Gene therapy

1. Gene Therapy
By Temesgen.o
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2. Outlines of the presentation
 Introduction
 Genetic basis of human disease
 Development of gene therapy
 Classification of gene therapy
 Gene delivery
 Benefits and limitation of gene therapy
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3. Objectives
After the presentation you will be able to:
Understand definition and mechanism of gene
therapy.
Explain different classification of gene therapy
Discuss potential advantage and disadvantage
of gene therapy.
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4. Introduction
 Gene therapy is the mechanism of using gene for
treatment or prevention of different genetic disease.
 Genes are segments of DNA, that code for specific
protein.
 The most common form of gene therapy include:
Inserting normal gene to replace abnormal gene
 Inactivating, or “knocking out,” a mutated
gene that is functioning improperly.
Introducing a new gene into the body to help
fight a disease.
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5. cont’d…
 Enhances the effect of a normally functioning
gene.
 Activates the gene that was shut down during
fetal grownth.
 Some of the disease treated by gene therapy includes:-
Cystic fibrosis
Severe combined immunodeficiency syndrome
Haemophilia
Many cancers
Sickle cell disease
HIV etc.
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6. Genetic Basis of Human Diseases
 Genetic disease is a disease that are inherited from ancestors
 Any defects in human genome including
chromosome,DNA,gene or allele will results in genetic
disease.
 Defects in human genome will occur by:-
 Mutation, Polymorphism
 Mutation can be caused by different disease, chemical damage
or mechanical damage to genetic material.
 Based on their genetic contribution, human diseases can be
classified as:
1.Monogenic,
2.Chromosomal,
3.Multifactorial
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7. Cont’d…
1.Monogenic disease
 Are caused by alterations in a single gene,
 Example:Huntingtons disease, tuberous sclerosis,cystic
fibrosis,Haemophilia,Sickle cell disease,Lebers heriditary
optic neuropathy etc.
2.Chromosomal disorder: As their name implies, are caused
by alterations in chromosomes. For instance, within an
individual's genome,
 some chromosomes may be missing ,extra chromosome
copies may be present, o certain portions of chromosomes
may be deleted or duplicated.
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8. cont’d…
 Chromosomal disorder may results in developmental
delay,intellectual disablity,infertility etc.
3.Multifactorial disorders
 The vast majority of human diseases can be categorized as
multifactorial.
 They are also referred to as complex diseases,
 Resulted by multiple gene defect, life style and environmental
factors.
Example:Cardiac disease,Diabetes,cancer etc.
 Those genetic disorders can be managed by:
 Enzyme replacement therapy
 Organ transplant
 Surgery
 Gene therapy1/3/2020 8

