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Antiviral Drugs, Vaccines and Gene Therapy


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Vaccines, Antiviral Drugs and Gene Therapy

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Antiviral Drugs, Vaccines and Gene Therapy

  2. 2. OVERVIEW • Antiviral drugs- definition, description, history, important antiviral drugs, flu antiviral therapy, AIDS antiviral drugs • Vaccines-definition, history, current status, important vaccines, immunological responses • Gene therapy- definition, history, mode of action, current status i
  4. 4. • Vaccines have provided considerable success in preventing viral disease. • But, they have modest or often no therapeutic effect for individuals who already are infected. • Consequently, our second arm of antiviral defense has been the development and use of antiviral drugs. • They can stop an infection once it has started. 2
  5. 5. However, despite almost 50 years of research, our arsenal of antiviral drugs remains dangerously small. Only about 30 antiviral drugs are available on the US market. Most against HIV and herpes viruses. 3
  6. 6. WHY WE HAVE SO FEW ANTIVIRAL DRUGS? 1. Compounds interfering with virus growth can adversely affect the host cell. • Side effects are common • Every step in viral life cycle engages host functions 4
  7. 7. 2. Many medically important viruses are dangerous, can't be tested in model systems, or can't be propagated. • Difficult or impossible to grow in the laboratory (hepatitis B and C, papilloma viruses) • Have no available animal model of human disease (smallpox virus, HIV ,measles virus) • Will kill investigators who aren't careful (Ebola virus , Lassa fever virus ,Small pox virus) 5
  8. 8. 3. Antiviral drug must block viral replication completely. • Partial inhibition is not acceptable for an antiviral drug. If a drug does not block virus replication completely, resistant viruses will emerge. 6
  9. 9. 4. Antiviral drug discovery is time consuming and expensive. 7
  10. 10. HISTORICAL PERSPECTIVE • The first modest search for antiviral drugs occurred in the early 1950’s. • Chemists looked at derivatives of the sulfonamide antibiotics. • In the 1960’s and 1970’s, drug companies launched huge “blind-screening” programs to find chemicals with antiviral activity. 8
  11. 11. BLIND SCREENING • Random chemicals and natural product mixtures tested for ability to block replication of a variety of viruses in cell culture systems. • The mechanism of how these compounds inhibit the virus is not given any importance. • Despite considerable efforts, very little success was achieved. • Amantadine was discovered which is effective for treatment of influenza A virus infections. 9
  12. 12. DISCOVERING ANTIVIRAL COMPOUNDS TODAY • New technology and recombinant DNA technology have made targeted discovery possible. • Blind screening procedures have lost popularity among pharmaceutical companies. 10
  13. 13. ANTIVIRAL AGENTS • Most antiviral drugs are antimetabolites. • Antimetabolites resemble structures of nitrogenous bases (purine and pyrimidine). • Antimetabolites are prodrugs that are activated by host cell enzymes or viral enzymes (mostly kinases). • Most of the antiviral drugs have ‘VIR’ hidden somewhere in their names. An antimetabolite is a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. 11
  14. 14. STEPS FOR VIRAL REPLICATION ① Adsorption and penetration into host cell ② Uncoating of viral nucleic acid ③ Synthesis of regulatory proteins ④ Synthesis of RNA or DNA ⑤ Synthesis of structural proteins ⑥ Assembly of viral particles ⑦ Release from host cell 12
  15. 15. MECHANISM OF ACTION AND ASSOCIATED DRUG MECHANISM OF ACTION EFFECTIVE DRUG Adsorption Enfuvirtide Penetration Alpha-Interferon Uncoating Amantadine Early protein synthesis No drug Nucleic acid synthesis Acyclovir Late protein synthesis Ritonavir-Protease inhibitor Packaging and assembly No drug Viral release Zanamivir-Neuraminidase inhibitor 1 3
  16. 16. ENFUVIRTIDE • Enfuvirtide is used along with other medications to treat human immunodeficiency virus (HIV) infection. • Enfuvirtide is in a class of medications called HIV entry inhibitors. • It works by decreasing the amount of HIV in the blood. Although enfuvirtide does not cure HIV, it may decrease chance of developing acquired immunodeficiency syndrome (AIDS) and HIV-related illnesses such as serious infections or cancer. • Taking these medications along with practicing safer sex and making other life-style changes may decrease the risk of transmitting the HIV virus to other people. 14
  17. 17. ALPHA-INTERFERON • Cells that have been infected with virus produce interferon, which sends a signal to other cells of the body to resist viral growth. • When first discovered in 1957, interferon was thought to be a single substance, but since then several types have been discovered, each produced by a different type of cell. • Alpha interferon is produced by white blood cells other than lymphocytes. • All interferons inhibit viral replication. 15
