Gene Therapy : Gene Therapy Supervised by Prof.Dr. Fadel A El-Shareef
Presented by : Belal A El-Dabour What’s gene ? : What’s gene ? • Gene is the biological unit of heredity.
A gene is a part of DNA molecule, and humans have 30.000 genes.
• Genes determine obvious traits, such as hair and eye color, as well as more subtle characteristics such as the ability of the blood to carry oxygen.
Genes carry instructions that allow the cells to produce specific proteins , such as enzymes.
DNARNA proteins Slide 3: Only certain genes in a cell are active at any given moment. As cell mature, many genes become permanently inactive.
The pattern of active and inactive genes in a cell and the resulting protein composition determine what kind of cell it is and what it can do and cannot do.
Genes When altered causes dysfunction of a protein .
When there is a mutation in the gene, then it will change the codon, which will change which amino acid is called for which will change the conformation of the protein which will change the function of the protein. Genetic disorders result from mutations in the genome. What is Gene Therapy : What is Gene Therapy It is a technique for correcting defective genes that are responsible for disease development
There are four approaches:
A normal gene inserted to compensate for a nonfunctional gene.
An abnormal gene traded for a normal gene
An abnormal gene repaired through selective reverse mutation
Change the regulation of gene pairs Gene therapy utilizes two theoriticaly possible directions : : Gene therapy utilizes two theoriticaly possible directions : 1. Somatic Gene Therapy : Transfer of a gene or genes into body cells other than germ cells with effect only on the patient.
The new genetic material cannot be passed on to offspring .
2. Germline gene therapy : Involve the genetic modification of germ cells. Such therapy would change the genetic make up of the egg or sperm of an individual and would be carried on to future generations . This would offer the possibility of removing an inherited disorder from a family line forever. The Beginning… : The Beginning… In the 1980s, Scientists began to look into gene therapy.
*They would insert human genes into a bacteria cell.
*Then the bacteria cell would transcribe and translate the information into a protein
*Then they would introduce the protein into human cells
Scientist took the logical step of trying to introduce genes straight into human
cells, focusing diseases caused by single-gene defects, such as cystic fibrosis,
hemophilia, muscular dystrophy and sickle cell anemia. However this has been
much harder than modifying simple bacteria, primarily because of the problems
involved in carrying large section of DNA and delivery it to the right site on the
genome How does gene therapy work : How does gene therapy work • In most gene therapy studies, a normal gene is inserted
into the genome to replace an “abnormal” disease causing gene.
This could be in vivo or ex vivo…
• A carrier molecule called a vector must be used to
deliver the therapeutic gene to the patients target cells.
The most common vector is a virus that has been
genetically altered to carry normal human DNA Continued … : Continued … Target cell such as the patient’s liver or lung cells are infected with the viral vector.
The viral vector then unload its genetic material containing the therapeutic human gene into the target cell.
The generation of a functional protein product from therapeutic gene restores the target cell to a normal state. The First Case : The First Case The first gene therapy was performed on September 14th, 1990
Ashanti DeSilva was treated for SCID
Sever combined immunodeficiency
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 Slide 10: Vectors : mainly viruses Why Viruses ? : Why Viruses ? A virus is the simplest organism there is—it is pretty much just genetic material wrapped up in a protein coat.
a virus can’t live on its own …
it survives and multiplies by parasitically attacking living cells and injecting its genetic material into cells.
The fact that many viruses are cell specific (e.g., a certain virus may only infect heart cells or lung cells) helps scientists target just the desired cells.
That makes it an ideal mechanism for getting genes into a cell.
a scientist might take the damaging, infecting portion of the DNA out of the virus and add to it the desired gene segment. Slide 12: Some of the different types of viruses as gene therapy vectors :
3. Adeno-associated viruses
4. Herpes simplex viruses Retroviruses : Retroviruses Created double stranded DNA copies from RNA genome
The retrovirus goes through reverse transcription using reverse transcriptase and RNA …
the double stranded viral genome integrates into the human genome using integrase .
integrase inserts the gene anywhere because it has no specific site
May cause insertional mutagenesis : One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)
vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety.
May trigger immune response . Slide 14: Retroviruses carrying healthy gene are mixed with unhealthy cells taken from a patient.
Retroviruses infect unhealthy cells with healthy gene, adding the geneto patient’s DNA.
Healthy cells then injected back to the patient. Adenoviruses : Adenoviruses Adenoviruses carry their genetic material in the form of DNA.
When these viruses infect a host, they introduce their DNA molecule into the host but not incorporated into the host genetic material.
These extra genes are not replicated, so when the host undergo cell division, the descendants of the cell will not have the extra gene.
This means that treatment with adenovirus will require regular doses to add the missing gene every time.
