Gene therapy

1. Gene therapy Class presentation Nanobiotechnology 2004

2. Gene therapy • 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 sick gene • 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 gene.

3. Why is gene therapy hard? The gene: • 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-gene diseases.

4. Vectors • Viral vectors: • DNA virus • RNA virus • Non-viral vectors • liposomes • polimers • nanodiveces

5. RNA vectors: • 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 retrovirus. • 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.

6. DNA vectors: • Adenovirus: • The DNA virus used most widely for in situ gene transfer vectors • 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. • AAV: • non-pathogenic virus that is widespread in the human population • Unfortunately appear to integrate in a nonspecific manner • room for about 4.8 kb of added DNA • Other types • HEPES • hybrid systems have been reported where an adenoviral vector is used to carry a retroviral vector into a cell that is normally inaccessible to retroviral transduction

7. 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 trasferred • 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 • major type: • cationic phosfolipids • cationic polymers

8. Liposomes • low immunogenicity but toxicity of lipids and low transfection efficiency. • 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.

9. Nano devices: • 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 function. • Ni-Au devices

10. What factors have kept gene therapy from becoming an effective treatment for genetic disease? • 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.

11. 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.

12. Clinical trials • 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.

13. Clinical 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).

14. 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.