  18. 18. AMANTADINE • Amantadine is a drug that has U.S. Food and Drug Administration approval for use both as an antiviral and an antiparkinsonian drug. • The mechanism of Amantadine's antiviral activity involves interference with a viral protein M2 (an ion channel), which is required for the viral particle to become "uncoated" once taken inside a cell by endocytosis. Influenza B does not possess M2 channels, and thus the drug is ineffective towards all Influenza B strains. • Amantadine has been associated with several central nervous system side effects. CNS side effects include nervousness, anxiety, agitation, insomnia and difficulty in concentrating. 1 6
  19. 19. VETERINARY MISUSE • In 2005, Chinese poultry farmers were reported to have used amantadine to protect birds against avian influenza. • According to international livestock regulations, amantadine is approved only for use in humans. • Chickens in China have received an estimated 2.6 billion doses of amantadine. • Avian flu (H5N1) strains in China and southeast Asia are now resistant to amantadine, although strains circulating elsewhere still seem to be sensitive. • If amantadine-resistant strains of the virus spread, the drugs of choice in an avian flu outbreak will probably be restricted to the scarcer and costlier zanamivir, which work by a differentmechanism. 17
  20. 20. ACYCLOVIR • Acyclovir is a guanosine analogue antiviral drug. • It is one of the most commonly used antiviral drugs. • It is used for the treatment of herpes simplex virus infections, as well as in the treatment of varicella zoster (chickenpox) • Acyclovir is converted by host cell kinases to acyclovir triphosphate that competitively inhibits and inactivates DNA polymerases and inhibit nucleic acid synthesis. • Overdose symptoms may include seizure (convulsions), hallucinations, and urinating less than usual or not at all. 18
  21. 21. RITOVIR • Ritovir is prescribed for HIV (Human Immunodeficiency Virus) infection either alone or combined with other antiviral agents. • It is a protease inhibitor. • It cleaves polyproteins. • Prevents late protein synthesis by inhibiting post translation modifications as segments of polyproteins after cleaved make a capsid. • Block protease to cleave polyprotein. • Useful in HIV because it produces a polyprotein which is cleaved later. HIV includes a protease, and so considerable research has been performed to find "protease inhibitors" to attack HIV at that phase of its life cycle. • No use in influenza. 19
  22. 22. ZANAMIVIR • Zanamivir is a neuraminidase inhibitor used in the treatment of influenza caused by influenza A and B viruses. • It was the first neuraminidase inhibitor commercially developed. • Zanamivir works by binding to the active site of the neuraminidase protein, rendering the influenza virus unable to escape its host cell and infect others. • It is also an inhibitor of influenza virus replication in vitro and in vivo. • In clinical trials, zanamivir was found to reduce the time-to- symptom resolution by 1.5 days if therapy was started within 48 hours of the onset of symptoms. 20
  26. 26. • Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognize the same host cell molecule, sialic acid. The Hemagglutinin protein facilitates viral attachment while neuraminidase is involved in viral release. • Only Influenza A has M2 protein. • Amantadine and Rimantadine block M2 protein. • Both of these drugs are ineffective against influenza B strain. • Zanamivir – a neuraminidase inhibitor is effective for both influenza A and B strain but it is costly compared to amantadine and rimantadine. • Start medicines within 2 days • Treatment time is 5 days 24
  27. 27. HIGH RISK GROUP • < 2 years • >65 years • Pregnant women • Chronic diseases • Severe diseases • Hospitalized 25
  28. 28. HIV LIFE CYCLE 26
  29. 29. INITIAL THERAPY • AZT is the first U.S. government-approved treatment for HIV, • AZT inhibits the enzyme (reverse transcriptase) that HIV uses to synthesize DNA, and thus prevents viral DNA from forming. • It slows HIV replication in patients, but does not stop it entirely. • HIV may become AZT-resistant over time, and therefore AZT is now usually used in conjunction with other anti-HIV drugs in the combination therapy called highly active antiretroviral therapy (HAART). • Early long-term higher-dose therapy with AZT was initially associated with side effects including anemia, neutropenia, hepatotoxicity, cardiomyopathy, and myopathy. All of these conditions were generally found to be reversible upon reduction of AZT dosages • When first prescribed, AZT was given in high doses, which commonly caused severe side-effects. Recommended doses are now much lower, and as a result, side-effects have lessened 27
  30. 30. CLASSES OF ANTI-HIV DRUGS • Nucleoside+ Nucleotide Reverse Transcriptase Inhibitor • Non Nucleoside Reverse Transcriptase Inhibitor • Protease Inhibitor • Fusion and attachment Inhibitor • Integrase Inhibitor 28