Adenoviruses can invade slower dividing cells, such as lung cells
Like retroviruses, immune response may reduce its effectiveness Adenovirus cont… : Adenovirus cont… http://en.wikipedia.org/wiki/Gene_therapy Adeno-associated Viruses : Adeno-associated Viruses Adeno-associated viruses are believed to occur naturally in humans, existing without causing disease or instigating in immune response from the body. This type of virus is being used, because it is non-pathogenic (most people carry this harmless virus).
• AAV are small viruses with a genome of single stranded DNA. They insert their material specifically into chromosome 19, at a specific site.
• Disadvantages in using AAV are the small amount of DNA it can carry (only 2 genes) and the difficulty in producing it.
gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. Continued … : Continued … Several trials with AAV are ongoing or in preparation, mainly trying to treat muscle and eye disease, the two tissues where the virus seems particularly useful.
hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.
Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year Herpes Simplex Viruses : Herpes Simplex Viruses Herpes simplex viruses : A class of double stranded DNA virus that usually used to target the nervous system because herpes simplex virus usually infects neurons.
Ex. Herpes simplex virus type 1 Non-viral Options : Non-viral Options Direct introduction of therapeutic DNA
*But only with certain tissue *Requires a lot of DNA .
Creation of artificial lipid sphere with aqueous core, liposome Carries therapeutic DNA through membrane .
98 percent of available intravenous drugs and 100 percent of oral medications cannot get from the blood to the brain
solution: Create a “molecular Trojan horse” to coat the new genes with lipids, and coat that with a chemical called propylene glycol (PEG) that keeps the coated genes from being absorbed by the liver and other tissues. Then, they can slip right from the blood and directly into the brain. Continued … : Continued … Introducing 47th artificial human chromosome into target cells. This chromosome would exist autonomously alongside the standard 46.
Could carry a lot of information .
But how to get the big molecule through membranes? Repaire therapy : : Repaire therapy : This approach is a lot more straightforward than conventional gene therapy , adding that conventional gene therapy is more difficult because researchers don’t have the ability to control the activity of any specific gene.
But by repairing messenger RNA rather than trying to replace a damaged gene, you are using the cell’s own regulatory mechanisms to produce normal protein .
This was used to cure patients with thalassemia by producing hemoglobin in the correct quantities .
Basically we tricked the body’s machinery for making red blood cells into producing normal hemoglobin.
even a small improvement in the production of normal hemoglobin will make a huge difference to these patients . GENE THERAPY AND RADIATION: A PROMISING COMBINATION : GENE THERAPY AND RADIATION: A PROMISING COMBINATION Nearly 30,000 men will die this year from prostate cancer, the leading cause of death among males after lung cancer, according to the American Cancer Society
a new therapy that combines gene therapy techniques with radiation offers some hope.
Essentially, the new approach entails injecting the adenovirus (cold virus) as the gene truck (viral vector) carrying new genes into the prostate. It spreads, infects, and apparently weakens tumor cells, in addition to carrying the genes to a wider group of prostate cells. Then, researchers bombard the tumor cells with radiation.
The combination looks effective. In the study, researchers treated fifteen men with advanced forms of prostate cancer.
All of them almost immediately showed a decrease in level of the prostate-specific antigen (PSA) protein, a common marker for prostate cancer. And a year later, ten of the men were entirely cancer free. What diseases could be treated withgene therapy : What diseases could be treated withgene therapy About 4000 diseases have been traced to gene disorders.
Current and possible candidates for gene therapy include cancer, AIDS, cystic fibrosis, Parkinsons, Alzheimer’s disease, Lou Gehrig’s disease, cardiovascular disease and arthritis.
more than 600 gene therapy clinical trials were under way in the US but only a handful of these are in advanced stages. What factors have kept gene therapy from becoming an effective treatment for genetic disease ? : What factors have kept gene therapy from becoming an effective treatment for genetic disease ? Short-lived nature of gene therapy.
Immune response .
Problem with viral vector : viruses may present a variety of potential problems to the patient – toxicity, immune and inflammatory response and also cause disease.
Multigene disorder : multigene or multifactorial disorders would be difficult to treat effectively using gene therapy.
High risk factor . What risk are associated with currentgene therapy trials ? : What risk are associated with currentgene therapy trials ? • Viruses can usually infect more than one type of cell, thus they might infect healthy cells.
• Another danger is that the new gene might be inserted in the wrong location in the DNA, possibly causing cancer or harmful mutations to the DNA.
There is a slight chance that the DNA could unintentionally be introduced into the patient’s reproductive cells, so it may be passed on if the patient has children after treatment.
The possibility that transferred genes could be “overexpressed” , producing so much of the missing protein as to be harmful .
the viral vector could cause inflammation or an immune reaction ; and that the virus could be transmitted from the patient to other individuals or into the environment. Slide 27: Gene therapy and gene-based medicine will revolutionize medicine over the next ten to twenty years. The big question is when.
Gene therapy pioneer French Anderson, 2001 Slide 28: http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg Slide 29: Thank You