Gene Therapy

Dr. Anum Gul

 Gene Therapy and its Classification
The gene therapy can be classified
into two major types:

⮚Somatic gene therapy

⮚Germline gene therapy

Somatic Gene Therapy
Somatic cell gene therapy involves the placement of a human gene into a living person's somatic
cells (cells that do not produce the eggs and sperm that in turn produce the next generation).

The therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte.
Any such modifications affect the individual patient only and are not inherited by offspring.

By using somatic gene therapy, somatic cells can be treated by inserting a vector loaded with the
correct gene into a person’s body.

Gene therapy, in its present stage only treats somatic cells in humans.
Somatic Gene Therapy
Affects only the targeted cells in the patient.

Short-lived because the cells of most tissues ultimately die and are replaced by
new cells.

Transporting the gene to the target cells or tissues is also problematic.

Appropriate and acceptable for many disorders, including cystic fibrosis,

muscular dystrophy, cancer, and certain infectious diseases.
Ex vivo & In vivo Somatic Gene Therapy
Germ Line Gene Therapy
⮚ Germline gene therapy is when DNA is transferred into the cells that produce
reproductive cells, eggs or sperm, in the body.
⮚ Potential for offering a permanent therapeutic effect for all who inherit the
target gene.
⮚ Possibility of eliminating some diseases from a particular family.
⮚ Also raises controversy:
▪ Some people view this type of therapy as unnatural and liken it to "playing
God”.
▪ Others have concerns about the technical aspects.
Importance of Vectors in Gene Therapy
Gene therapy utilizes the delivery of DNA into cells, which can be accomplished
by a number of methods.

The two major classes of methods:

⮚ Viral Vector

⮚ Non-viral Vector
Virus
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.
Types of Virus
Many gene therapy clinical trials rely on retroviruses or adenoviruses to deliver
the desired gene.

Other viruses used as vectors include adeno-associated viruses, lentiviruses, pox

viruses, alphaviruses, and herpes viruses.

Differ in how well they transfer genes to the cells they recognize and are able to
infect, and whether they alter the cell’s DNA permanently or temporarily.
Properties of a Viral Vector
Safety: Although viral vectors are occasionally created from pathogenic viruses,
they are modified in such a way as to minimize the risk of handling them.

This usually involves the deletion of a part of the viral genome critical for viral
replication. Such a virus can efficiently infect cells but, once the infection has taken
place, requires a helper virus to provide the missing proteins for production of new
virions.

Low toxicity: The viral vector should have a minimal effect on the physiology of
the cell it infects.
Properties of a Viral Vector
Stability: Some viruses are genetically unstable and can rapidly rearrange their
genomes.

Cell type specificity: Most viral vectors are engineered to infect a wide a range
of cell types as possible. However, sometimes the opposite is preferred. The viral
receptor can be modified to target the virus to a specific kind of cell.

Identification: Viral vectors are often given certain genes that help identify
which cells took up the viral genes. These genes are called markers. A common
marker is resistance to a certain antibiotic.
Retroviruses
Retroviruses are one of the mainstays of current gene therapy approaches.

The recombinant retroviruses such as the Moloney murine leukemia virus (MMLV)
have the ability to integrate into the host genome in a stable fashion.

They contain a reverse transcriptase to make a DNA copy of the RNA genome, and
an integrase that allows integration into the host genome.

They have been used in a number of FDA-approved clinical trials such as the SCID.
Retroviruses
Retroviral vectors can either be replication-competent or replication-defective.

Replication-defective vectors are the most common choice in studies because

the viruses are defective for one or more functions that are essential for viral
genome replication or synthesis and assembly of viral particles.

These virus are capable of infecting their target cells and delivering their viral
payload, but then fail to continue the typical lytic pathway that leads to cell lysis
and death.
Retroviruses
Replication-competent viral vectors contain all necessary genes for virion
synthesis and continue to propagate themselves once infection occurs.

Because the viral genome for these vectors is much lengthier, the length of the
actual inserted gene of interest is limited compared to the possible length of the
insert for replication-defective vectors.
Retroviruses Drawback
The primary drawback to use of retroviruses such as the Moloney retrovirus
involves the requirement for cells to be actively dividing for transduction.

As a result, cells such as neurons are very resistant to infection and transduction by
retroviruses.

Moreover, severe oncogenic retroviruses often arise as the result of attaining

sequences derived from cellular proto-oncogenes provided an additional stimulus.
Lentiviruses
Lentiviruses are a subclass of Retroviruses.

They are sometimes used as vectors for gene therapy thanks to their ability to
integrate into the genome of non-dividing cells, which is the unique feature of
Lentiviruses as other Retroviruses can infect only dividing cells.