  31. 31. 29
  32. 32. References herapy 64.html 30
  34. 34. WHAT IS A VACCINE? A vaccine is any preparation used as a preventive inoculation to confer immunity against a specific disease, usually using a harmless form of the disease agent, such as killed or weakened bacteria or viruses. The purpose of which is to stimulate antibody production. 32
  35. 35. SOME BASICS Most often the terms vaccination and immunization are used interchangeably but their meanings are not exactly the same. • A vaccine is a product that produces immunity from a disease and can be administered through needle injections, by mouth, or by aerosol. • Vaccination is when a vaccine is administered to a person (usually by injection). • Immunization is what happens in one’s body after they have been vaccinated. The vaccine stimulates one’s immune system so that it can recognize the disease and offer protection from future infection. 33
  36. 36. HISTORICAL MILESTONES Vaccination is a miracle of modern medicine. In the past 50 years, it’s saved more lives worldwide than any other medical product or procedure. • 429 BC: Thucydides notices that people who survive smallpox do not get re-infected As long ago as 429 BC, the Greek historian Thucydides observed that those who survived the smallpox plague in Athens did not become re-infected with the disease. • 900 AD: Chinese discover variolation The Chinese were the first to discover and use a primitive form of vaccination called variolation. • 1700s: Variolation spreads around the world Variolation eventually spread to Turkey, and arrived in England in the early 18th century. At this time, smallpox was the most infectious disease in Europe. 34
  37. 37. 1796: Edward Jenner discovers vaccination British physician, Dr. Edward Jenner, discovered vaccination in its modern form and proved to the scientific community that it worked. 1803: Royal Jennerian Institute founded Support for vaccination grew. Jenner was awarded government funding, and in 1803 the Royal Jennerian Institutewas founded. 1870s: Violent opposition to vaccination Although vaccination was taken up enthusiastically by many, there was some violent opposition as it became more widespread. People felt that it took away their civil liberties, particularly now that it was compulsory. 1880s: A vaccine against rabies Louis Pasteur improved vaccination, and developed a rabies vaccine. 1890: Emil von Behring discovers the basis of diphtheria and tetanus vaccines German scientist, Emil von Behring, was awarded the first Nobel Prize in Physiology orMedicine. 35
  38. 38. 1879 First Laboratory Vaccine Louis Pasteur produced the first laboratory-developed vaccine: the vaccine for chicken cholera. 36
  39. 39. An image from the Florentine Codex compiled in Mexico in the 1500’s showing the devastating effects of Smallpox on the native population. Edward Jenner with James Phipps. 37
  40. 40. 1920s: Vaccines become widely available By the end of the 1920s, vaccines for diphtheria, tetanus, whooping cough and tuberculosis were all available. 1955: Polio vaccination begins Polio vaccination was introduced in the UK and it dramatically reduced the number of cases. 1956: WHO fights to eradicate smallpox The first attempt to use the smallpox vaccine on a global scale began when the World Health Organization (WHO) decided to try and eradicate smallpox across the world. 1980: Smallpox eradicated from the world Smallpox was declared eradicated in 1980. It was one of the most remarkable achievements in the history of medicine. 2008: Cervical cancer scientist awarded Nobel Prize Professor Harald zur Hausen discovered that cervical cancer was caused by a virus, making it possible to develop a vaccine for the disease. 38
  41. 41. 2008: NHS vaccinates girls against cancer In England, the NHS cervical cancer vaccination programme began whereby all 12-13 year-old-girls are offered HPV vaccination to protect them against cervical cancer. This is the first time that a routine universal vaccine was been given to prevent a type of cancer. 2013: NHS vaccinates against shingles and rotavirus The NHS vaccination programme sees the introduction of rotavirus vaccination for babies and a shingles vaccine for over-70s. A children's flu vaccine is launched which is given as a nasal spray rather than an injection. 39
  42. 42. MECHANISM OF ACTION Vaccines Produce: • Humoral immunity (B cell response) i.e. most bacterial vaccines OR • Cell-mediated immunity (T cell response) i.e. live vaccines such as MMR and BCG 40
