GENE THERAPY

A Partial Fulfillment in Science, Technology and

Society I

Submitted by:

Sherylyn Gatil Paringit

Daniela Regina Colapo Gonzales

Lara Mae Maneja Latina

A.Y. 2019 – 2020

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Gene Therapy is an experimental technique for correcting defective genes that are
responsible for disease development. The most common form of gene therapy involves
inserting a normal gene to replace an abnormal gene. Other approaches used are:

 Replacing a mutated gene that causes disease with a healthy copy of the gene.
 Inactivating, or “knocking out”, a mutated gene that is functioning improperly.
 Introducing a new gene into the body to help fight a disease.

Researchers are studying a Gene Therapy for several diseases, such as:

 Severe combined immune deficiencies (SCID)

 Hemophilia
 Parkinson’s disease
 Cancer
 HIV

History and Development of Gene Therapy

 1960 – The concepts of Gene Therapy were introduced

 1970 – Friedmann and Roblin author of a paper in Science entitled “Gene Therapy
for Human Genetic Diseases?” cite the first attempt to perform Gene Therapy
 1990
o The first improved gene therapy case at the National Institute of Health,
United Kingdom. It was performed on a four-year-old girl named Ashanti Da
Silva. It was a treatment for a genetic defect that left her with an immune
system deficiency
o New gene therapy approach repairs errors in messenger RNA derived from
defective genes. This technique has the potential to treat the blood disorder
Thalassaemia, Cystic fibrosis, and some cancers
o Sickle cell disease is successfully treated in mice
 1992 – Doctor Caludio Bordignon working at the Vita-Salute San Raffaele
University in Milan, Italy performed the first procedure of gene therapy using a
hematopoietic stem cells as vectors to deliver genes intended to correct hereditary
diseases
 1999 – Death of Jesse Gelsinger in a gene-therapy experiment resulted in a
significant setback to gene therapy research in the US

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  2006 – Scientists at the National Institutes of Health in Bethesda, Maryland have
successfully treated metastatic melanoma in two patients. This study constitutes
one of the first demonstrations that gene therapy can be effective in treating cancer
 2007 – 2011 – Research is still ongoing and the number of diseases that has been
treated successfully by gene therapy increases
o Retinal Disease
o Color Blindness
o Adrenoleukodystrophy
 2011 – Medical community accepted that it can cure HIV, Gero Hutter has cured
a man from HIV using gene therapy

Types of Gene Therapy

Germ Line Gene Therapy is the process of genetically modifying sperm or egg cells to
create a new offspring. It is a type of gene therapy where new DNA is inserted into cells
using a vector, like a virus. The new DNA replaces only a faulty DNA to cure genetic
diseases. Some people view this type of therapy as unnatural and liken it to “playing God”.
While others have concerns about the technical aspects.

Somatic Gene Therapy is the transfer of genes into the somatic cells of the patient, such
as cells of the bone marrow, and hence the new DNA does not enter the eggs or sperm.

 Somatic cells are nonreproductive

 Somatic cell therapy is viewed as a more conservative, safer approach because it
affects only the targeted cells in the patient and is not passed on to future
generations.
o Problems of Somatic Cell Therapy:
 Effects are short-lived. Because the cells of most tissues ultimately
die and are replaced by new cells, repeated treatments over the
course of the individual’s life span are required to maintain the
therapeutic effect.
 Transporting the gene to the target cells or tissue

Regardless of these difficulties, somatic cell gene therapy is appropriate and acceptable
for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain
infectious diseases.

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 Types of Somatic Gene Therapy

 Ex Vivo – which means exterior (where cells are modified outside the body and
then transplanted back in again)
- This gene therapy involves the genetic modification of cells outside of
the body to produce therapeutic factors and their subsequent
transplantation back into patients

 In Vivo – which means interior (where genes are changed in cells still in the body)
- This gene transfer strategies administer the gene therapy vector either
directly to the target organ or deliver it via the vascular system into
vessels feeding that organ.
- Challenges that need to overcome for in vivo gene therapy strategies:
o Induction of immunity by the gene transfer vector
o Transport of the gene therapy vector to the targeted cells / organs
o Efficient binding of the vector to the cell
o Translocation of the genetic material to the nucleus
o Toxicity and immunity induced by expression of virus and / or
transgene peptides

Basic Process of Gene Therapy

A gene cannot be directly inserted into a person’s cell. It must be delivered to the cell
using a carrier, or vector. Vector systems can be divided into:

1. Viral Vectors
- A tool commonly used by molecular biologists to deliver genetic material
into cells
- Can be performed in vivo or in vitro
- Viruses have evolved specialized molecular mechanisms to efficiently
transport their genomes inside the cells they infect
- Delivery of genes by a virus is termed transduction and the infected cells
are described as transduced

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2. Non-Viral Vectors
- Methods of non-viral gene delivery have also been explored using
physical (carrier-free gene delivery) and chemical approaches (synthetic
vector-based gene delivery)
- Physical Methods – employ physical force that permeated the cell
membrane and facilitates intercellular gene transfer
o Needle Injection – the genetic material of interest is
administered through a needle carrying syringe into tissue or
systematic injection from a vessel
o Ballistic DNA – Other terms used are Bombardment, Micro
Projectile Gene Transfer or Gene Gun. This method was first
used as gene transfer technique to plants. The method is based
on the principle of delivering DNA coated heavy metal particles
by crossing target tissue at a certain speed.
o Electroporation – Other terms used are gene electro injection,
gene electro transfer, electrically mediated gene therapy, electro
gene transfer. Applying an electric field that is greater than the
membrane capacitance will cause charges of opposite polarity to
line up on either side of cell membrane thus performing a
potential difference at a specific point on the cell surface.
o Sonoporation – is a noninvasive site-specific technique which
utilizes ultrasound wave to temporarily permeabilize the cell
membrane to allow cellular uptake of DNA.
o Photoporation – utilizes single laser pulse to generate transient
pores on a cell membrane to allow DNA to enter the cell

o Magnetofection – is based on the hypothesis of magnetically

targeted drug delivery. The technique is based on coupling
therapeutic gene to magnet nanoparticle.
o Hydroporation – also called hydrodynamic gene transfer. The
technique uses hydrodynamic pressure to penetrate the cell
membrane.
o Mechanical Massage – is based on the hypothesis that
mechanical massage of liver generates transient membrane
defects for a few minutes, which facilitates plasmid DNA to enter
hepatic cells by diffusion.

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- Chemical Carriers – broadly classified into inorganic particles, lipid
based, polymer based, and peptide based.
o Inorganic particles – generally nanoparticles that can be
engineered by varying in size, shape and porosity in order to
escape from reticulo endothelial system or to protect an
entrapped molecule from degradation. Carbon nanotubes,
fullerenes, supra molecular system are most studied in this
category.
 Calcium phosphate – first particles to be used in this
system. It is biocompatible and biodegradable. Calcium
plays a vital role in endocytosis and has the advantage of
being readily absorbed and it poses high binding affinity.
 Silica – the major component of widely used materials like
sand and glass by humans. Its relative ease of
functionalizing makes its attractive to use as gene delivery
vehicle.
 Gold – modifying the surface of gold with DNA can be
used to transfect the cell by using photo thermal effect.
Thermal denaturation induces by photo thermal effect
helps to control the denaturation induced by photo thermal
effect helps to control the release of gene.
- Synthetic / Natural Biodegradable:
o Cationic Lipids – capable of delivering nucleic acids, including
genes, siRNA or antisense RNA into cells, thus

potentially resulting in their functional expression. These vectors

are considered as an alternative for virus-based delivery
systems, which may suffer from immunological and mutational
hazards.
o Lipid Nano Emulsion – is a dispersion of one immiscible liquid
in another stabilized by emulsifying agent. It is superior to
liposomes in scaling up, and stability, thus making it more serum
resistant.
o Solid Lipid Nanoparticles – developed to overcome the
limitations of other colloidal carriers, such as emulsions,
liposomes and polymeric nanoparticles because they have
advantages like good release profile and targeted drug delivery

