gene therapy

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GENE THERAPY ‘The use of genes as medicine’ Mrs. Rani Ashok Assoc. Prof. of Zoology Lady Doak College, Madurai – 2 Email:


Introduction Definition Approaches Types Challenges

Gene Therapy:

Gene Therapy insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease.


Approaches Introduce genes directly into human cells Genetically-mediated therapy – Antisense therapy Introduce genetically engineered gene Replacement strategy based on homologous recombination Gene-knockout mediated gene therapy


TYPES OF GENE THERAPY BASED ON THE CELLS INVOLVED GERM LINE GENE THERAPY SOMATIC CELL GENE THERAPY germ cells, i.e., sperm or eggs , are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. Change due to therapy would be heritable and would be passed on to later generations Therapeutic genes are transferred into the somatic cells of Modifications and effects - restricted to the individual patient only; not heritable


TYPES OF GENE THERAPY BASED ON THE CELLS INVOLVED GERM LINE GENE THERAPY SOMATIC CELL GENE THERAPY Ethical problems associated minimizes the usage Persistent difficulty in introducing genes into germ cells High frequency of insertional mutations and teratogenic consequences are observed Functional technical expertise is available Genes are usually tissue specific


TYPES OF GENE THERAPY BASED ON FUNCTION GENE EXPRESSION THERAPY GENE BLOCKING THERAPY altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. gene modulation seeks to alter the expression of an endogenous gene (perhaps through the introduction of a gene encoding a novel modulatory protein) Antisense therapy Ribozyme therapy Genetically modified enzymes which cleave the mutant mRNA sequence




TYPES OF GENE THERAPY BASED ON MODE OF GENE ENTRY IN-VIVO GENE THERAPY EX-VIVO GENE THERAPY in vivo (in VEE-voh), is to inject the vector directly into the patient, aiming to target the affected cells. ex vivo (ex VEE-voh), is to deliver the gene to cells that have been removed from the body and are growing in culture. After the gene is delivered, integration and activation are confirmed, and the cells are put back into the patient. less likely to trigger an immune response, because no viruses are put into patients. They also allow researchers to make sure the cells are functioning properly before they're put in the patient.

Two methods of gene introduction approaches:

Two methods of gene introduction approaches Gene Augmentation Therapy Corrective Gene Therapy Random insertion of healthy counterpart of defective gene in genome so that its product could be made available Suitable for recessive disorders and single gene mutations; not suitable for dominant disorders No recombination event is required Work as long as appropriate regulatory controls of expression is provided Technically feasible approach and is similar to transplantation approach Directive insertion of healthy gene at specific site to displace defective gene is required Possible for dominant disorders Directive insertion necessitates the recombination event Ideal for errant genes that produce destructive of interfering substance Extensive study is a prerequisite for direct gene insertion at correct position


Challenges The condition in question must be well understood The underlying faulty gene must be identified and a working copy of the gene involved must be available The specific cells in the body requiring treatment must be identified and accessible A means of efficiently delivering working copies of the gene to these cells must be available

Criteria of ideal gene delivery method :

Criteria of ideal gene delivery method it should protect the transgene against degradation by nucleases in intercellular matrices it should bring the transgene across the plasma membrane and into the nucleus of target cells it should have no detrimental effects.

Non-viral gene delivery systems:

Non-viral gene delivery systems Physical Methods needle injection electroporation gene gun ultrasound hydrodynamic delivery Chemical Methods Cationic Lipid-Mediated Gene Delivery Cationic Polymer-Mediated Gene Transfer Lipid-Polymer Hybrid System

Needle Injection:

Needle Injection





Gene Gun:

Gene Gun



Hydrodynamic delivery:

Hydrodynamic delivery

Cationic lipid-mediated gene delivery:

Cationic lipid-mediated gene delivery

Cationic polymer-mediated gene delivery:

Cationic polymer-mediated gene delivery

Lipid-polymer hybrid gene delivery:

Lipid-polymer hybrid gene delivery


Advantages and Limitations of Current Non-viral Gene Delivery Systems Method Route of Gene Delivery Advantages Limitations Needle injection Intratissue Simplicity and safety Low efficiency Gene gun Topical Good efficiency Tissue damage in some applications Electroporation Topical Intratissue High efficiency Limited working range; need for surgical procedure for nontopical applications Hydrodynamic delivery Systemic Intravascular High efficiency, simplicity, effectiveness for liver gene delivery Extremely effective in small animals; surgical procedure may be needed for localized gene delivery Ultrasound Topical Systemic Good potential for site- specific gene delivery Low efficiency in vivo Cationic lipids Topical Intratissue Systemic Airway High efficiency in vitro; low to medium high for local and systemic gene delivery Acute immune responses; limited activity in vivo Cationic polymers Topical Intratissue Systemic Airway Highly effective in vitro; low to medium high for local and systemic gene delivery Toxicity to cells; acute immune responses Lipid/polymer hybrids Intratissue Systemic Airway Low to medium-high efficiency in vitro and in vivo; low toxicity Low activity in vivo


DISADVANTAGES OF GENE THERAPY Short-lived nature of gene therapy - 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. Patients will have to undergo multiple rounds of gene therapy.


DISADVANTAGES OF GENE THERAPY Contd … Immune response - The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Ihe immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.


DISADVANTAGES OF GENE THERAPY Contd … 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.


DISADVANTAGES OF GENE THERAPY Contd … Multigene disorders - Multigene or multifactorial disorders such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes would be especially difficult to treat effectively using gene therapy.

Gene delivery - Vectors:

Gene delivery - Vectors Harmless viral vectors Stem cells

Advantages of viral vectors: :

Advantages of viral vectors: They're very good at targeting and entering cells. Some target specific types of cells. They can be modified so that they can't replicate and destroy cells.

Drawbacks of viral vectors:

Drawbacks of viral vectors They can carry a limited amount of genetic material. Therefore, some genes may be too big to fit into some viruses. They can cause immune responses in patients, leading to two potential problems: Patients may get sick. The immune system may block the virus from delivering the gene to the patient's cells, or it may kill the cells once the gene has been delivered.


Retroviruses RNA viruses Create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Eg. Human immunodeficiency virus (HIV)


Adenoviruses Double-stranded DNA genomes Cause respiratory, intestinal, and eye infections in humans. Causative agent of common cold is an adenovirus.

Adeno-associated viruses :

Adeno -associated viruses Small, single-stranded DNA viruses Insert their genetic material at a specific site on chromosome 19.

Herpes simplex viruses :

Herpes simplex viruses Double-stranded DNA viruses Infect a particular cell type - neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.

Stem Cells:

Stem Cells Bone marrow contains stem cells that give rise to many types of blood cells. Bone marrow transplants are used to treat many genetic disorders, especially those that involve malfunctioning blood cells. Ideally, a "matched" donor, often a relative, donates bone marrow to the patient. The match decreases the chances that the patient's immune system will reject the donor cells.

Stem Cells:

Stem Cells However, it's not always possible to find a match. In these cases, the patient's own bone marrow cells can be removed and the faulty gene corrected with gene therapy. The corrected cells can then be returned to the patient.

Success stories:

Success stories

Immunodeficiency diseases:

Immunodeficiency diseases 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 genes. After the genes have been delivered, the stem cells are returned to the patient. Because the cells are treated outside the patient's body, the virus will infect and transfer the gene to only the desired target cells.

Successful Gene Therapy for Severe Combine Immunodeficiency:

Successful Gene Therapy for Severe Combine Immunodeficiency Infants with severe combined immunodeficiency are unable to mount an adaptive immune response, because they have a profound deficiency of lymphocytes. severe combined immunodeficiency is inherited as an X-linked recessive disease, which for all practical purposes affects only boys. In the other half of the patients with severe combined immunodeficiency, the inheritance is autosomal recessive — and there are several abnormalities in the immune system when the defective gene is encoded on an autosome.

Severe Combine Immunodeficiency Continued:

Severe Combine Immunodeficiency Continued A previous attempt at gene therapy for immunodeficiency was successful in children with severe combined immunodeficiency due to a deficiency of adenosine deaminase. In these patients, peripheral T cells were transduced with a vector bearing the gene for adenosine deaminase. The experiment was extremely labor intensive, because mature peripheral-blood T cells were modified rather than stem cells, and the procedure therefore had to be repeated many times to achieve success.

Successful One Year Gene Therapy Trial For Parkinson's Disease :

Successful One Year Gene Therapy Trial For Parkinson's Disease Neurologix a biotech company announced that they have successfully completed its landmark Phase I trial of gene therapy for Parkinson's Disease. This was a 12 patient study with four patients in each of three dose escalating cohorts. All procedures were performed under local anesthesia and all 12 patients were discharged from the hospital within 48 hours of the procedure, and followed for 12 months. Primary outcomes of the study design, safety and tolerability, were successfully met. There were no adverse events reported relating to the treatment.

Parkinson's Disease Cont.:

Parkinson's Disease Cont. The gene transfer procedure utilized the AAV (adeno-associated virus) vector, a virus that has been used safely in a variety of clinical gene therapy trials, and the vehicle that will be used in all of the company's first generation products, including epilepsy and Huntington's disease. In its Parkinson's disease trial, Neurologix used its gene transfer technology.

Blood disease:

Blood disease Patients with beta-Thalassemia have a defect in the beta-globin gene, which codes for an oxygen-carrying protein in red blood cells. Because of the defective gene, patients don't have enough red blood cells to carry oxygen to all the body's tissues. Many who have this disorder depend on blood transfusions for survival. 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. Seven years after the procedure, he was still doing well without blood transfusions. A similar approach could be used to treat patients with sickle cell disease


Hemophilia People with hemophilia are missing proteins that help their blood form clots. Those with the most-severe forms of the disease can lose large amounts of blood through internal bleeding or even a minor cut. 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.

Fat metabolism disorder :

Fat metabolism disorder In 2012, Glybera became the first viral gene-therapy treatment to be approved in Europe. The treatment uses an adeno-associated virus to deliver a working copy of the LPL (lipoprotein lipase) gene to muscle cells. The LPL gene codes for a protein that helps break down fats in the blood, preventing fat concentrations from rising to toxic levels.

Hereditary blindness :

Hereditary blindness 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 eyes. Encouraging results from animal models (especially mouse, rat, and dog) show that gene therapy has the potential to slow or even reverse vision loss.

Hereditary blindness :

Hereditary blindness 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).

Hereditary blindness :

Hereditary blindness 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.

Cancer :

Cancer 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.

Cancer :

Cancer 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.

Ethical Issues of Gene therapy:

Ethical Issues of Gene therapy

Ethical Issues:

Ethical Issues Because gene therapy involves making changes to the body’s set of basic instructions, it raises many unique ethical concerns. The ethical questions surrounding gene therapy include: How can “good” and “bad” uses of gene therapy be distinguished? Who decides which traits are normal and which constitute a disability or disorder?

Ethical Issues Contd …:

Ethical Issues Contd … Will the high costs of gene therapy make it available only to the wealthy? Could the widespread use of gene therapy make society less accepting of people who are different? Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?


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