Antiviral Drugs, Vaccines and Gene Therapy

ANTIVIRAL DRUGSVACCINES

GENE THERAPY

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OVERVIEW

• Antiviral drugs- definition, description, history, importantantiviral drugs, flu antiviral therapy, AIDS antiviral drugs

• Vaccines-definition, history, current status, importantvaccines, immunological responses

• Gene therapy- definition, history, mode of action, currentstatus

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ANTIVIRAL DRUGS

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MOMINA SHAHID

• Vaccines have provided considerable success inpreventing viral disease.

• But, they have modest or often no therapeuticeffect for individuals who already are infected.

• Consequently, our second arm of antiviraldefense has been the development and use ofantiviral drugs.

• They can stop an infection once it has started.

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However, despite almost 50 years ofresearch, our arsenal of antiviral drugsremains dangerously small.

Only about 30 antiviral drugs areavailable on the US market.Most against HIV and herpes viruses.

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WHY WE HAVE SO FEW ANTIVIRAL DRUGS?

1. Compounds interfering with virus growth canadversely affect the host cell.

• Side effects are common

• Every step in viral life cycle engages host functions

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2. Many medically important viruses are dangerous, can'tbe tested in model systems, or can't be propagated.

• Difficult or impossible to grow in thelaboratory (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)

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3. Antiviral drug must block viral replicationcompletely.

• Partial inhibition is not acceptable for an antiviraldrug. If a drug does not block virus replicationcompletely, resistant viruses will emerge.

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4. Antiviral drug discovery is time consumingand expensive.

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HISTORICAL PERSPECTIVE

• The first modest search for antiviral drugs occurred inthe early 1950’s.

• Chemists looked at derivatives of the sulfonamideantibiotics.

• In the 1960’s and 1970’s, drug companies launchedhuge “blind-screening” programs to find chemicalswith antiviral activity.

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BLIND SCREENING

• Random chemicals and natural product mixtures tested forability to block replication of a variety of viruses in cellculture systems.

• The mechanism of how these compounds inhibit the virusis not given any importance.

• Despite considerable efforts, very little success wasachieved.

• Amantadine was discovered which is effective fortreatment of influenza A virus infections. 9

DISCOVERING ANTIVIRAL COMPOUNDS TODAY

• New technology and recombinant DNAtechnology have made targeted discoverypossible.

• Blind screening procedures have lostpopularity among pharmaceutical companies.

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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

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

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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

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ENFUVIRTIDE

• Enfuvirtide is used along with other medications to treathuman immunodeficiency virus (HIV) infection.

• Enfuvirtide is in a class of medications called HIV entryinhibitors.

• It works by decreasing the amount of HIV in the blood.Although enfuvirtide does not cure HIV, it may decreasechance of developing acquired immunodeficiencysyndrome (AIDS) and HIV-related illnesses such as seriousinfections or cancer.

• Taking these medications along with practicing safer sexand making other life-style changes may decrease the riskof transmitting the HIV virus to other people. 14

ALPHA-INTERFERON

• Cells that have been infected with virus produce interferon,which sends a signal to other cells of the body to resist viralgrowth.

• When first discovered in 1957, interferon was thought to bea single substance, but since then several types have beendiscovered, each produced by a different type of cell.

• Alpha interferon is produced by white blood cells otherthan lymphocytes.

• All interferons inhibit viral replication.

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AMANTADINE

• Amantadine is a drug that has U.S. Food and DrugAdministration approval for use both as an antiviral andan antiparkinsonian drug.

• The mechanism of Amantadine's antiviral activity involvesinterference with a viral protein M2 (an ionchannel), 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 thedrug is ineffective towards all Influenza B strains.

• Amantadine has been associated with several centralnervous system side effects. CNS side effects includenervousness, anxiety, agitation, insomnia and difficulty inconcentrating. 1

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VETERINARY MISUSE

• In 2005, Chinese poultry farmers were reported to haveused 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 billiondoses of amantadine.

• Avian flu (H5N1) strains in China and southeast Asia arenow resistant to amantadine, although strains circulatingelsewhere still seem to be sensitive.

• If amantadine-resistant strains of the virus spread, thedrugs of choice in an avian flu outbreak will probably berestricted to the scarcer and costlier zanamivir, which workby a different mechanism.

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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 acyclovirtriphosphate that competitively inhibits and inactivates DNApolymerases and inhibit nucleic acid synthesis.

• Overdose symptoms may include seizure (convulsions),hallucinations, and urinating less than usual or not at all.

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RITOVIR

• Ritovir is prescribed for HIV (Human ImmunodeficiencyVirus) infection either alone or combined with otherantiviral agents.

• It is a protease inhibitor.

• It cleaves polyproteins.

• Prevents late protein synthesis by inhibiting posttranslation modifications as segments of polyproteins aftercleaved make a capsid.

• Block protease to cleave polyprotein.

• Useful in HIV because it produces a polyprotein which iscleaved later. HIV includes a protease, and so considerableresearch has been performed to find "protease inhibitors"to attack HIV at that phase of its life cycle.

• No use in influenza.19

ZANAMIVIR

• Zanamivir is a neuraminidase inhibitor used in thetreatment of influenza caused by influenza A and Bviruses.

• It was the first neuraminidase inhibitor commerciallydeveloped.

• Zanamivir works by binding to the active site of theneuraminidase protein, rendering the influenza virusunable to escape its host cell and infect others.

• It is also an inhibitor of influenza virus replication invitro and in vivo.

• In clinical trials, zanamivir was found to reduce the time-to-symptom resolution by 1.5 days if therapy was startedwithin 48 hours of the onset of symptoms.

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ANTIVIRAL THERAPY FOR FLU

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INFLUENZA A STRAIN STRUCTURE

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INFLUENZA B STRAIN STRUCTURE

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• 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 hostcell molecule, sialic acid. The Hemagglutinin proteinfacilitates viral attachment while neuraminidase is involvedin viral release.

• Only Influenza A has M2 protein.

• Amantadine and Rimantadine block M2 protein.

• Both of these drugs are ineffective against influenza Bstrain.

• Zanamivir – a neuraminidase inhibitor is effective for bothinfluenza A and B strain but it is costly compared toamantadine and rimantadine.

• Start medicines within 2 days

• Treatment time is 5 days24

HIGH RISK GROUP

• < 2 years

• >65 years

• Pregnant women

• Chronic diseases

• Severe diseases

• Hospitalized

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HIV LIFE CYCLE

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INITIAL THERAPY

• AZT is the first U.S. government-approved treatment for HIV,

• AZT inhibits the enzyme (reverse transcriptase) that HIV uses tosynthesize 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 isnow usually used in conjunction with other anti-HIV drugs inthe combination therapy called highly active antiretroviraltherapy (HAART).

• Early long-term higher-dose therapy with AZT was initiallyassociated with side effects includinganemia, neutropenia, hepatotoxicity, cardiomyopathy,and myopathy. All of these conditions were generally found to bereversible upon reduction of AZT dosages

• When first prescribed, AZT was given in high doses, whichcommonly caused severe side-effects. Recommended doses arenow much lower, and as a result, side-effects have lessened

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CLASSES OF ANTI-HIV DRUGS

• Nucleoside+ Nucleotide Reverse Transcriptase Inhibitor

• Non Nucleoside Reverse Transcriptase Inhibitor

• Protease Inhibitor

• Fusion and attachment Inhibitor

• Integrase Inhibitor

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References

http://en.wikipedia.org/wiki/Highly_active_antiretroviral_therapy

http://www.aidsmap.com/Side-effects/page/1730907/

http://www.nlm.nih.gov/medlineplus/druginfo/meds/a682064.html

http://www.cdc.gov/flu/

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VACCINES

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SEHRISH QAMAR

WHAT IS A VACCINE?

A vaccine is any preparation used as a preventiveinoculation to confer immunity against a specificdisease, usually using a harmless form of the diseaseagent, such as killed or weakened bacteria or viruses.The purpose of which is to stimulate antibodyproduction.

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SOME BASICS

Most often the terms vaccination and immunization are usedinterchangeably but their meanings are not exactly the same.

• A vaccine is a product that produces immunity from a disease andcan be administered through needle injections, by mouth, or byaerosol.

• Vaccination is when a vaccine is administered to a person (usuallyby injection).

• Immunization is what happens in one’s body after they havebeen vaccinated. The vaccine stimulates one’s immune system sothat it can recognize the disease and offer protection from futureinfection.

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HISTORICAL MILESTONES

Vaccination is a miracle of modern medicine. In the past 50 years, it’s savedmore lives worldwide than any other medical product or procedure.

• 429 BC: Thucydides notices that people who survive smallpox do notget re-infected

As long ago as 429 BC, the Greek historian Thucydides observed that thosewho survived the smallpox plague in Athens did not become re-infected withthe disease.

• 900 AD: Chinese discover variolation

The Chinese were the first to discover and use a primitive form of vaccinationcalled variolation.

• 1700s: Variolation spreads around the world

Variolation eventually spread to Turkey, and arrived in England in the early18th century. At this time, smallpox was the most infectious disease in Europe.

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1796: Edward Jenner discovers vaccinationBritish physician, Dr. Edward Jenner, discovered vaccination in its modernform and proved to the scientific community that it worked.

1803: Royal Jennerian Institute foundedSupport for vaccination grew. Jenner was awarded government funding, and in1803 the Royal Jennerian Institute was founded.

1870s: Violent opposition to vaccinationAlthough vaccination was taken up enthusiastically by many, there was someviolent opposition as it became more widespread. People felt that it took awaytheir civil liberties, particularly now that it was compulsory.

1880s: A vaccine against rabiesLouis Pasteur improved vaccination, and developed a rabies vaccine.

1890: Emil von Behring discovers the basis of diphtheria and tetanusvaccinesGerman scientist, Emil von Behring, was awarded the first Nobel Prize inPhysiology or Medicine.

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1879

First LaboratoryVaccineLouis Pasteurproduced thefirst laboratory-developedvaccine: thevaccine forchicken cholera.

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An image from the Florentine Codexcompiled in Mexico in the 1500’sshowing the devastating effects ofSmallpox on the native population.

Edward Jenner withJames Phipps.

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1920s: Vaccines become widely availableBy the end of the 1920s, vaccines for diphtheria, tetanus, whoopingcough and tuberculosis were all available.

1955: Polio vaccination beginsPolio vaccination was introduced in the UK and it dramatically reduced thenumber of cases.

1956: WHO fights to eradicate smallpoxThe first attempt to use the smallpox vaccine on a global scale began when theWorld Health Organization (WHO) decided to try and eradicatesmallpox across the world.

1980: Smallpox eradicated from the worldSmallpox was declared eradicated in 1980. It was one of the most remarkableachievements in the history of medicine.

2008: Cervical cancer scientist awarded Nobel PrizeProfessor Harald zur Hausen discovered that cervical cancer was caused by avirus, making it possible to develop a vaccine for the disease.

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2008: NHS vaccinates girls against cancerIn England, the NHS cervical cancer vaccination programme beganwhereby all 12-13 year-old-girls are offered HPV vaccination to protect themagainst cervical cancer. This is the first time that a routine universal vaccinewas been given to prevent a type of cancer.

2013: NHS vaccinates against shingles and rotavirusThe NHS vaccination programme sees the introduction of rotavirusvaccination for babies and a shingles vaccine for over-70s. A children's fluvaccine is launched which is given as a nasal spray rather than an injection.

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MECHANISM OF ACTION

Vaccines Produce:

• Humoral immunity (B cell response) i.e. mostbacterial vaccines

OR

• Cell-mediated immunity (T cell response) i.e. livevaccines such as MMR and BCG

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Humoral Immunity primarilyproduces antibodies in the bloodcirculation as a sensing orrecognizing function of theimmune system to the presence offoreign antigens in the body.

Cell Mediated Immunityprimarily destroys, digests andexpels foreign antigens out of thebody through the activity of itscells found in the thymus, tonsils,adenoids, spleen, lymph nodesand lymph system throughout thebody. This process of destroying,digesting and discharging foreignantigens from the body is knownas the acute inflammatoryresponse and is often accompaniedby the classic signs ofinflammation: fever, pain, malaiseand discharge of mucus, pus, skinrash or diarrhea.

