1. ©1999 Timothy G. Standish Pharmaceutical Biotechnology PHG 424 Mounir M. Salem, Ph.D. mounirmsalem@yahoo.com King Saud University College of Pharmacy Departments of Pharmaceutics/ Pharmacognosy

2. ©1999 Timothy G. Standish Gene Therapy

3. ©1999 Timothy G. Standish Gene Therapy Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. it’s the proteins that perform most life functions and even make up the majority of cellular structures. Introduction

4. ©1999 Timothy G. Standish Gene Therapy When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result. Each of us carries about half a dozen defective genes. We remain blissfully unaware of this fact unless we, or one of our close relatives, are amongst the many millions who suffer from a genetic disease. Introduction….

5. ©1999 Timothy G. Standish What is Gene Therapy It is a technique for correcting defective genes that are responsible for disease development 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 reverse mutation 4.Change the regulation of gene pairs Gene Therapy

6. ©1999 Timothy G. Standish Gene Therapy is Experimental Advances in understanding and manipulating genes have set the stage for scientists to alter a person's genetic material to fight or prevent disease. Gene therapy is an experimental treatment that involves introducing genetic material (DNA or RNA) into a person's cells to fight disease. Gene Therapy

7. ©1999 Timothy G. Standish 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 Gene Therapy

8. ©1999 Timothy G. Standish The First Case The first gene therapy was performed on September 14 th, 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  Gene Therapy

9. ©1999 Timothy G. Standish 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 from the therapeutic gene causing the cell to return to a normal state Gene Therapy

10. ©1999 Timothy G. Standish http://encarta.msn.com/media_461561269/Gene_Therapy.html Gene Therapy

11. ©1999 Timothy G. Standish Principles of Gene therapy A normal gene may be inserted into a non-specific location within the genome to replace a non-functional gene. This approach is most common. An abnormal gene could be swapped for a normal gene through homologous recombination. The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered. Gene Therapy

12. ©1999 Timothy G. Standish Gene Therapy Depends on Delivery of Corrective Genes Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Gene Therapy

13. ©1999 Timothy G. Standish Viruses are used as Delivery Tolls Viruses are used as vectors to introduce the genetic material inside the bodies. These viruses are inactivated, they are not able to reproduce Adenoviruses most common Herpes viruses DNA tumor viruses Retroviruses RNA tumor viruses most common Gene Therapy

14. ©1999 Timothy G. Standish Why Viruses? Viruses through the time of evolution have evolved to infect the cells with great specificity Viruses tend to be very efficient at transfecting their own DNA into the host cell genome. This allows them to produce new viral particles at the period of synthesis of the cell

15. ©1999 Timothy G. Standish Majority are Trails Gene therapy is being studied in clinical trials (research studies with people) for many different types of cancer and for other diseases.cancer It is not currently available outside a clinical trials. Gene Therapy

16. ©1999 Timothy G. Standish What Gene therapy can Achieve Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or “knocking out,” a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease. Gene Therapy

17. ©1999 Timothy G. Standish Uses of gene therapy Replace missing or defective genes; Deliver genes that speed the destruction of cancer cells; Supply genes that cause cancer cells to revert back to normal cells ?? Deliver bacterial or viral genes as a form of vaccination; Provide genes that promote or impede the growth of new tissue; and; Deliver genes that stimulate the healing of damaged tissue. Gene Therapy

18. ©1999 Timothy G. Standish Delivering desired Genes Gene Therapy ES = Embryonic stem cells. HLA= human leukocyte antigen. SCNT = somatic-cell nuclear transfer

19. ©1999 Timothy G. Standish Gene Therapy Corrects Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes: Gene Therapy

20. ©1999 Timothy G. Standish Steps in Gene Therapy Gene Therapy AAV = adeno-associated virus

21. ©1999 Timothy G. Standish

22. Manipulation corrects the Defective Genes Gene Therapy

23. ©1999 Timothy G. Standish Gene Therapy delivers Proteins Today, gene therapy is the ultimate method of protein delivery, in which the delivered gene enters the body's cells and turns them into small "factories" that produce a therapeutic protein for a specific disease over a prolonged period. Gene Therapy

24. ©1999 Timothy G. Standish Antisense therapy Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off". Gene Therapy

