GENE THERAPY D of PG Studies

Objectives- Introduction Gene therapy and use Chromosome intro. Steps of gene therapy Types Problems Current status Conclusion References GENE THERAPY

Gene & Genome- Gene - A segment of DNA found on a chromosome that codes for a particular protein. Humans have approximately 100,000 genes. Genome - The sum of all genes that code for a particular organism. GENE THERAPY

Genes- Are carried on a chromosome The basic unit of heredity Encode how to make a protein DNARNA proteins Proteins carry out most of life’s function. When altered causes dysfunction of a protein

A. What is gene therapy? Why is it used? Gene therapy = Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized  to improve a genetic disorder

Structure of a Chromosome GENE THERAPY 6

Many human diseases are caused by defective genes Disease Genetic defect hemophilia A absence of clotting factor VIII hemophilia B absence of clotting factor IX cystic fibrosis defective chloride channel protein muscular dystrophy defective muscle protein (dystrophin) sickle-cell disease defective beta globin GENE THERAPY

The Beginning… In the 1980s, Scientists began to look into gene therapy. They would insert human genes into a bacterial cell. Then the bacterial cell would transcribe and translate the information . Then they would introduce the protein into human cells. GENE THERAPY 8

What is Gene Therapy It is a technique for correcting defective genes that are responsible for disease development. OR The treatment of disease by either replacing damaged or abnormal genes with normal ones, or by providing new genetic instructions to help fight disease. GENE THERAPY

How is Gene Therapy Carried Out? Modification of somatic cells by transferring desired gene sequences into the genome. Somatic cells necessary to ensure that inserted genes are not carried over to the next generation.

How It Works A vector delivers the therapeutic gene into a patient’s target cell The target cells become infected with the viral vector The vector’s genetic material is inserted into the target cell Functional proteins are created causing the cell to return to a normal state A vector is a carrier molecule, usually a virus The target cells are usually in the liver or lung GENE THERAPY 11

How It Works……. GENE THERAPY 12

Steps in Gene Therapy

The First Case, The first gene therapy was performed on September 14th, 1990. Ashanti DeSilva was treated for SCID. Doctors removed her white blood cells,inserted the missing gene into the WBC, and then put them back into her blood stream. This strengthened her immune system, Only worked for a few months. GENE THERAPY 14

Approaches: Germline therapy A gene is inserted into the DNA of the germline cells (egg or sperm) so that the offspring of the patient will have the inserted gene. The egg is a very large cell, relatively easy to manipulate and inject with DNA. Inherited by the next generation. GENE THERAPY

Somatic Gene Therapy Somatic cells include all the non-reproductive cells in the human body. Not inherited by the next generation. e.g:- bone marrow cells, blood cells, skin cells etc. GENE THERAPY

Technique of Somatic Therapy GENE THERAPY

Gene transfer Vectors - Gene therapy delivery vehicles, or carriers, that encapsulate therapeutic genes for delivery to cells. Examples Biological (Viral vectors, mammalian chromosomes etc) Physical (Microinjection, Gene gun, naked ‘DNA’, Electroporation etc) Chemical (Liposomes, Oligonucleotides etc) GENE THERAPY

In vivo = delivery of genes takes place in the body Different Delivery Systems are Available In vivo = delivery of genes takes place in the body Ex vivo = delivery takes place out of the body, and then cells are placed back into the body GENE THERAPY

In vivo GENE THERAPY GENE THERAPY

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

Viruses Replicate by inserting their DNA into a host cell. Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait. The virus is engineered so that it cannot reproduce. Four different types 1) Retroviruses 2) Adenoviruses 3) Adeno-associated Viruses 4) Herpes Simplex Viruses GENE THERAPY 22

General drawbacks of Viral Vectors Can't "expand" to fit a piece of genetic material larger than it is naturally built to carry, some genes may be too big to fit into a certain type of virus. Viruses can cause immune responses in patients, resulting in two potential outcomes: Patients may get sick. A patient's immunity to a virus may limit treatments. GENE THERAPY

