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Tina Rostinawati, M.Si

Gene therapy :is the insertion of genes

into individuals cells and tissue to treat
the disease, and heredity diseases in

In the 1980s ,human genes to be
sequence and clone (insulin)
On September 1990, the first approved
gene therapy procedur on four-year old
Ashanti DeSilva (SCID=Severe Combined

In Ashantis gene therapy procedur,

doctors removed white blood cell from the
childs body, let the cells grown in the lab,
inserted the missing gene into the cell,
and then infused the genetically modified
blood cells back into the patients

Types of gene therapy

1. Ex vivo : where cells are modified
outside the body and then transplanted
back in again
2. In vivo : where genes are changed in
cells still in the body

A normal gene may be inserted into a
nonspecific location within the genome to
replace a nonfunctional gene. This
approach is most common
An abnormal gene could be swapped for
normal gene through homologous

The abnormal gene could be repaired

through reverse mutation, which returns
the gene to its normal function
The regulation (the degree to which a
gene is turned on or off) of particular gene
could be altered

Vector in gene therapy

1. Viral Vector
-Adeno-associated viruses
-Envelope protein pseudotyping of viral

2. Non-viral methods
Naked DNA
Lipoplexe and polyplexes
3.Hybrid methods

The Ideal Vector for Gene

High concentration of virus allowing many cells to
be infected or transduced
Convenience and reproducibility of production
Ability to transduce dividing and non-dividing cells
Ability to integrate into a site-specific location in the
host chromosome, or to be successfully maintained
as stable episome
A transcriptional unit that can respond to
manipulation of its regulatory elements
Ability to target the desired type of cell
No components that elicit an immune response

All viruses attack their hosts and introduce
their genetic material into the host cell as
part of their replication cycle
This genetic material contains basic
instruction of how to produce more
copies of these viruses.
Molecular biologists realized that viruses
like this could be used as vehicles to carry
good genes into a human cell.

Removing the genes in the virus that

cause disease
Replacing those genes with genes
encoding the desired effect

The Genetic material is in the form RNA
The Genetic material of their hosts is in the form
When a retrovirus infects a host cell, it will
introduce its RNA together with some enzymes
into the cell
This RNA molecule from the retrovirus must
produce a DNA copy from its RNA molecule
before it can be considered for part of the
genetic material of the host cell

The process of producing a DNA copy

from an RNA molecule is termed reverse
It is carried out by one of the enzymes
carried in the virus, called reverse
After this DNA copy is produced and is
free in the nucleus of the host cell, it must
be incorporated into the genome of the
host cell

This process is done by another enzyme

carried in the virus called integrase
The problems of gene therapy using retrovirus
The integrase enzyme can insert the genetic material of
the virus in any arbitary position in the genome of the
If genetic material happens to be inserted in the middle
of one of the original genes of the host cell, this gene will
be disrupted (insertional mutagenesis)
If the gene happens to be one regulating cell division,
uncontrolled division (i.e., cancer) can occur.
This problem has recently begun to be addressed by
utilizing zinc finger nucleases or by including certain
sequence such the beta-globin locus control region

The genetic material is in the form ds DNA
Causing respiratory (especially the common
cold), intestinal, and eye infections in humans
When these viruse infect a host cell, they
introduce their DNA molecule into the host.
The genetic material of the adenoviruses is not
incorporated into the host cells genetic material.
The DNA molecule is left free in the nucleus of
the host cell

These extra genes are not replicated

when the cell is about to undergo cell
Treatment with adenovirus will require
This vector system has shown real
promise in treating cancer

Adeno-associated viruses (AAV)

Small virus with genome of ss DNA.
Inserting genetic material at a specific site on
chromosome 19
Most people treated with AAV will not build an
immune response
AAV vector are used to deliver genes to the
brain (can infect non-dividing((quiescent)) cells,
such as neurons in which their genomes be
expressed for a long time

A few disadvantages to using AAV

The small amount of DNA it can carry (low
capacity) and difficulty in producing it
In recent human trials, CD8+ immune cells
have recognised the AAV infected cells,
this action appears to be triggered by part
of the capsid of outer coat of the type 2

Envelope protein pseudotyping of viral

The viral vectors described above have natural
host cell population that they infect most efficiently.
Retrovirus have limited natural host cell range, and
adenovirus or AAV are able to infect a relatively
broader range
many vectors have been developed in which the
endogenous viral envelope protein have been
replaced by either envelope proteins from other
viruses, or by chimeric protein.