25. ©1999 Timothy G. Standish Antisense Therapy Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods Gene Therapy

26. ©1999 Timothy G. Standish

27. Making the new Genetic Material Functional Gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is used to introduce the therapeutic gene into the patient's target cells. The most common vector that is used is a virus that has been genetically altered to carry normal human DNA. Gene Therapy

28. ©1999 Timothy G. Standish Somatic and Germ Line Gene Therapy Gene therapy can target somatic (body) or germ (egg and sperm) cells. In somatic gene therapy the recipient's genome is changed, but the change is not passed on to the next generation; whereas with germ line gene therapy the newly introduced gene is passed on to the offspring. Gene Therapy

29. ©1999 Timothy G. Standish Safety Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. Gene Therapy

30. ©1999 Timothy G. Standish Making safe Protocols Low toxicity:  The viral vector should have a minimal effect on the physiology of the cell it infects. Stability:  Some viruses are genetically unstable and can rapidly rearrange their genomes. Gene Therapy

31. ©1999 Timothy G. Standish Cell type specificity Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell. Gene Therapy

32. ©1999 Timothy G. Standish Lentivirus Lentivirus (lenti-, Latin for "slow") is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Gene Therapy

33. ©1999 Timothy G. Standish Retroviruses Retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse- transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme Gene Therapy

34. ©1999 Timothy G. Standish Vectors deliver the Genetic Materials The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. Gene Therapy

35. ©1999 Timothy G. Standish Adenoviruses As opposed to lenti viruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. Adenoviral vectors are occasionally used in in vitro experiments. Gene Therapy

36. ©1999 Timothy G. Standish Choosing non infective Adenovirus Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity. Gene Therapy

37. ©1999 Timothy G. Standish Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy Gene Therapy Adeno-associated viruses

38. ©1999 Timothy G. Standish Limitation of Direct Gene Induction The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA. Gene Therapy

39. ©1999 Timothy G. Standish Nonviral approach Nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane Gene Therapy

40. ©1999 Timothy G. Standish Nonviral Vectors: Liposome's less Immunogenic DNA/lipid complexes are easy to prepare and there is no limit to the size of genes that can be delivered. Because carrier systems lack proteins, they may evoke much less immunogenic responses. More importantly, the cationic lipid systems have much less risk of generating the infectious form or inducing tumorigenic mutations because genes delivered have low integration frequency and cannot replicate or recombine. Gene Therapy

41. ©1999 Timothy G. Standish Nanoengineered substances Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate) Gene Therapy

42. ©1999 Timothy G. Standish Transfection and Nanoengineering Transfection is the process of introducing nucleic acids into cells by non-viral methods. The term "transformation" is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells; "transduction" is often used to describe virus-mediated DNA transfer. Gene Therapy

43. ©1999 Timothy G. Standish Problems of Large Gene It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cells. Gene Therapy

44. ©1999 Timothy G. Standish Gene Therapy Uses AIDS Virus to Fight AIDS In the study, immune cells were removed from the patients' bodies, modified with a disabled AIDS virus known as a lentivirus, and then intravenously returned. The genetically altered cells disseminated anti-HIV material and prevented HIV from reproducing ( 07 November, 2006) Gene Therapy

45. ©1999 Timothy G. Standish Technical Difficulties in Gene Therapy Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells. Delivery of genes for cancer therapy may also be complicated by the disease being present at several sites. Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have been more successful Gene Therapy

46. ©1999 Timothy G. Standish 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 Gene Therapy

47. ©1999 Timothy G. Standish What are the ethical issues surrounding gene therapy? How can “good” and “bad” uses of gene therapy be distinguished? Who decides which traits are normal and which constitute a disability or disorder? 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?

48. ©1999 Timothy G. Standish The Future of Gene Therapy Current uses of gene therapy focus on treating or curing existing conditions. In the future, the focus could shift to prevention. As more of the human genome is understood, medicine will know more about which genes contribute to or cause disease. With that knowledge in hand, gene therapy could be used to head off problems before they occur.

49. ©1999 Timothy G. Standish Creating 47th Chromosome Researchers are also experimenting with introducing a 47 th artificial chromosome to the body. It would exist autonomously along side of the other 46, not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic information and the body’s immune system would not attack it.