Studies have shown that lentivirus vectors have a lower tendency to integrate in
places that potentially cause cancer than retroviral vectors.
Adenoviruses
Adenoviruses are viruses that contain their genetic material in the form of DNA
when these viruses infect host cells, they introduce their DNA into the host cells
for replications.

They infect non-dividing and dividing cells of different types.

Genetic material of the adenoviruses is not integrated into the host cells genetic
material and the adenovirus DNA molecule is left free in the nucleus of the host
cell (episomal integration).
 Gene Therapy Using an Adenovirus

The genetic messages in this extra DNA molecule

are transcribed just like any other gene and the only
difference is that these extra DNA does not get
replicated when the host cell is about to undergo
cell division.
Therefore, the progenies of that cell will not have
the extra DNA in their nucleus.
Adeno-Associated Viruses
Adeno-associated viruses (AAV) belong to the parvovirus family—small viruses
with a genome of single-stranded DNA.

AAV can infect both dividing and non-dividing cells and may incorporate its
genome into that of the host cell.

Moreover, AAV mostly stays as episomal (replicating without incorporation into

the chromosome).
 Adeno-Associated Viruses
⮚ AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus.

⮚ In non-dividing cells, these concatemers remain intact for the life of the host cell.

⮚ In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is
not replicated along with the host cell DNA

⮚ There are a few disadvantages of using AAV in gene therapy as the DNA of AAV is
small and it is difficult to carry and to produce DNA.
Adeno-Associated Viruses
Nevertheless, this type of virus is being employed because it is nonpathogenic in
function.

Furthermore, in contrast to adenoviruses, most patients treated with AAV may

not build an immune response to remove the virus.

Several clinical trials with AAV are in progress, primarily trying to treat muscle
and eye diseases, the two tissues where the virus seems to be particularly useful.
Hybrids
Hybrid vectors are vector viruses that are genetically engineered to have qualities of more than
one vector.

Viruses are altered to avoid the shortcomings of typical viral vectors, which may have limited
loading capacity, immunogenicity, genotoxicity, and fail to support long-term adequate
transgenic expression.

Through the replacement of undesirable elements with desired abilities, hybrid vectors may in
the future outperform standard transfection vectors in terms of safety and therapeutic
efficiency.
Non-Viral Mediated Gene Therapy
Besides virus-mediated gene-delivery systems, there are several non-viral options for gene
delivery.

It has been reported that non-viral methods have certain advantages over viral methods, with a
simple way to produce in large scale and low host immunogenicity.

One of the major reasons why scientists were not serious about using non-viral approach of gene
therapy was because of its low levels of transfection and expression of the gene, nevertheless,
recent developments in vector technology have yielded molecules and techniques with
transfection efficiencies similar to those of viruses.
Non-Viral Mediated Gene Therapy
Physical Methods
• Electroporation
• Gene gun
• Sonoporation

Chemical Method
• Lipoplexes
• Polyplexes
Physical Methods
Electroporation

In this method, DNA molecules pass through cell membrane by using short pulses of
high voltage and it has been suggested that this shock is thought to cause temporary
formation of pores in the cell membrane, allowing DNA molecules to pass through.

It has been suggested that electroporation is generally efficient and works across a
broad range of cell types; nonetheless, a high rate of cell death following
electroporation has been reported which has limited its application.
 Physical Methods
Gene Gun

In this technique, DNA is coated with gold particles and loaded into a device which
generates a force to achieve penetration of DNA–gold into the cells.

Sonoporation

It has been reported that DNA can be delivered into cells by using ultrasonic
frequencies and the process of acoustic vibration is believed to disturb the cell
membrane and permit DNA to move into the cells
Lipoplexes
To make DNA delivery most effective, DNA molecules must be protected from any structural
and functional damage.

In the beginning, anionic and neutral lipids were used to protect DNA from damage by
construction of lipoplexes and, consequently, cationic lipids are first used to condense
negatively charged DNA molecules so as to facilitate the encapsulation of DNA into
liposomes.

The use of cationic lipids significantly enhanced the stability of lipoplexes and also as a result
of their charge, cationic liposomes network with the cell membrane.
Lipoplexes
Furthermore, endocytosis is generally believed as the major route by which cells
uptake lipoplexes.

Endosomes are formed as the consequences of endocytosis.

Nevertheless, if genes cannot be released into cytoplasm by breaking the

membrane of endosome, they will be sent to lysosomes where all DNA will be
broken before they attain their functions.
Polyplexes
In addition to lipoplexes, there are complexes that can also be used in gene transfection,
such as polyplexes.