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A vaccination consists of introducing a disease agent ordisease antigen into an individual’s body withoutcausing the disease. If the disease agent provoked thewhole immune system into action it would cause all thesymptoms of the disease. The symptoms of a disease areprimarily the symptoms (fever, pain, malaise, loss offunction) of the acute inflammatory response to thedisease. So the trick of a vaccination is to stimulate theimmune system just enough so that it makes antibodiesand remembers the disease antigen but not so muchthat it provokes an acute inflammatory response by thecellular immune system and makes us sick with thedisease we are trying to prevent.

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VACCINES PRODUCING HUMORAL IMMUNITY

• B cells are a type of lymphocyte (white bloodcells) capable of producing antibodies.

• B cells with the right receptor shape recognisea vaccine antigen and bind to it

• The B cells are activated to produce a clone ofantibodies with the same specificity

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VACCINES PRODUCING HUMORAL IMMUNITY

• The B cells mature and become “plasma”cells (capable of excreting 2000 moleculesantibody/second) and “memory” cells

• If the “memory” cells encounter theantigen again they will change into plasmacells and produce large numbers of specificantibodies

• The size, specificity and speed of theresponse will increase with repeated exposure

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HELP FROM T CELLS IN THE HUMORAL RESPONSE

A certain type of T cell (helper or CD4 cell) can helpB cells differentiate into clones. (Where this is anessential element for a particular vaccine this istermed a “T cell dependent” response)

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PRIMARY IMMUNE RESPONSE

• Primary immuneresponse developsin the weeksfollowing firstexposure to anantigen. MainlyIgM antibody

• Secondaryimmune responseis faster and morepowerful.PredominantlyIgG antibody

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DIFFERENT TYPES OF VACCINES

Vaccines are made using several different processes. The differentvaccine types each require different development techniques.

Live, Attenuated VaccinesAttenuated vaccines can be made in several different ways. Some of themost common methods involve passing the disease-causing virusthrough a series of cell cultures or animal embryos (typically chickembryos). Using chick embryos as an example, the virus is grown indifferent embryos in a series. With each passage, the virus becomesbetter at replicating in chick cells, but loses its ability to replicate inhuman cells. When the resulting vaccine virus is given to a human, itwill be unable to replicate enough to cause illness, but will still provokean immune response that can protect against future infection.

Examples: Measles, mumps, rubella, Varicella (chickenpox),Influenza and Rotavirus

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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

Meningococcal49

Vaccine type Other available vaccines

Live, attenuated

Zoster (shingles)

Yellow fever

Inactivated/Killed Rabies

Subunit/conjugate Human papillomavirus (HPV)

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Killed or Inactivated Vaccines:Vaccines of this type are created by inactivating a pathogen, typicallyusing heat or chemicals such as formaldehyde or formalin. Thisdestroys the pathogen’s ability to replicate, but keeps it “intact” sothat 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 ofpathogen can be made by inactivating the toxin that causes diseasesymptoms. As with organisms or viruses used in killed or inactivatedvaccines, this can be done via treatment with a chemical such asformalin, or by using heat or other methods. Immunizations createdusing inactivated toxins are called toxoids.

Examples: Diphtheria, tetanus 51

Subunit andConjugate Vaccines:

Both subunit and conjugatevaccines contain only piecesof the pathogens theyprotect against. Subunitvaccines use only part of atarget pathogen to provoke aresponse from the immunesystem. This may be done byisolating a specific proteinfrom a pathogen andpresenting it as an antigenon its own. Another type ofsubunit vaccine can becreated via geneticengineering.Examples: Acellularpertussis vaccine andinfluenza vaccine.

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VACCINE PREVENTABLE DISEASES

Some vaccine-preventable diseases arelisted 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

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DISEASE ERADICATION

• When a disease stops circulating ina region, it’s considered eliminatedin that region. Polio, for example,was eliminated in the UnitedStates by 1979 after widespreadvaccination efforts.

• If a particular disease is eliminatedworldwide, it’s considerederadicated. To date, only oneinfectious disease that affectshumans has been eradicated. In1980, after decades of efforts by theWorld Health Organization, theWorld Health Assembly endorseda statement declaring smallpoxeradicated.