  43. 43. Humoral Immunity primarily produces antibodies in the blood circulation as a sensing or recognizing function of the immune system to the presence of foreign antigens in the body. Cell Mediated Immunity primarily destroys, digests and expels foreign antigens out of the body through the activity of its cells found in the thymus, tonsils, adenoids, spleen, lymph nodes and lymph system throughout the body. This process of destroying, digesting and discharging foreign antigens from the body is known as the acute inflammatory response and is often accompanied by the classic signs of inflammation: fever, pain, malaise and discharge of mucus, pus, skin rash or diarrhea. 41
  44. 44. A vaccination consists of introducing a disease agent or disease antigen into an individual’s body without causing the disease. If the disease agent provoked the whole immune system into action it would cause all the symptoms of the disease. The symptoms of a disease are primarily the symptoms (fever, pain, malaise, loss of function) of the acute inflammatory response to the disease. So the trick of a vaccination is to stimulate the immune system just enough so that it makes antibodies and remembers the disease antigen but not so much that it provokes an acute inflammatory response by the cellular immune system and makes us sick with the disease we are trying to prevent. 42
  45. 45. VACCINES PRODUCING HUMORAL IMMUNITY • B cells are a type of lymphocyte (white blood cells) capable of producing antibodies. • B cells with the right receptor shape recognise a vaccine antigen and bind to it • The B cells are activated to produce a clone of antibodies with the same specificity 43
  46. 46. 44
  47. 47. VACCINES PRODUCING HUMORAL IMMUNITY • The B cells mature and become “plasma” cells (capable of excreting 2000 molecules antibody/second) and “memory” cells • If the “memory” cells encounter the antigen again they will change into plasma cells and produce large numbers of specific antibodies • The size, specificity and speed of the response will increase with repeated exposure 45
  48. 48. HELP FROM T CELLS IN THE HUMORAL RESPONSE A certain type of T cell (helper or CD4 cell) can help B cells differentiate into clones. (Where this is an essential element for a particular vaccine this is termed a “T cell dependent” response) 46
  49. 49. PRIMARY IMMUNE RESPONSE • Primary immune response develops in the weeks following first exposure to an antigen. Mainly IgM antibody • Secondary immune response is faster and more powerful. Predominantly IgG antibody 47
  50. 50. DIFFERENT TYPES OF VACCINES Vaccines are made using several different processes. The different vaccine types each require different development techniques. Live, Attenuated Vaccines Attenuated vaccines can be made in several different ways. Some of the most common methods involve passing the disease-causing virus through a series of cell cultures or animal embryos (typically chick embryos). Using chick embryos as an example, the virus is grown in different embryos in a series. With each passage, the virus becomes better at replicating in chick cells, but loses its ability to replicate in human cells. When the resulting vaccine virus is given to a human, it will be unable to replicate enough to cause illness, but will still provoke an immune response that can protect against future infection. Examples: Measles, mumps, rubella, Varicella (chickenpox), Influenza and Rotavirus 48
  51. 51. Vaccine type Vaccines of this type on U.S. Recommended Childhood (ages 0-6) Immunization Schedule Live, attenuated Measles, mumps, rubella (MMR combined vaccine) Varicella (chickenpox) Influenza (nasal spray) Rotavirus Inactivated/Killed Polio (IPV) Hepatitis A Toxoid (inactivated toxin) Diphtheria, tetanus (part of DTaP combined immunization) Subunit/conjugate Hepatitis B Influenza (injection) Haemophilus influenza type b (Hib) Pertussis (part of DTaP combined immunization) Pneumococcal Meningococcal 49
  52. 52. Vaccine type Other available vaccines Live, attenuated Zoster (shingles) Yellow fever Inactivated/Killed Rabies Subunit/conjugate Human papillomavirus (HPV) 50
  53. 53. Killed or Inactivated Vaccines: Vaccines of this type are created by inactivating a pathogen, typically using heat or chemicals such as formaldehyde or formalin. This destroys the pathogen’s ability to replicate, but keeps it “intact” so that the immune system can still recognize it. Examples: Polio (IPV), Hepatitis A Toxoids: Some bacterial diseases are not directly caused by a bacterium itself, but by a toxin produced by the bacterium. One example is tetanus: its symptoms are not caused by the Clostridium tetani bacterium, but by a neurotoxin it produces. Immunizations for this type of pathogen can be made by inactivating the toxin that causes disease symptoms. As with organisms or viruses used in killed or inactivated vaccines, this can be done via treatment with a chemical such as formalin, or by using heat or other methods. Immunizations created using inactivated toxins are called toxoids. Examples: Diphtheria, tetanus 51
