Gen e T h erap y

1. Gene Therapy

2. 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 they are altered, this causes dysfunction of a protein • When there is a mutation in the gene, then it will change the codon, so amino acid is changed and the conformation of the protein will change the function of the protein. Genetic disorders result from mutations in the genome.

3. http://www.accessexcellence.org/RC/VL/GG/genes.html

4. What is Gene Therapy? Gene therapy is firstly defined by UK Gene Therapy Advisory Committee (GTAC). It was described as the introduction of the genetic material to the somatic cells of patients for therapeutic,prophylactic or diagnostic reasons.

5. What is Gene Therapy? • It is a technique for correcting defective genes that are responsible for disease development • Gene therapy (GT) = Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product

7. There are four approaches: 1- A normal gene inserted to compensate for a nonfunctional gene. 2- An abnormal gene traded for a normal gene 3- An abnormal gene repaired through selective mutation 4- Change the regulation of gene pairs

8. Why is it used? • GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized

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

10. A vector (a carrier molecule) delivers the therapeutic gene into a patient’s target cell • The target cells (usually in liver or lungs) become infected with the vector (mostly viral) • The vector’s genetic material is inserted into the target cell • Functional proteins are created from the therapeutic gene causing the cell to return to a normal state

12. Target cell: • Cell has a long half-life • High potential of proliferation • Gene transfer to stem cells. • Recently, bone marrow is used as stem or precusor cells in gene transfer.

13. Different Delivery Systems are Available • In vivo versus ex vivo • 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

14. In vivo techniques usually utilize viral vectors • Virus = carrier of desired gene • Virus is usually “crippled” to disable its ability to cause disease • Viral methods have proved to be the most efficient to date • Many viral vectors can stable integrate the desired gene into the target cell’s genome

15. Approaches to Gene Transfer 1- Cultured cells are taken from the patient, then the gene is transferred to these cells and they are given again to the patient. 2- The transfer of the gene directly or by the other appropriate cells to the extracelluler fluid, in vivo.

16. DNA Transfer to Cells: Viral Vectors • An ideal vector for gene therapy must be safety, must be prepared easily, it must be transferred to the appropriate cell easily and must be expressed lifelong.Retrovirus, adenovirus and adenoassociate virüs. • Retrovirus-based vectors carrying the RNA genome are the most widely used vectors.

17. The most important disadvantage of retroviral vectors is they can not integrate to DNA of undivided cells, so the can not be used in most of the tissues. • The most important advantage of viral vectors is they can enter to all target cells.

18. 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 • Four different types

19. 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

20. Picture  http://encarta.msn.com/media_461561269/Gene_Therapy.html

21. Adenoviruses • Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans • The inserted DNA is not incorporate into genome • Ex. Common cold

22. Adenovirus http://en.wikipedia.org/wiki/Gene_therapy

23. Adeno-associated Viruses • Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19 • From parvovirus family- causes no known disease and doesn't trigger patient immune response. • gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. • 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.

24. Herpes Simplex Viruses • Double stranded DNA viruses that infect neurons • Ex. Herpes simplex virus type 1 http://www.ucmp.berkeley.edu/alllife/virus.html

25. Non-viral Options • There are many different nonviral methods for gene therapy. The most common is liposome associated DNA transfection. • Lipofection(liposome transfection), is a method which the genetic material is inserted into the cell by liposomes.Liposomes easily combine with the cell membrane, because both of them have phospholipid structure.

26. Non-viral Options • Direct introduction of therapeutic DNA • But only with certain tissue • Requires a lot of DNA • Chemically linking DNA to molecule that will bind to special cell receptors • DNA is engulfed by cell membrane • Less effective 

27. Limitations and Risks of Gene Therapy • Gene delivery • Limited tropism of viral vectors • Dependence on cell cycle by some viral vectors (i.e. mitosis required) • Duration of gene activity • It can be transient or stable

28. Patient can give negative reaction to the vector or the transferred gene. • The transferred gene can integrate to the patient’s DNA and it can activate a protooncogene or can impair the structure of a tumor suppressor gene and cause cancer. • It can damage the structure of a basic gene. But these types of fatal mutations are so rare and they cause death in only one cell.

29. Patient safety • Immune hyperresponsiveness (hypersensitivity reactions directed against viral vector components or against transgenes expressed in treated cells) • Integration is not controlled  oncogenes may be involved at insertion point  cancer?

30. Ethic Problems:Almost all government agencies and religious organizations received a positive decision on the implementation of gene therapy for genetic diseases. Except from the gene transfer to germline, there is no different ethical role of somatic gene therapy from the other new therapy approaches. • Some candidate diseases for gene therapy: Thalassemia, hemophilia, some types of immune failure, etc.

31. 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

33. The First Case • The first gene therapy was performed on September 14th, 1990 • Ashanti DeSilva was treated for SCID • Severe 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 

34. Current Status • Up to date, cancer is by far the most common disease treated by genetherapy. • It composes over 60% of all ongoing clinical gene therapy trialsworldwide, followed by monogenetic and cardiovascular diseases.

36. Currently, more than 1800 approved gene therapy clinical trialsworldwide have been conducted or are still ongoing. • Adenoviral vectors,retroviral vectors, plasmids have been the most commonlyused gene transfer vectors in clinical trials

38. Problems with Gene Therapy • Short Lived • Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time • Would have to have multiple rounds of therapy • Immune Response • new things introduced leads to immune response • increased response when a repeat offender enters • Viral Vectors • patient could have toxic, immune, inflammatory response • also may cause disease once inside • Multigene Disorders • Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene • May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis

39. Safety and Ethical Aspects Gene therapy is a therapeutic model and it will be a standard for many diseases. But, still there are many questions that must be discussed. The most important one is the safety of gene therapy in humans and the potantial effects on children.

40. Generally there are two types of gene therapy: germ line and somatic gene therapy. The difference between them is; in somatic gene therapy genetic material is inserted to some target cells and is not inherited to the other generations. • Germ line gene therapy is inherited to the other generations. • Today only somatic gene therapy is legal.

41. One of the arguements about gene therapy is to form uncontrolled new genetic changes and to inherit these cahnges to other generations. The other methods actually also cause particular genetic changes. For example, some mutagenic drugs, radiation therapy, e.t.c.

42. To use gene therapy in fatal diseases (cancer, SCID, etc) is more acceptable. There can be some ethical problems to use gene therapy at Down syndrome or at some mental retardation syndromes.

43. Unsuccessful Gene therapies • Jesse Gelsinger, a gene therapy patient who lacked ornithine transcarbamylase activity, died in 1999. • Within hours after doctors shot the normal OTC gene attached to a therapeutic virus into his liver, Jesse developed a high fever. His immune system began raging out of control, his blood began clotting, ammonia levels climbed, his liver hemorrhaged and a flood of white blood cells shut down his lungs. • One problem with gene therapy is that one does not have control over where the gene will be inserted into the genome. The location of a gene in the genome is of importance for the degree of expression of the gene and for the regulation of the gene (the so-called "position effect"), and thus the gene regulatory aspects are always uncertain after gene therapy

45. 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.

46. Severe Combine Immunodeficiency • 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.

47. Currently, the first gene based products have entered the marketand it is very likely that gene therapy will find its place in specificareas of clinical medicine. • The development of genemedicine products should emphasize the importance of appropriatepre-clinical models, include the use of bigger, non-rodent, animalmodels that would support the evaluation of the efficacy and safetyof gene therapy products. • Ultimately it is thepatient who decides, whether she/he wants to be treated with genetherapy.

48. http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpghttp://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg