gene-therapy vectors used in the eye are based on AAV (adeno-associated virus).
In one small trial of patients with a form of degenerative blindness called LCA (Leber congenital amaurosis), gene therapy greatly improved vision for at least a few years. However, the treatment did not stop the retina from continuing to degenerate. In another trial, 6 out of 9 patients with the degenerative disease choroideremia had improved vision after a virus was used to deliver a functional REP1 gene.
Several promising gene-therapy treatments are under development for cancer. One, a modified version of the herpes simplex 1 virus (which normally causes cold sores) has been shown to be effective against melanoma (a skin cancer) that has spread throughout the body. The treatment, called T-VEC, uses a virus that has been modified so that it will (1) not cause cold sores; (2) kill only cancer cells, not healthy ones; and (3) make signals that attract the patient's own immune cells, helping them learn to recognize and fight cancer cells throughout the body. The virus is injected directly into the patient's tumors. It replicates (makes more of itself) inside the cancer cells until they burst, releasing more viruses that can infect additional cancer cells.
A completely different approach was used in a trial to treat 59 patients with leukemia, a type of blood cancer. The patients' own immune cells were removed and treated with a virus that genetically altered them to recognize a protein that sits on the surface of the cancer cells. After the immune cells were returned to the patients, 26 experienced complete remission.
Avoiding the immune response: The role of the immune system is to fight off intruders. Sometimes new genes introduced by gene therapy are considered potentially-harmful intruders. This can spark an immune response in the patient, that could be harmful to them. Scientists therefore have the challenge of finding a way to deliver genes without the immune system ‘noticing’. This is usually by using vectors that are less likely to trigger an immune response Delivering the gene to the right place and switching it on: it is crucial that the new gene reaches the right cell delivering a gene into the wrong cell would be inefficient and could also cause health problems for the patient even once the right cell has been targeted the gene has to be turned on cells sometimes obstruct this process by shutting down genes that are showing unusual activity Many genetic disorders that can be targeted with gene therapy are extremely rare. Gene therapy often requires an individual, case-by-case approach. This may be effective, but may also be very expensive.
Strategies of gene therapy
Strategies of gene therapy
• Gene therapy is when DNA is introduced into a patient to treat a genetic
disease. The new DNA usually contains a functioning gene to correct the effects
of a disease-causing mutation.
• Gene therapy is a strategy used to treat disease by correcting defective genes or
modifying how genes they are expressed.
• Gene therapy will enable patients to be treated by inserting genes into their
cells rather than administering drugs or subjecting them to surgery.
• A gene that is inserted directly into a cell usually does not function. Instead, a
carrier called a vector is genetically engineered to deliver the gene.
• Gene therapy holds promise for treating a wide range of diseases, such as
cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS.
TYPES OF GENE THERAPY
• Somatic gene therapy: transfer of a section of DNA to any cell of the body that
doesn’t produce sperm or eggs. Effects of gene therapy will not be passed onto the
patient’s children. Somatic cells are nonreproductive, Often the effects of somatic
cell therapy are short-lived. Somatic gene therapy can be broadly split into two
• Ex vivo: where cells are modified outside the body and then transplanted back in
again. cells from the patient’s blood or bone marrow are removed and grown in the
• In vivo: where genes are changed in cells still in the body.
• Germline gene therapy: transfer of a section of DNA to cells that
produce eggs or sperm. Effects of gene therapy will be passed onto the
patient’s children and subsequent generations.
• The therapy alters the genome of future generations to come, the use of
germline therapy due to fears over unknown risks and long-term effects
in future generations is inhibited in various countries.
• In addition, the therapy is very costly.
Gene therapy using ADENOVIRUS VECTOR
• A gene that is inserted directly into a cell usually does not
function, instead a vector is used i.e. viruses such as
retroviruses, integrate their genetic material (including the new
gene) into a chromosome in the human cell.
• The vector can be injected or given intravenously (by IV)
directly into a specific tissue in the body, where it is taken up by
• If the treatment is successful, the new gene delivered by the
vector will make a functioning protein.