Non-Viral Vectors More efficient to deliver a gene using a non-viral vector, which has fewer size constraints and which won't generate an immune response Typically circular DNA molecules- Plasmids Bacteria use plasmids to transfer genes from cell to cell Easily and efficiently store and replicate genes of interest from any organism GENE THERAPY

Chromosome No immune response. Could carry a lot of information i.e one or more therapeutic genes. GENE THERAPY

Viral Vectors General advantages of viral vectors They are very good at targeting and entering cells. Some viral vectors might be engineered to target specific types of cells. They can be modified so that they can't replicate and destroy the cell. GENE THERAPY

Gene guns Gene guns Isolate normal DNA fragment. Make very small spheres of a heavy metal like gold. Coat gold with DNA. Fire particles at cells at high speed so particles enter cells. May be used directly on tissues or organs in situ. Other techniques… a) Electoporation b)Liposomes GENE THERAPY

Problems with Gene Therapy Short Lived Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy for long time. Have multiple rounds of therapy. Viral Vectors Patient could have toxic, immune, inflammatory response GENE THERAPY 28

Multigene Disorders Immune Response PROBLEMS CONT….. Multigene Disorders Hard to treat because need to introduce more than one gene. Immune Response New things introduced leads to immune response May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis. GENE THERAPY

Current Status FDA hasn’t approved any human gene therapy product for sale Reasons: In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for ornithine transcarboxylase deficiency. January 2003, halt to using retrovirus vectors in blood stem cells because children developed leukemia-like condition after successful treatment for X-linked severe combined immunodeficiency disease. GENE THERAPY 30

Successful Gene Therapy Severe Combine Immunodeficiency Infants with severe combined immunodeficiency are unable to mount an adaptive immune response, because they have a profound deficiency of lymphocytes. Attempt of gene therapy for immunodeficiency was successful in children with severe combined immunodeficiency due to a deficiency of adenosine deaminase. GENE THERAPY 31

cancer Multiple gene therapy strategies have been developed to treat a wide variety of cancers. Phase III trial for head and neck cancer Phase III gene vaccine trials for prostate cancer and pancreas cancer. Additionally, numerous Phase I and Phase II clinical trials for cancers in the brain, skin, liver, colon, breast and kidney are being conducted . GENE THERAPY

Gene Therapy in Heart Failure Gene therapy in HF must be aimed at correcting key molecular mechanisms in cardiac tissue. This requires, introduction of DNA/RNA that targets specific cardiomyocyte processes that alter HF outcomes. GENE THERAPY

Challenges in Gene Therapy Gene delivery and activation. Introducing changes into the germline. Immune response. Disrupting important genes in target cells. Success requires -efficient delivery, - the correct gene, -the correct cells, -the correct tissue, GENE THERAPY

Conclusion The viral and non viral gene delivery strategies have been fairly successful in cell culture systems and animal models The therapeutic success of GT in human still remains questionable. With the explosive increase in the availability of information on human genome, several genetic disorder have become candidates for gene therapy. GENE THERAPY 35

Conclusion…. The field is still at its infancy, the need of the hour is to initiate more studies in different systems on the various aspects. In spite of various drawbacks, gene therapy is witnessing a rapid growth and hope fully the progress would continue. GENE THERAPY

References Vyas S. P. , Dixit V. K., Pharmaceutical Biotechnology 1st edition, CBS Publisher & distributors 2001, New Delhi,401-435 D’Souza J.I. , Killedar S. J., Biotechnology & Fermentation Process 3rd edition, Nirali Prakashan Pune. 2007, 7.1-7.32 K.Sambamurthy, Ashutosh Kar.“ Pharmaceutical Biotechnology”,2nd Edn.,New Age International Publishers123-135. U.Satyanarayana Biotechnology 3rd edition uppala author publishers. GENE THERAPY

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