Smallpox eradicationcampaign

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DISEASE ERADICATION

• The eradication of smallpox raised hopes that thesame could be accomplished for other diseases,with many named as possibilities: polio, mumps,and Guinea worm disease, among others. Malariahas also been considered, and its incidence hasbeen reduced drastically in many countries. Itpresents a challenge to the traditional idea oferadication, however, in that having malaria doesnot result in lifelong immunity against it (assmallpox and many other diseases do).

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Red is measles,green is whoopingcough, yellow ispolio, and blue isrubella.

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Factors Influencing Vaccination uptake in Pakistan

Pakistan, one of the three endemic polio reservoirs, is posing aserious threat to the success of the Global Polio EradicationInitiative to eradicate polio completely. Some of the hurdles knownto 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

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REFERENCES

www.unicef.org

www.historyofvaccines.org

www.vaccines.com

http://www.immune.org.nz/types-vaccines

http://wenliang.myweb.uga.edu/mystudy/immunology/ScienceOfImmunology/NotesImages/Topic247NotesImage2.gif

http://www.who.int/i

http://philipincao.crestonecolorado.com/index_htm_files/How%20Vaccinations%20Work.pdfmmunization/documents/Elsevier_Vaccine_immunology.pdf

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GENE THERAPY

NEELAM PERVEEN 60

GENES• Are carried on a chromosome

• The basic unit of heredity

• Encode how to make a protein-DNARNAproteins

• Proteins carry out most of life’s function.

• When altered causes dysfunction of a protein

• When there is a mutation in the gene, then itwill change the codon, which will changewhich amino acid is called for which willchange the conformation of the protein whichwill change the function of the protein.Genetic disorders result from mutations in thegenome.

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GENE THERAPY• It is a technique for correcting defective genes that are

responsible for disease development. Its the elaboration of therecombinant DNA technology that brought gene therapy intothe realm of feasibility.

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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.

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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

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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.

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SUCCESSFUL GENE THERAPY FOR SEVERE COMBINE IMMUNODEFICIENCY

• Infants with severe combined immunodeficiencyare unable to mount an adaptive immune response,because they have a profound deficiency oflymphocytes.

• severe combined immunodeficiency is inherited asan X-linked recessive disease, which for all practicalpurposes affects only boys. In the other half of thepatients with severe combined immunodeficiency,the inheritance is autosomal recessive — and thereare several abnormalities in the immune systemwhen the defective gene is encoded on anautosome. 66

SEVERE COMBINE IMMUNODEFICIENCY CONT.

• A previous attempt at gene therapy forimmunodeficiency was successful inchildren with severe combinedimmunodeficiency due to a deficiency ofadenosine deaminase. In these patients,peripheral T cells were transduced with avector bearing the gene for adenosinedeaminase. The experiment was extremelylabor intensive, because mature peripheral-blood T cells were modified rather thanstem cells, and the procedure therefore hadto be repeated many times to achievesuccess.

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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 causingthe cell to return to a normal state

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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

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SCID CONT.

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GENE THERAPYIn vivo Ex vivo

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IN VIVO GENE THERAPY

• The genetic material is transferred directly into thebody of the patient

• More or less random process

• Small ability to control

• Less manipulations

• Only available option for tissues that can not be grownin vitro; or if grown cells can not be transferred back

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EX VIVO GENE THERAPY

• The genetic material is first transferred into the cells grownin vitro

• Controlled process

• Genetically altered cells are selected and expanded

• More manipulations

• Cells are then returned back to the patient

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HOW TO FIX A PROBLEM? USE VECTORS

• A virus is found which replicates by inserting its genes intothe host cell's genome. This virus has three genes - A, B andC.

• Gene A encodes a protein which allows this virus to insertitself into the host's genome.

• Genes B and C actually cause the disease this virus isassociated with.

• Replace B and C with a beneficial gene. Thus, the modifiedvirus could introduce your 'good gene' into the host cell'sgenome without causing any disease.

