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
6.
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
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?
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?
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. AdenovirusesWild 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.
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.
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.
Hinweis der Redaktion
Is the most common approach
The abnormal gene would be swapped by homologous recombination
Would cause a return to normal function
Control expression of genes. Similar to epistasis, when one gene affects the expression of another gene.
A vector is a carrier molecule, usually a virus
The target cells are usually in the liver or lung