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Gene therapy

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Gene therapy Gene therapy: to correct a genetic defect by transferring of a functional normal copy of the gene into cells Examples of diseases caused by genetic defect – PowerPoint PPT presentation

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Title: Gene therapy


1
Gene therapy
  • Gene therapy
  • to correct a genetic defect by transferring of a
    functional normal copy of the gene into cells
  • Examples of diseases caused by genetic defect
  • Ornithine transcarbamylase (OTC deficiency)
  • Hemophilia (blood coagulation factors VIII or IX)
  • SCID( severe combined immunodeficiency)
  • Muscular dystrophy
  • Cystic fibrosis
  • Sickle cell anemia

2
Application of gene therapy
  • Genetic disorder (deficiency) OTC
  • Cancer
  • Genetic predisposition
  • Mutation in oncogene or tumor suppressor gene
  • Autoimmunity diseases rheumatoid arthritis
  • Delivery of counteracting gene
  • Diseases involve several genes and the
    environmental interact diabetes

3
Factors to be considered in Gene therapy
  • How to deliver genes to specific cells, tissue
    and whole animals? (methods of delivery)
  • How much and how long the introduced gene will be
    expressed?
  • The site and dose of gene delivery
  • Is there any adverse immunological consequence of
    both delivery vehicle (Virus) and the gene in
    animals?
  • Is there any toxic effects?

4
Methods of gene delivery
  • Viral Vectors
  • Adenovirus
  • Retrovirus
  • Lentivirus
  • Adeno-associated virus (AAV)
  • Herpes simplex virus (HSV)
  • Non-viral vector based
  • Naked DNA (plasmid DNA) injection or genegun
  • Liposomes (cationic lipids) mix with genes
  • Ex-vivo
  • In vivo

5
Why use viral vectors
  • Virus are obligate intracellular parasites
  • Very efficient at transferring viral DNA into
    host cells
  • Specific target cells depending on the viral
    attachment proteins (capsid or glycoproteins)
  • Gene replacement non-essential genes of virus
    are deleted and exogenous genes are inserted

6
Generation of viral vector for gene therapy
  • Replication-competent virus
  • Replication-defective virus
  • Amplicon doesnt encode structural proteins
  • Cant replicate beyond the first cycle of
    infection
  • Elements needed to generate amplicon
  • Transfer Vector plasmid (promoter, gene of
    interest, ori, packaging signal)
  • Packaging vector (cosmid or cell lines) provide
    the viral structural proteins for packaging of
    transfer vector
  • Helper virus (packaging of transfer vector)
    deleted Packaging signal sequence

7
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8
Adenoviral vectors
  • Non-enveloped ds DNA, 36 kilobases
  • Early proteins (E1A, E1B, E2,E3 and E4), late
    proteins (L1-L5)
  • Causes a benign respiratory infections in human
  • Serotypes 2 and 5 are commonly used as vectors

9
Early generations of adenoviral vectors
(replication defective)
10
Death of 18-year old Jesse Gelsinger
  • Liver disease OTC deficiency (genetic disease)
  • University of Pennsylvania
  • High dose of adenoviral vector (E1 and E4 genes
  • deleted ) carrying the normal copy of OTC gene
    was administered
  • Suspected cause of death
  • Toxicity of high titer adenoviral vector
  • High immunogenicity of adenoviral vector (an
    immune revolt)

11
Gutless Adenoviral vector (Amplicon)
12
Modification of the tropism of adenovirus vector
  • Adenovirus fiber binds to CAR (coxsakie and
    adenovirus receptor, CAR), receptor which is
    ubiquitous
  • Modify the fiber protein

13
Characteristics of adenoviral vector
  • Advantages
  • High titers
  • Both dividing and non-dividing cells
  • Wide tissue tropism
  • Easily modify tissue tropism
  • Disadvantages
  • Transient expression ( not good for genetic
    diseases)
  • Highly immunogenic
  • High titers of virus can be toxic
  • More suitable for cancer immunotherapy

14
Retroviral vector
  • Moloney murine leukemia virus (MuLV)
  • Generation of replication defective retroviral
    vector
  • Transfer plasmid vector
  • Gene of interest
  • Long terminal repeats(LTR) promoter, polyA,
    integration, replication and reverse
    transcription
  • Primer binding site (PBS) (origin of replication)
  • RNA packaging signal
  • Poly purine tract (important for replication)
  • Packaging vector
  • Cell line stably transfected with plasmid
    constructs containing Gag/pol and Env

15
Generation of retroviral vector
16
Pseudotyped retroviral vector
17
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18
Characteristics of retroviral vector
  • Advantages
  • Integration permanent expression
  • Pseudotyped virus
  • Disadvantages
  • Only infecting dividing cells
  • Insertional mutagenesis (tumor formation)
  • Activate oncogenes
  • Inhibit tumor suppressor genes

19
A case of leukemia in a SCID child treated with a
retroviral vector
  • SCID disease or Bubble boy disease ( T cell
    deficiency)
  • Overall quite successful, over 1000 peoples
    received retroviral gene therapy
  • A French babys treated with retroviral vector 3
    years ago
  • A leukemia-like illness developed this summer.
  • Nine other children treated same time show no
    sign of leukemia
  • But the side effect isnt a big enough risk yet
    that genetic experiments for children with an
    often fatal immune disease should stop
  • People receiving retroviral gene therapy should
    be warned about the risk of developing leukemia

20
Lentiviral vectors
  • Infection of non-dividing cells (hepatocytes,
    neurons)
  • HIV, a human lethal pathogen
  • Delete accessory genes
  • Provide an envelope from a non-retrovirus (VSV)
  • Develop vectors from lentiviruses of non-human
    pathogens
  • SIV, FIV, EIAV etc

21
Herpesvirus vectors
  • Herpes simplex virus 1, mild disease in human, no
    risk
  • Linear ds DNA, 152 kb, about half of the total 81
    genes are non-essential for virus replication
  • 40-50 kb of foreign DNA can be accommodated
  • Neurotropic virus, target to nervous system
  • Replication defective amplicon particles

22
Adeno-associated virus vectors
  • Non-pathogenic human parvovirus, non-enveloped ss
    DNA virus, 4.6 kilobases
  • Dependent on a helper virus ( adenovirus or
    herpesvirus) for replication (dependovirus)
  • AAV-2 mostly used for vector

23
Generation of adeno-associated virus vector
24
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25
Characteristics of AAV vector
  • Advantages
  • Integration and persistent expression
  • No insertional mutagenesis
  • Infecting dividing and nondividing cells
  • Safe
  • Disadvantages
  • Size limitation, 4.9 kb
  • Low titer of virus, low level of gene expression

26
Comparison of different viral vectors
Viral vector titers manupilation of
immunogenicity infecting of tropism
non-dividing cells Adenovirus 1011 terrific v
ery high yes Retrovirus 107 good low only
lentivirus Herpesvirus 107 not so
good low yes AAV 107 not so good low yes
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