9. Development of Gene Therapy
 The goal of gene therapy was to identify the genes responsible
for disease and its exact location
 In addition to that gene therapy is used to know mechanisms
of disease so that new treatment approaches and preventative
measures can be proposed.
 After the completion of Human Genome Project(HGP) in 2003
providing complete human DNA sequencing ,many genetic
researches were conducted including gene therapy.
 But the idea of gene therapy was started before the completion
of HGP in the early 1970’s.
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10. Cont’d…
 The first approved gene therapy took place in US on 14
September 1990, at the National Institutes of Health
(NIH), under the direction of William French Anderson.
 Four-year-old Ashanti DaSilva received treatment for a
genetic defect that left her with SCID(a severe combined
immuno deficiency caused by adenosine deaminase
deficiency).
 Medical community accepted that it can cure HIV as in
2008, Gero Hutter has cured a man from HIV using gene
therapy.
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11. Classification of gene Therapy
Based on the mechanism, gene therapy can be
divided as:
1) Gene replacement therapy
2) Gene deactivation therapy
3) Transgenesis
4)Gene Enhancement or activation therapy
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12. Cont’d…
1.Gene replacement therapy: is a gene therapy in
which defective gene is replaced by normal gene.
2. Gene deactivation therapy: associated with gene
silencing and reducing gene expression.e.g. deactivation
of the gene of cancerous cell.
3. Transgenesis:process of introducing a gene (referred
to as a transgene) from one organism into the genome of
another organism.
.The aim is that the resulting transgenic organism will
express the gene and exhibit some new characteristic.
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13. Cont’d…
4.Gene Enhancement or activation therapy:is the process
of activating gene by transferring gene activating molecule
called transcription factors.
 Based on site of gene introduction gene therapy can be
divided as:
A) Somatic cell therapy
B) Germ line therapy
A) Somatic cell therapy
 Therapeutic genes transferred into the somatic cells.
 Will not be inherited to later generations.
 E.g.. Introduction of genes into bone marrow cells, blood
cells,skin cells etc
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14. Cont’d…
B)Germ line therapy
 Therapeutic genes transferred into the germ cells
 It is heritable and passed on to later generations.
E.g.. Genes introduced into eggs and sperms
 There also another mechanisms in which gene therapy is
delivered:
Suicide gene therapy:Provide ‘suicide’ genes to target cancer
cells for destruction.
 Antisense gene therapy
Provides a single stranded gene in an ’antisense’
(backward) orientation to block the production of harmful
proteins by turning off that genes.e.g.AIDS/HIV
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15. Gene Delivery
 Gene delivery is the process by which gene is
transferred to the target cell or tissue.
 The carriers used to transduce genes are called
vector.
 Vectors are needed since the genetic material has to
be transferred across the cell membrane and
preferably in to the cell nucleus.
 Generally gene delivery can occur in two ways:
1.Viral vector
2.Non viral vector
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16. Cont’d…
1.Viral vector
 Are recombinant viruses
 Virus bind to their hosts and introduce their genetic
material into the host cell.
 Plausible strategy for gene therapy, by removing the
viral DNA and using the virus as a vehicle to deliver
the therapeutic DNA.
 The viruses used are altered to make them safe,
although some risks still exist with gene therapy
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17. Viral vector gene deliver can be conducted:
1. Ex vivo/invitro
2. Invivo
Ex vivo:cells are modified outside the body
and then transplanted back in again
E.g. SCID therapy
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18. Cont’d…
 called ex vivo because the cells are treated outside the
organism.
 In vivo
 genes are changed in cells when the cells are still in
the body.
 Called in vivo because the gene is transferred to cells
 Is used when individual cells cannot be cultured in
vitro in sufficient numbers (e.g. brain cells)
E.g. Cystic fibrosis therapy
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21. Cont’d…
Common viral vectors include:
Retroviruses
Adeno viruses
Adeno associated viruses
Herpes simplex viruses
Lentviruses
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22. Cont’d…
 A)Adenovirus
• medium-sized (90–100 nm),
• Non-enveloped (naked),
• Composed of a nucleocapsid and a double-stranded
linear DNA genome.
• There are over 51 different serotypes in humans,
which are responsible for 5–10% of upper respiratory
infections in children, and many infections in adults
as well.
• Adenoviral DNA does not integrate into the genome
and is not replicated during cell division.
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23. Cont’d…
• The genes of the virus are removed so they lose
the ability to divide.
• The human genes are inserted and the vector is
transfected in the culture containing the
sequences for replication.
• The virus thus replicates in the cell culture.
• The packed viruses are then introduced in to
the patient.
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24. Cont’d…
B)Adeno associated virus-
• It is also DNA virus.
• It has no known pathogenic effect and has wide
tissue affinity.
• AAV enters host cell, becomes double stranded
and gets integrated into chromosome
• AAV is not currently known to cause disease and
consequently the virus causes a very mild immune
response.
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25. Cont’d…
C) Retroviruses
• The recombinant retroviruses have the ability to
integrate into the host genome.
• Can carry a DNA of small siz
D) Lentiviruses
• Subclass of Retroviruses.
• Are slowly dividing virus
• 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 via viral integrase enzyme.
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26. Cont’d…
E)Herpes simplex virus
• Viruses which have natural tendency to infect a
particular type of cell.
• The Herpes simplex virus is a human neurotropic
virus.
• This is mostly examined for gene transfer
in the nervous system.
Viral gene delivery has some limitations like
• Immune reaction
• Infection of normal cell
• Harmful mutation due to insertion of DNA to wrong
site etc.
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27. Non Viral Vector System
1.Spontaneous uptake by endocytosis complexed
with liposome.
2.Gene gun methods
3.Electroporation
4.Naked DNA injection
5.Polyplexes
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28. Liposomal method 28

29. Cont’d…
2.Gene gun methods
originally designed for plant transformation,
Is a device for injecting cells with genetic
material.
The payload is an elemental particle of a heavy
metal coated with plasmid DNA.
Gene guns have also been used to deliver DNA
vaccines.
DNA is introduced by accelerating the DNA-
Particle complex in partial vacuum.
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31. 3.Electroporation
Electroporation designates the use of short
high-voltage pulses to overcome the barrier of
the cell membrane.
can be used to load cells with a variety of
different molecules,like DNA transfer.
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32. 4. Naked DNA
• Direct introduction of pure DNA construct into target cell or
tissue by microinjection .
5. polyplexes
polyplexes, have the ability to protect the DNA
from undesirable degradation during the transfection process.
• Plasmid DNA can be covered with lipids in an organized
structure like a micelle or a liposome.
• Complexes of polymers with DNA are called polyplexes.
Limitation of non viral vector
 Non viral vectors have poor chromosomal integration
 Less effective invivo
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33. Benefits and Limitation of Gene Therapy
Some advantages of gene therapy include:
 Used to manage different inherited immune
deficiency disease like SCID,Sickle cell
disease,albinism etc
 Is used for discovery and development of new drugs.
Like anti virals
 Effective for prevention of disease
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34. Cont’d…
• Limitations of gene therapy includes:
 It is Hard to rapidly integrate therapeutic DNA into genome.
 Some vectors may not accommodate full length of genes.
 Immune hyper responsiveness(in vector or in target cells).
 Viral vectors may cause infection.
 If integration is not controlled oncogenes might be inserted
and cause cancer.
 Difficult to treat multigene disease e.g. cardiac disease,
diabetes etc.
 Ethical consideration for germ line gene therapy.
 Unaffordable
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35. THANK YOU!
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