  54. 54. Subunit and Conjugate Vaccines: Both subunit and conjugate vaccines contain only pieces of the pathogens they protect against. Subunit vaccines use only part of a target pathogen to provoke a response from the immune system. This may be done by isolating a specific protein from a pathogen and presenting it as an antigen on its own. Another type of subunit vaccine can be created via genetic engineering. Examples: Acellular pertussis vaccine and influenza vaccine. 52
  55. 55. VACCINE PREVENTABLE DISEASES Some vaccine-preventable diseases are listed below: • Cholera • Diphtheria • Hepatitis A • Hepatitis B • Influenza • Rabies • Rubella • Smallpox • Tetanus • Influenza Disease • Invasive Meningococcal Disease • Japanese Encephalitis • Measles • Mumps • Pertussis • Pneumococcal • Poliomyelitis • Typhoid • Varicella • Yellow Fever 53
  56. 56. DISEASE ERADICATION • When a disease stops circulating in a region, it’s considered eliminated in that region. Polio, for example, was eliminated in the United States by 1979 after widespread vaccination efforts. • If a particular disease is eliminated worldwide, it’s considered eradicated. To date, only one infectious disease that affects humans has been eradicated. In 1980, after decades of efforts by the World Health Organization, the World Health Assembly endorsed a statement declaring smallpox eradicated. Smallpox eradication campaign 54
  57. 57. DISEASE ERADICATION • The eradication of smallpox raised hopes that the same could be accomplished for other diseases, with many named as possibilities: polio, mumps, and Guinea worm disease, among others. Malaria has also been considered, and its incidence has been reduced drastically in many countries. It presents a challenge to the traditional idea of eradication, however, in that having malaria does not result in lifelong immunity against it (as smallpox and many other diseases do). 55
  58. 58. Red is measles, green is whooping cough, yellow is polio, and blue is rubella. 56
  59. 59. Factors Influencing Vaccination uptake in Pakistan Pakistan, one of the three endemic polio reservoirs, is posing a serious threat to the success of the Global Polio Eradication Initiative to eradicate polio completely. Some of the hurdles known to retard the campaign include: i. The war against terrorism ii. Misconceptions about polio vaccine iii. Religious misinterpretations iv. Frustration among vaccinators v. Lack of awareness vi. Social considerations vii. Natural calamities viii.Inaccessibility Inefficient vaccines and weak health management is found at the hub of majority of the challenges. 57
  60. 60. 58
  61. 61. REFERENCES nceOfImmunology/NotesImages/Topic247NotesImage2.gif ow%20Vaccinations%20Work.pdfmmunization/documents/ Elsevier_Vaccine_immunology.pdf 59
  63. 63. 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 • 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. 61
  64. 64. GENE THERAPY • It is a technique for correcting defective genes that are responsible for disease development. Its the elaboration of the recombinant DNA technology that brought gene therapy into the realm of feasibility. 62
  65. 65. Classes • There are two basic "classes" of gene therapy.  Somatic cell gene therapy : Somatic cell gene therapy changes/fixes/replaces genes in just one person. The targeted cells are the only ones affected, the changes are not passed on to that person's offspring.  Germ line gene therapy: · Germ line gene therapy makes changes in the sperm or egg of an individual. The changes that are made, adding or subtracting genes from the person's DNA, will be passed on to their offspring. This type of gene therapy raises a lot of ethical questions because it impacts the inheritance patterns of humans. 63
  66. 66. APPROACHES • 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 64
  67. 67. AIM • Gene insertion therapy aims to insert a good copy of the gene or the desired gene without regard to the presence of the deleterious gene. • It does not attempt to eliminate or delete the bad gene. The objective here is to insert the non defective or desired gene in such a way that it makes enough product to compensate for the inability of the defective resident gene to produce such a product. • The celebrated cases of the first human gene therapy trial involving adenosine deaminase deficiency is an example of this approach. 65
  68. 68. 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. 66
  69. 69. SEVERE COMBINE IMMUNODEFICIENCY CONT. • 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. 67
  70. 70. 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 68
  71. 71. 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  69
  72. 72. SCID CONT. 70
  73. 73. GENE THERAPY In vivo Ex vivo 71
  74. 74. IN VIVO GENE THERAPY • The genetic material is transferred directly into the body of the patient • More or less random process • Small ability to control • Less manipulations • Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back 72
  75. 75. EX VIVO GENE THERAPY • The genetic material is first transferred into the cells grown in vitro • Controlled process • Genetically altered cells are selected and expanded • More manipulations • Cells are then returned back to the patient 73