A

B C a beneficial geneA

virus modified virus

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VIRUSES

• Replicate by inserting their DNA into a host cell

• Gene therapy can use this to insert genes that encode for adesired protein to create the desired trait

• Four different types

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RETROVIRUSES

• Created double stranded DNA copies from RNAgenome• 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 celldivision causes cancer from uncontrolled cell division)

• vectors used are derived from the humanimmunodeficiency virus (HIV) and are beingevaluated for safety

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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

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ADENOVIRUS cont.

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

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ADENO-ASSOCIATED VIRUSES

• Adenoassociated virus, a virus much smaller than the adenovirusbut usually isolated with adenovirus as that virus needed for thereproduction of adeno-associated virus, can also infect humancells, 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 insertgenetic material at a specific point on chromosome 19

• From parvovirus family- causes no known disease and doesn'ttrigger 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 produceFactor IX and thus prevent bleeding.• Study by Wilson and Kathy High (University of Pennsylvania),

patients have not needed Factor IX injections for more than ayear 79

HERPES SIMPLEX VIRUSES

• Double stranded DNA viruses that infectneurons

• Herpes virus, with some members causingcold sores in humans, has the proclivity toinfect cells of the nervous system andtherefore may provide the vehicle to deliverdesired genes to this otherwise generallyinaccessible system.

• Other herpes viruses preferentially infecthuman cells in the blood, and vectorsbased on them could be utilized to delivergenes to the immune cells.

• Ex. Herpes simplex virus type 180

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 specialcell 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

CURRENT STATUS

• FDA has not approved any human gene therapyproduct for sale

• Reason:

January 2003, halt to using retrovirus vectors in bloodstem cells because children developed leukemia-likecondition after successful treatment for X-linkedsevere combined immunodeficiency disease 82

GENE THERAPY IN PAKISTAN

• Breast cancer is one of the most common cancers among women around theworld. It accounts for 22.9% of all the cancers and 18% of all female cancersin the world.

• One million new cases of breast cancer are diagnosed every year. Pakistanhas more alarming situation with 90,000 new cases and ending up into40,000 deaths annually. The risk factor for a female to develop breast canceras compared with male is 100 : 1.

• The traditional way of treatment is by surgery, chemotherapy orradiotherapy. Advanced breast cancer is very difficult to treat with any of thetraditional treatment options. A new treatment option in the form of genetherapy can be a promising treatment for breast cancer.

• Gene therapy provides treatment option in the form of targeting mutatedgene, expression of cancer markers on the surface of cells, blocking themetastasis and induction of apoptosis, etc.

• Gene therapy showed very promising results for treatment of variouscancers. All this is being trialed, experimented and practiced outside ofPakistan. Therefore, there is an immense need that this kind of work shouldbe started in Pakistan. There are many good research institutes as well aswell-reputed hospitals in Pakistan working over it. 83

POPULAR CULTURE• Gene therapy is the basis for the

plotline of the film I Am Legend

• I Am Legend is a 2007American post-apocalyptic sciencefiction horror film directedby Francis Lawrence andstarring Will Smith. Smithplays virologist Robert Neville,who is immune to a man-made virus originally created tocure cancer. He works to create aremedy while defending himselfagainst mutants created by thevirus.

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POPULAR CULTURE CONT.

• Gene therapy is the basis for theplotline of the film Rise of thePlanet of the Apes

• In the 2011 film Rise of the Planetof the Apes, a fictional genetherapy called ALZ-112 was a drugthat was a possible curefor Alzheimer's disease, thetherapy increased the host'sintelligence and made their irisesgreen, along with the revisedtherapy called 113 which increasedintelligence in apes yet was adeadly, internal virus in humans.

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PROBLEMS WITH GENE THERAPY

• Short Lived• Hard to rapidly integrate therapeutic DNA into genome and

rapidly dividing nature of cells prevent gene therapy from longtime

• 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 morethan one gene

• May induce a tumor if integrated in a tumor suppressor genebecause insertional mutagenesis 86

References

• http://openreads.com/chapter/one-world-chapter-7-advances-in-genetics-gene-therapy/

• http://my.safaribooksonline.com/book/biotechnology/0131010115/gene-therapy/ch07#X2ludGVybmFsX0h0bWxWaWV3P3htbGlkPTAtMTMtMTAxMDExLTUlMkZjaDA3bGV2MXNlYzMmcXVlcnk9

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