50. ©1999 Timothy G. Standish Several Diseases have Genetic basis Gene mutations probably play a role in many of today's most common diseases, such as heart disease, diabetes, immune system disorders, and birth defects. These diseases are believed to result from complex interactions between genes and environmental factors. When genes for diseases have been identified, scientists can study how specific environmental factors, such as food, drugs, or pollutants interact with those genes.

51. ©1999 Timothy G. Standish Gene therapy of pain: emerging strategies and future directions Gene therapy to alleviate pain could appear surprising and perhaps not appropriate when opioids and other active molecules are available. However, the possibility of introducing a therapeutic protein into some targeted structures, where it would be continuously synthesised and exert its biological effect in the near vicinity of, or inside the cells, might avoid some drawbacks of "classical" drugs.

52. ©1999 Timothy G. Standish Pain – Cancer a major research area Numerous other molecules involved in pain processing or associated with chronic pain have been identified and the gene- based techniques might be particularly adapted for the evaluation of the possible therapeutic interest of these new potential targets

53. ©1999 Timothy G. Standish Cancer Gene Therapy …Using Tumor Suppressor Genes.

54. ©1999 Timothy G. Standish Overview Gene Therapy p53 Using Gene Therapy to Treat Lung Cancer Problems

55. ©1999 Timothy G. Standish Types of Viruses… Retrovirus Adinovirus Lentiviruses Poxviruses and Herpes Viruses

56. ©1999 Timothy G. Standish Adenovirus 36 kb Double Stranded DNA Genome Entry through CAR receptor and integrin co-receptor

57. ©1999 Timothy G. Standish E1A E3 E1B E2AE4E2B L1L2 L4 L3L5 Latest Generation Adenoviral Vector “Gutless”; Helper-dependent; Minimal Ad Therapeutic Transgene Stuffer DNA  ITR

58. ©1999 Timothy G. Standish Which Virus to Use? Depends how well they transfer the genes to cells which cells they can recognize and infect and whether they alter the cell’s DNA permanently or temporarily

59. ©1999 Timothy G. Standish Cells removed from body Transgene delivered Cells cultured Cells returned to the body Ex VivoIn Vivo Transgene delivered directly into host Strategies for Transgene Delivery

60. ©1999 Timothy G. Standish Which cells are the target cells Both Healthy and Cancerous cells can be a target Ex of targeting Healthy cells –One way is by replacing a missing or altered gene with a “normal” one

61. ©1999 Timothy G. Standish Cont: Which cells are the target cells Ex of targeting Cancer Cells Scientists can target cancer cells with genes that can be used to destroy the cells. In this technique, cancer cells are introduced to what is called “suicide genes”

62. ©1999 Timothy G. Standish Naked DNA Target Cell Therapeutic Protein AAV Retrovirus/Lentivirus Adenovirus Nucleus Gene Therapy Principles

63. ©1999 Timothy G. Standish Adenovirus Cell Entry

64. ©1999 Timothy G. Standish p53 Pathway

65. ©1999 Timothy G. Standish

66. Using Gene Therapy to Treat Lung Cancer In this clinical trial the scientist used gene therapy in combination with radiation therapy so they can treat lung cancer in 19 different patients

67. ©1999 Timothy G. Standish Treatment: Gene therapy and Radiation. Intratumoral needle injections of Ad-p53 on days 1, 18 and 32 of the treatment. tumors ≥ 4 cm where injected with 10 ml tumors ‹ 4 cm were injected with 3 ml Radiation therapy

68. ©1999 Timothy G. Standish Results 17/19 patients made it through the entire therapy complete response in 2 patients (11%) partial response in 4 patients (21%) stable disease in 1 patient (5%) progressive disease in 11 patients (57%)

69. ©1999 Timothy G. Standish

70. Results Not That Good 57% of the patients showed that the cancer progressed to worse stages Why?

71. ©1999 Timothy G. Standish Major Problems that Scientists Must Overcome Identify more efficient ways to deliver the genes to the patients’ genetic material Develop vectors that can specifically focus on the targeted cells Ensure that vectors will successfully insert the desired genes into each of these target cells

72. ©1999 Timothy G. Standish Cont. Major Problems that Scientists Must Overcome Deliver genes to a precise location in the patient’s DNA Ensure that transplanted genes are precisely controlled by the body’s normal physiologic signals