Most polyplexes consist of cationic polymers and their production is regulated by ionic
interactions.

The mechanism by which polyplexes, particularly those displaying a high proton

buffering capacity, release their nucleic acid cargo from the endosome, is thought to rely
on a so-called “proton sponge effect”, in essence an osmotically induced rupturing of the
endosomal membrane.
Proton Sponge Effect
Lipoplexes & Polyplexes
Hybrid methods
In view of having various shortcomings in gene transfer technologies, there have
been efforts to make hybrid technology that combines two or more techniques
together.

It has been reported that virosomes are a hybrid that is developed by combining
liposomes with an inactivated HIV or influenza virus. This hybrid method has
shown to have more efficient gene transfer in respiratory epithelial cells than
either by viral or by liposomal methods alone.
Challenges of Non-viral Mediated Gene Therapy
⮚Low transfection efficiency of target cells, especially in vivo, and to the transient nature of
transgene expression.

⮚Not particularly suitable for the treatment of pathological conditions that require long-term
transgene expression, such as neurodegenerative disorders and inherited or acquired genetic
diseases.

⮚Optimal application is in immunotherapy for cancer and infectious diseases, as a transient

expression of the transgene might be sufficient to trigger effective and durable host immune
responses.
Applications of Gene Therapy
First approved gene therapy case took place in 1999, where gene therapy was
performed on a four-year-old girl named Ashanti DeSilva as she was suffering
from immune system deficiency.

The gene therapy was conducted, though benefits were only temporary, but it
created lots of interest among the scientific community around the world.
Severe Combined Immune Deficiency (SCID)
SCID was one of the first genetic disorders to be treated successfully with gene
therapy, proving that the approach could work.

However, the first clinical trials ended when the viral vector triggered leukemia
(a type of blood cancer) in some patients.

Since then, researchers have begun trials with new, safer viral vectors that are
much less likely to cause cancer.
Adenosine deaminase (ADA) deficiency
ADA deficiency is another inherited immune disorder that has been successfully treated
with gene therapy.

In multiple small trials, patients' blood stem cells were removed, treated with a retroviral
vector to deliver a functional copy of the ADA gene, and then returned to the patients.

For the majority of patients in these trials, immune function improved to the point that
they no longer needed injections of ADA enzyme. Importantly, none of them developed
leukemia.
Hereditary blindness
In one small trial of patients with a form of degenerative blindness called LCA
(Leber congenital amaurosis), gene therapy greatly improved vision for at least a
few years.

However, the treatment did not stop the retina from continuing to degenerate. In
another trial, 6 out of 9 patients with the degenerative disease choroideremia had
improved vision after a virus was used to deliver a functional REP1 gene.
 Cancer
Several promising gene-therapy treatments are under development for cancer.

One, a modified version of the herpes simplex 1 virus (which normally causes cold sores) has been
shown to be effective against melanoma (a skin cancer) that has spread throughout the body. The
treatment, called Talimogene laherparepvec (T-VEC), uses a virus that has been modified so that it will

(1) not cause cold sores;

(2) kill only cancer cells, not healthy ones; and

(3) make signals that attract the patient's own immune cells, helping them learn to recognize and fight
cancer cells throughout the body.
Cancer
Parkinson's disease
⮚Patients with Parkinson's disease gradually lose cells in the brain that produce the signaling
molecule dopamine. As the disease advances, patients lose the ability to control their
movements.

⮚A small group of patients with advanced Parkinson's disease were treated with a lentiviral
vector (a subclass of retrovirus) to introduce three genes into cells in a small area of the brain.

⮚These genes gave cells that don't normally make dopamine the ability to do so. After
treatment, all of the patients in the trial had improved muscle control.
Advantages of Gene Therapy
• Give a chance of a normal life to baby born with genetic disease.
• Give hope of healthy life to cancer patient.
• For certain disease that do not have any cure except gene therapy, it could save
many lives.
 Disadvantages of Gene Therapy

⮚ The genetic testing, screening and research in finding the availability of certain
gene is very controversial.
⮚ May increase rate of abortion if prenatal test regarding baby with genetic
disease is done.
⮚ The cost is very high, and the patient might need an insurance to cover the
treatment.
Technical and Regulatory Issues with Gene Therapy

⮚ Short-lived nature of gene therapy

⮚ Immune response

⮚ Problems with viral vectors

⮚ Multigene disorders
Ethical Questions Surrounding Gene Therapy
⮚ How can “good” and “bad” uses of gene therapy be distinguished?
⮚ Who decides which traits are normal and which constitute a disability or
disorder?
⮚ Will the therapy only benefit the wealthy due to its high cost?
⮚ Could the widespread use of gene therapy make the 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?