Gene therapy involves techniques that modify or manipulate genes to treat or prevent diseases. The first gene therapy treatment occurred in 1990 for severe combined immunodeficiency. There are four main approaches to gene therapy: inserting a normal gene to compensate for a defective one, replacing an abnormal gene with a normal one, repairing an abnormal gene, or altering gene regulation. Viruses are commonly used as vectors to deliver therapeutic genes into target cells, with retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses being some of the most widely used viral vectors, each with advantages and limitations.

1. By
Farshid Mokhberi
Shahid Behehsti University Of Medical Sciences and
Health Service

2. 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

3. 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

4. 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 

5. How It Works
 A vector delivers the therapeutic gene into a patient’s
target cell
 The target cells become infected with the 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

7. Principle of gene therapy
 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.

8. Approaches of gene therapy
 1. Gene modification
 Replacement therapy
 Corrective Gene therapy
 2. Gene transfer
 Physical
 Chemical
 Biological
 3. Gene transfer in specific cell line
 Somatic gene therapy
 Germ line gene therapy

9. Vectors in gene therapy
Some of the different types of viruses used as gene
therapy vectors:
Retroviruses
Adenoviruses
Adeno -associated viruses
Herpes simplex viruses

10. Viral methods for gene therapy

11. Non-viral methods
 Injection of Naked DNA
 Physical & chemical Methods to Enhance
Delivery
 Electroporation
 Gene Gun
 Sonoporation
 Magnetofection
 Oligonucleotides

12. Electroporation
 Is a method that uses short pulses of high voltage to carry
DNA across the cell membrane.
 This shock is thought to cause temporary formation of
pores in the cell membrane, allowing DNA molecules to
pass through.
 Electroporation is generally efficient and works across a
broad range of cell types.
 However, a high rate of cell death following
electroporation has limited its use, including clinical
applications.

13. Gene Gun
 DNA is coated with gold particles and loaded into a
device which generates a force to achieve penetration
of DNA/gold into the cells.
 Example: If the DNA is integrated in the wrong place
in the genome, for example in a tumor suppressor
gene, it could induce a tumor.
 This has occurred in clinical trials for X-linked severe
combined immunodeficiency (X-SCID) patients.

14. 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?

15. Delivery of gene by direct and cell
based methods

16. Advantages of gene therapy
 In case of ‘silence’ a gene. In the case of someone with
HIV, which had not yet developed into AIDS, scientists
could save them the pain and suffering of the disease by
using gene therapy to ‘silence’ the disease before its onset.
 Gene therapy has the potential to eliminate and prevent
hereditary diseases such as cystic fibrosis and is a possible
cure for heart disease, AIDS and cancer.
 These sceptics would almost certainly choose gene
therapy, especially if it was the last hope for them or one
of their loved ones – as is the case for many gene therapy
patients.

17. Disadvantages of Gene Therapy
 Short-lived nature of gene therapy.
 Immune response - Genes injected with a virus may
trigger an immune response against the virus.
Problems with viral vectors (once inside the patient,
the viral vector could recover its ability to cause
disease).
 Multigene disorders - The genetic material might not
get into the right cell, or the right place in the cell’s
DNA.

18. Ethical issues surrounding gene
therapy
 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?

19. Application of gene therapy
Parkinson’s diseases
Alzheimer’s disease
Cyctic fibrosis
Diabetic Neuropathy

20. Viral vectors
The use of viral vectors as a tool for clinical gene
therapy did not emerge until the 1980s.
Mammals have equally evolved highly complex
mechanisms to protect themselves against invading
pathogens such as viral gene transfer vectors.

21. 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

22. Remember!
The success of in vivo gene therapy not only depends
on the ability to control the immune response toward
the input vector, but also to the therapeutic transgene.

23. 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

24. Retroviruses
 Retroviruses were the first type of vector ever used for
gene therapy and are now the second most common
vector used in clinical trials.
 All retroviruses integrate their DNA into the host
genome, leading to long-term expression of the target
gene.
 This useful trait is also the major problem with
retrovirus vectors.

25. problem with retrovirus vectors
 Insertion of new DNA into the middle of an existing
open reading frame would disrupt function.
 Insertion of new DNA with viral promoters could
induce transcription of nearby protooncogenes.
 Another problem is that some retroviruses can only
infect dividing cells.

26. How to solve their problem
Performing the transduction in vitro and screening
for tumor cells before re-injection can help with these
issues,
No one plasmid contains all the genes to produce the
vector, as each one only produces a single component
of the virus.
no plasmid contains viral replication genes, so the
final vector is not capable of selfreplication.

27. Lentiviruses
They are also capable of targeting non-dividing cells.
There is some concern that lentiviruses derived from
HIV could undergo homologous recombination wild
type virus.

28. 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

29. AdenovirusesWild type adenoviruses are common, being responsible
for roughly 10% of upper respiratory infections.
their large DNA capacity has made them very popular
vectors.
Accounting for almost a quarter of all clinical trials.

30. Adenoviruses
Adenoviruses do not integrate their DNA,
instead they become episomes.
This removes any possibility of inducing
Cancer.
the new DNA will eventually be degraded, requiring
re-injection.

31. Problems
Most people have antibodies for them.
If the adenovirus dosage is too high this can cause a
severe immune reaction.
Short-term immunosuppression can help, but can
lead to opportunistic infections

32. Tactic to solve the problems
Some promise is to use different adenovirus
serotypes, or replacing the virus antigens with those
of a different serotype.
changing the viral promoters are retained in the
vector can reduce the immune response.

33. adeno-associated virus (AAV)AAV is a small, nonenveloped single-stranded DNA
virus.
This virus infects humans, and is from the Parvoviridae
family.
AAV has a high safety profile (because it does not induce
a large inflammatory response).

34. adeno-associated virus (AAV)
Can transduce (transfer into a cell) a wide variety of
tissues and cells in vivo.
long-term expression can be achieved without
integration.

35. adeno-associated virus (AAV)
Is growing in popularity due to its lack of
pathogenicity and immune response
AAV does not cause any disease and cannot replicate
without coinfection from an adenovirus.
Without this helper virus it integrates into the host
genome at a site that does not seem to be tumorigenic
in humans.

36. adeno-associated virus
It provokes almost no immune response in most.

37. AAV vector problem
Its small size limits the length of the gene that can be
inserted to under 5 kb.
This excludes some genes, but is enough for other
useful ones.

38. herpes simplex virus (HSV)
HSV genome is large, measuring 152 kb.
It is possible to insert additional genes of ~10 kb in
size into the intact viral genome.
There are three main classes of HSV-1 genes, namely
immediate-early (IE or a) genes, the early (E or b)
genes and the late (L or g) genes.

39. herpes simplex virus (HSV)
After various non-essential DNA sequences have been
removed it is possible to insert or ‘package’ ~30 kb of
foreign genetic material into the virion.
Can live in neurons in a latent state that does not
appear to affect normal cellular physiology
This has sparked interest in this virus as a potential
vector in the treatment of neurological disorders.

40. HSV vector problem
Damaging effects
Long-term gene expression in HSV vectors

41. HSV vector problem
The direct introduction of HSV into the brain as
would be required for testing genes or in gene therapy
procedures will result in a lethal encephalitis due to
viral replication.
During the onset of latent infection the virus shut
down

42. HSV vector problem
Shut down of gene expression also occurs for any
exogenous genes, other virus promoters such as the
immediate early promoter of cytomegalovirus or a
variety of cellular promoters.
This results in expression of the foreign gene being
observed for only a few days at the most.