Such chimera would consist of these parts of the

viral protein necessary for incorporation into the
virion as well as sequences meant to interact
with specific host cell proteins.
Viruses in which the envelope protein have been
replaced as described are referred to as
pseudotype viruses
Example : The lentivirus Simian
Immunodeficiency virus coated with the
envelope proteins, G-protein, from Vesicular
Stomatitus virus. This vector is referred to as
V VG-pseudotyped lentivirus infects an almost
universal set of the cell .

Non-viral methods
Non-viral methods present certain
advantages over viral methods
simple large scale production
low host immunogenecity
Low levels of transfection and expression of
the gene held non-viral methods at a

Naked DNA
Simplest method of non-viral transfection
Delivery of the naked DNA such as
electroporation and the use of a gene
gun, which shoots DNA coated particles
into the cell using high pressure gas

Oligodeoxynucleotides (antisense mRNA

and siRNA)

To inactivate the genes involved in the disease

Using antisense mRNA specific to the target
gene to disrupt the transcription of the faulty
Using small catalytic molecules of RNA called
siRNA to cleave specific unique sequences in
the mRNA transcript of the faulty gene,
disrupting translation of the faulty mRNA, and
therefore expression of the gene

Lipoplexes and polyplexes

The DNA must be protected from damage
and its entry into ther cell must be
Plasmid DNA that covered with lipids in an
organized structure like a micelle or a

Three types of lipoplexes

anionic (negatively charged)
neutral or cationic (positively charged)
Anionic and neutral are compatible with
body fluids
Cationic lipids naturally complex with the
negatively charged DNA, interact with the
cell membrane, enocytosis of the lipoplex
occurs and the DNA is released into the

The cationic lipids also protect against

degradation of the DNA by the cell
The most common use of lipoplexes has been in
gene transfer into cancer cell, where the
supplied genes have activated tumor suppressor
control in the cell and decrease the activity of
Complexes of polymers with DNA
Polyplexes cannot release their DNA load into
the cytoplasm
Co-transfection with endosome-lytic agents (to
lyse the endosome that ia made during
endocytosis, the process by which the
polyplexes enters the cell


Hybrid methods
Hybrid methods developed that
combine two or more techniques.
Virosome are one example; they
combine liposomes with an inactivated
HIV or influenza virus.

Recent developments in gene therapy

Scinentist at the National Institutes of Health
have successfully treated metastatic melanoma
in two patients using killer T cells genetically
retargeted to attack the cancer cellc (October
HSR-TIGET group utilized a newly uncovered
network of genes regulated by molecules known
as microRNA that selectively turn off the identity
of their therapeutic gene in cells of the immune
system and prevent the gene from being found
and destroyed (In May 2006)

University of California, research team

gets genes into the brain using liposome
coated in a polymer called PEG. This
method has potential for treating
Parkinsons disease. (March 20, 2003)

Problems and ethics

Short-lived nature of gene therapy, the
therapeutic DNA introduced into target
cells must remain functional and the cells
containing the therapeutic DNA must be
long-lived and stable
Immune respone- Anytime a foreign object
is introduced into human tissue, the
immune system is designed to attack the

Problem with viral vectors-Viruses, while the

carrier of choice in most gene therapy studies,
present a variety of potential problems to the
patienttoxicity, immune and inflammatory
respones, and gene control and targetting
tissues.The viral vector . Once inside the
patient,may recover is ability to cause disease
Multigenes or multifactorial disorders such as
heart disease, Alzheimers disease, and
diabetes would be especially difficult to treat
effectively using gene therapy
Chance of inducing a tumor-If the DNA is
integrated in the wrong place in the genome, for
example in tumor suppressor gene,it could
induce a tumor