Strategies of gene therapy
• Gene augmentation therapy
• Targeted killing of specific cells
• Targeted mutation correction
• Targeted inhibition of gene expression
I. GENE AUGMENTATION THERAPY
• It is used to treat diseases caused by a mutation that stops a
gene from producing a functioning product, such as a protein.
• This therapy adds DNA containing a functional version of the
lost gene back into the cell.
• The new gene produces a functioning product at sufficient
levels to replace the protein that was originally missing.
• This is only successful if the effects of the disease are reversible
or have not resulted in lasting damage to the body.
• For example, this can be used to treat loss of function disorders such as
cystic fibrosis by introducing a functional copy of the gene to correct the
II.Targeted killing of specific cells
• Artificial cell killing and immune system assisted cell killing have been popular in the
treatment of cancers.
• The aim is to insert DNA into a diseased cell that causes that cell to die.
• Genes are directed to the target cells and then expressed so as to cause cell killing.
• It can be done by two ways:
1. Direct killing: the inserted DNA contains a “suicide” gene that produces a highly toxic
product which kills the diseased cell.
2. Indirect killing: uses immune-stimulatory genes to provoke or enhance an immune
response against the target cell. The inserted DNA causes expression of a protein that
marks the cells so that the diseased cells are attacked by the body’s natural immune
• It is essential with this method that the inserted DNA is targeted
appropriately to avoid the death of cells that are functioning normally
III. Targeted mutation correction
• The repair of a genetic defect to restore a functional allele.
• Technical difficulties have meant that it is not sufficiently reliable
to warrant clinical trails.
• In principle, it can be done at different levels: at the gene level
or at the RNA transcript level.
IV. TARGETED INHIBITION OF GENE
• suitable for treating infectious diseases and some cancers.
• The basis of this therapy is to eliminate the activity of a gene
that encourages the growth of disease-related cells.
• If disease cells display an inappropriate expression of a gene
a variety of different systems can be used specifically to block
the expression of a single gene at the DNA, RNA or Protein
• For example, cancer is sometimes the result of the over-activation of an oncogene. So, by
eliminating the activity of that oncogene through gene inhibition therapy, it is possible to
prevent further cell growth and stop the cancer in its tracks.
• Successes represent a variety of approaches—different vectors, different target
cell populations, and both in vivo and ex vivo approaches for treating a variety
• Immune deficiencies
• Hereditary blindness
• Blood disease
• Several inherited immune deficiencies have been treated
successfully with gene therapy.
• Most commonly, blood stem cells are removed from patients, and
retroviruses are used to deliver working copies of the defective
• Severe Combined Immune Deficiency (SCID) and Adenosine
deaminase (ADA) deficiency.
• Gene therapies are being developed to treat several different
types of inherited blindness, especially degenerative forms,
where patients gradually lose the light-sensing cells in their
• Most gene-therapy vectors used in the eye are based on AAV
• In one small trial of patients with a form of degenerative
blindness called LCA (Leber congenital amaurosis), gene
therapy greatly improved vision for at least a few years.
Hemophilia and Cancer
• People with hemophilia are missing proteins that help their blood form
• In a small trial, researchers successfully used an adeno-associated viral
vector to deliver a gene for Factor IX, the missing clotting protein, to
liver cells. After treatment, most of the patients made at least some
Factor IX, and they had fewer bleeding incidents.
• CANCER: herpes simplex 1 virus was used (which normally causes cold
sores) has been shown to be effective against melanoma that has spread
throughout the body.
• Patients with beta-Thalassemia have a defect in the beta-globin gene, which
codes for an oxygen-carrying protein in red blood cells.
• In 2007, a patient received gene therapy for severe beta-Thalassemia. Blood
stem cells were taken from his bone marrow and treated with a retrovirus to
transfer a working copy of the beta-globin gene.
• The modified stem cells were returned to his body, where they gave rise to
healthy red blood cells.
Challenges of gene therapy
• Delivering the gene to the right place and switching it on.
• Avoiding the immune response.
• Making sure the new gene doesn’t disrupt the function of other genes.
• cost of gene therapy.