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 with excellent physical stability. It is currently the choice of
delivery system for SiRNA
o Peptide Based – are considered advantageous over other non-
viral vectors in tight compact and protecting DNA, target specific
cell receptor, disrupting endosomal membrane and delivering
genetic cargo into nucleus.
o Polymer based vectors – Cationic polymers mix with DNA to
form nanosized complex called polyplexes. Polyplexes are more
stable than lipoplexes. Polymers are categorized into:
 Natural Polymers – proteins, peptides, polysaccharides
 Chitosan – a natural polymer based on cationic
polysaccharide. One of the most studied non-viral
vectors. It nontoxic even at high concentrations.
 Poly (DL-Lactide) (PLA) and Poly (DL-Lactide-
co-glycoside) (PLGA) – are biodegradable
polyesters undergo bulk hydrosis thus providing
sustained delivery. PLGA is approved by FDA as
vehicle for protein delivery.
 Synthetic Polymers – Polyethylenemine (PEI),
dendrimers, and polyphosphoesters
 Polyethylenimine (PEI) – considered as a gold
standard for in vivo and invitro gene transfer.
 Dendrimers – Dendrimer molecules are
symmetrical in size and shape with terminal group
functionality. It binds to genetic material when
positively charged peripheral groups interact with
nucleic acids in physiological pH due. Due to
nanometric size it can interact effectively with cell
membranes, organelles and proteins. The terminal
amino group and positive charge density determine
the toxicity profile.
 Polymethacrylate – are vinyl-based polymer able
to condense polynucleotides into nanometer size
particle. But the transfection is limited due to their
low ability to interact with membranes.

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Gene Therapy Successes

Successes represent a variety of approaches – different vectors, different target cell

populations, and both in vivo and ex vivo approaches – to treating a variety of disorders.

1. Immune deficiencies – Several inherited immune deficiencies have been treated

successfully with gene therapy. Most commonly, blood stem cells are removed
from patients, and retroviruses are used to deliver working copies of the defective
genes.
a. Severe Combined Immune Deficiency (SCID) – one of the first genetic
disorders to be treated successfully with gene therapy, proving that the
approach could work. However, the first clinical trial ended when the viral
vectors triggered leukemia in some patients.
b. Adenosine deaminase (ADA) deficiency – another inherited immune
disorder that has been successfully treated with gene therapy. In multiple
small trials, patient’s 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 most of the patients, immune function improved
to the point that they no longer needed injections of ADA enzyme.

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2. Hereditary blindness – gene therapies are being developed to treat several
different types of inherited blindness – especially degenerative forms, where
patients gradually lose the light-sensing cells in their eyes. Encouraging results
from animal models (especially mouse, rat and dog) show that gene therapy has
the potential to slow or even reverse vision loss.
3. Hemophilia – in a small trial, researchers successfully used an adeno-associated
viral vector to deliver a gene for Factor IX, the missing clotting protein, to liver cells.
After treatment, most of the patients made at least some Factor IX, and they had
fewer bleeding incidents.
4. Blood disease – in 2007, a patient received gene therapy for severe beta-
Thalassemia. Blood stem cells were taken from his bone marrow and treated with
a retrovirus to transfer a working copy of the beta-globin gene. The modified stem
cells were returned to his body, where they gave rise to healthy red blood cells.
Seven years later after the procedure, he was still doing well without blood
transfusions.

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5. Cancer – Several promising gene-therapy treatments are under development for
cancer.
a. A modified version of the herpes simplex 1 virus has been shown to be
effective against melanoma (a skin cancer) that has spread throughout the
body. T-VEC treatment 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. The virus is injected
directly into the patient’s tumors. It replicates inside the cancer cells until
they burst, releasing more viruses that can infect additional cancer cells.

6. Parkinson’s Disease – a small group of patients with advance Parkinson’s

disease were treated with a retroviral vector to intorduce 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 do. After treatment, all of the patients in the trial has
improved muscle control.

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Advantages of Gene Therapy

1. Gene therapy provides hope for those who may not have had any in the past.
2. Gene therapy could change the perspectives that people have about disease.
3. Gene therapy could offer the potential of new discoveries.
4. Gene therapy coul be used in different ways to improve life.
5. Gene therapy does not just apply to human treatment options.
6. Gene therapy allows us to use technology to improve the quality of life for people.
7. Gene therapy allows us to treat the “untreatable” diseases.

Disadvantages of Gene Therapy

1. Gene therapy does not have a reliable delivery method.

2. Gene therapy is a expensive procedure.
3. Gene therapy requires ongoing treatments to be effective.
4. Gene therapy may not be ablr to adapt to a changing world.
5. Gene therapy might only delay the inevitable.
6. Gene therapy will shift society toward new polarization.
7. Gene therapy could change the way we think about competition.

References

http://www.genetherapynet.com/types-of-gene-therapy.html

https://www.sciencedirect.com/topics/neuroscience/ex-vivo-gene-transfer

https://www.sciencedirect.com/topics/neuroscience/in-vivo-gene-transfer

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347098/

https://link.springer.com/article/10.1007/s00249-006-0092-4

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4616893/

https://learn.genetics.utah.edu/content/genetherapy/success/

https://connectusfund.org/14-advantages-and-disadvantages-of-gene-therapy

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