  76. 76. HOW TO FIX A PROBLEM? USE VECTORS A B C A a beneficial gene virus modified virus • A virus is found which replicates by inserting its genes into the host cell's genome. This virus has three genes - A, B and C. • Gene A encodes a protein which allows this virus to insert itself into the host's genome. • Genes B and C actually cause the disease this virus is associated with. • Replace B and C with a beneficial gene. Thus, the modified virus could introduce your 'good gene' into the host cell's genome without causing any disease. 74
  77. 77. 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 75
  78. 78. 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 76
  79. 79. ADENOVIRUSES • Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans • The inserted DNA is not incorporate into genome • Not replicated though  • Has to be reinserted when more cells divide • Ex. Common cold 77
  80. 80. ADENOVIRUS cont. 78
  81. 81. ADENO-ASSOCIATED VIRUSES • Adenoassociated virus, a virus much smaller than the adenovirus but usually isolated with adenovirus as that virus needed for the reproduction of adeno-associated virus, can also infect human cells, and its genes are integrated into the host cell chromosome, therefore allowing for the long-term, stable expression of the gene. • 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. • Low information capacity • 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. • Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year 79
  82. 82. HERPES SIMPLEX VIRUSES • Double stranded DNA viruses that infect neurons • Herpes virus, with some members causing cold sores in humans, has the proclivity to infect cells of the nervous system and therefore may provide the vehicle to deliver desired genes to this otherwise generally inaccessible system. • Other herpes viruses preferentially infect human cells in the blood, and vectors based on them could be utilized to deliver genes to the immune cells. • Ex. Herpes simplex virus type 1 80
  83. 83. 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 • Chemically linking DNA to molecule that will bind to special cell receptors • DNA is engulfed by cell membrane • Less effective  • Trying to introduce a 47th chromosome • Exist alongside the 46 others • Could carry a lot of information • But how to get the big molecule through membranes? 81
  84. 84. CURRENT STATUS • FDA has not approved any human gene therapy product for sale • Reason: 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 82
  85. 85. GENE THERAPY IN PAKISTAN • Breast cancer is one of the most common cancers among women around the world. It accounts for 22.9% of all the cancers and 18% of all female cancers in the world. • One million new cases of breast cancer are diagnosed every year. Pakistan has more alarming situation with 90,000 new cases and ending up into 40,000 deaths annually. The risk factor for a female to develop breast cancer as compared with male is 100 : 1. • The traditional way of treatment is by surgery, chemotherapy or radiotherapy. Advanced breast cancer is very difficult to treat with any of the traditional treatment options. A new treatment option in the form of gene therapy can be a promising treatment for breast cancer. • Gene therapy provides treatment option in the form of targeting mutated gene, expression of cancer markers on the surface of cells, blocking the metastasis and induction of apoptosis, etc. • Gene therapy showed very promising results for treatment of various cancers. All this is being trialed, experimented and practiced outside of Pakistan. Therefore, there is an immense need that this kind of work should be started in Pakistan. There are many good research institutes as well as well-reputed hospitals in Pakistan working over it. 83
  86. 86. POPULAR CULTURE • Gene therapy is the basis for the plotline of the film I Am Legend • I Am Legend is a 2007 American post-apocalyptic science fiction horror film directed by Francis Lawrence and starring Will Smith. Smith plays virologist Robert Neville, who is immune to a man-made virus originally created to cure cancer. He works to create a remedy while defending himself against mutants created by the virus. 84
  87. 87. POPULAR CULTURE CONT. • Gene therapy is the basis for the plotline of the film Rise of the Planet of the Apes • In the 2011 film Rise of the Planet of the Apes, a fictional gene therapy called ALZ-112 was a drug that was a possible cure for Alzheimer's disease, the therapy increased the host's intelligence and made their irises green, along with the revised therapy called 113 which increased intelligence in apes yet was a deadly, internal virus in humans. 85
  88. 88. 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 86
  89. 89. References • gene-therapy/ • ch07#X2ludGVybmFsX0h0bWxWaWV3P3htbGlkPTAtMTMtM TAxMDExLTUlMkZjaDA3bGV2MXNlYzMmcXVlcnk9 87
  90. 90. 88