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1. Department of Zoology
Name: Muhammad Mohsin
Roll no: 851
MOLECULAR GENETICS
ASSIGNMENT
SUBMITTED TO
Madam Bushra
Topic: Gene Therapy
 Introduction
 Approaches
 Vectors in gene therapy
 Physical methods
 Chemical methods
 Non -viral method
 Types
 Advantages and disadvantages

2. Gene therapy.
The medical approach that involves the modification or replacing of defective genes in a
person’s cells to prevent genetic disorders and diseases. Manipulating of the non-
functional or the malfunctional genes is termed as the gene therapy.
This technique has the ability to treat those diseases that cannot be treated with
conventional medicine. The first successful gene therapy clinical trial was conducted by a
team of researchers led by Dr.W. French Anderson in 1990 that aimed to treat a young
boy named Ashanti DeSilva who had a genetic disorder named as severe combined
immunodeficiency (SCID). They use a retroviral vector to introduce the missing gene in
the patient blood stem cells. Promising results have been achieved in case of various
genetic diseases including blood disorders, immune deficiency, metabolic disorders, and
various types of cancers.
Approaches.
 Gene replacement therapy: This approach involves the introduction of a
functional copy of a defective gene into patient cell. It aims to restore the missing
or faulty gene.
 Gene addition therapy: In case where the missing or nonfunctional protein is
causing the disease, an additional copy of the correct gene is introduced.
 Adjustment of the gene expression of the genes: by editing of the specific genes
their expression can be controlled.
Vectors in gene therapy.
1. Retroviruses.
Retroviruses are a type of RNA virus that can reverse transcribe their RNA
genome into DNA and integrate into host cell’s DNA. This feature makes them
useful in gene therapy where they can deliver therapeutic genes into cells for
treatment of genetic disorders. The problem while using the retroviruses in gene
therapy is that the integrase enzyme can insert the viral genome into the host cell
that can lead to the insertional mutation. If
this insertion takes place at the time of cell
division than it can cause to uncontrolled cell
division that can cause cancer. Zinc finger
nucleases are type of engineered DNA
cutting enzymes used in gene therapy to
precisely modify an organism’s genome.
2. Adenoviruses.
This class of viruses have a double stranded DNA and they cause various
infectious diseases in human including respiratory, intestinal and ocular infections

3. When they infect a cell, they insert their DNA into
the host. However, their DNA is not inserted into the
organism’s genome. In case of adenoviruses the
when the host cell is divided the foreign genes are
not replicated and the cells generated by division do
not have any additional genes. So, using the
adenoviruses for treatment in gene therapy the
growing cell population requires re injection.
3. Adeno associated viruses.
Adeno associated viruses is a small class of
viruses that cause diseases in human. They have gained
significant importance due to their potential use as gene therapy vectors. Using AAVs in
gene therapy various therapeutical genes are
transferred for the aim of treatment of various diseases.
Benefits of using the AAVs for the treatment is the non-
pathogenicity of the virus for human in the absence of
their helper viruses such as adenoviruses and the herpes
viruses. They have the ability to transfer their genetic
material specifically at the chromosome 19.
4. Herpes simplex viruses.
These viruses have a double stranded DNA and they infect the neural cells.
Herpes simplex virus is a neurotropic virus that is mainly used to transfer the gene in the
nervous system. HSV-1 is commonly used for the gene therapy in it’s mutant form as it is
unable to replicate.
Physical methods
i. Electroporation.
Electroporation is a technique used in gene therapy to introduce genetic material
into cells. It involves applying short bursts of electrical pulses to create temporary
pores in the cell membrane, allowing DNA and other molecules to enter. It’s
valuable tool in the field of gene therapy due to its efficiency in delivering the
gene. However high rate of cell death has limited its clinical use.
ii. Gene gun.
The gene gun also known as biolistic particle delivery system is a technique used
to introduce foreign genetic material into cell or tissues. In this process gene is
coated with gold particles and then placed inside a device which provide the

4. required force to enter the cell. This method has been used in the clinical trials of
treatment of the patients having X-linked severe immunodeficiency (X-SCID).
However, it’s important to note that gene gun has been a valuable tool, it has
certain limitations such as potential cell damage and variable efficiency.
iii. Sonoporation.
It is an ultrasound mediated technique that use ultrasound waves to temporarily
create pores in cell membrane, allowing the uptake of the genetic material.
Sonoporation is considered as a noninvasive technique and offer advantages in
terms of cell viability compared to other methods that offer more damage to the
cell.
iv. Magnetofection.
In this technique DNA is complexed with magnetic particles and a magnet is placed
under cellular culture so that the DNA can be exposed to the one cell layer
compound. This method combines the principle of magnetic field and
nanoparticles to enhance the uptake of
substance into the cell, making it particularly
useful in gene therapy. The gene is extracted
from the magnetic particles by the
intermolecular restriction enzymes.
Magnetofection has the limitations such as the
toxicity of nano particles and need for
optimization to ensure the efficient delivery.
Chemical methods.
I. Oligonucleotides.
Synthetic oligonucleotides are used in the inactivation of the undesirable
genes that are causing the genetic disorders. By using the specific antisense
of the target gene, the transcription of the specific gene is stopped. Small
interfering RNAs (siRNA) are also used that breakdown the sequence of the
gene mRNA thus preventing its translation i.e., it’s expression.
II. Lipoplex and polyplex.
The combination of the DNA and polymers is called as the polyplex. The
polymers often used are cationic in nature so that their positive charge
interact with the negatively charged genetic material. Lipoplex are also
used as vectors and are prepared by the DNA and the lipids. These are
anionic or neutral in nature. The lipid fuse with the cell membrane and
release the genetic material in the cell.

5. III. Dendrimers
Dendrimers are the spherical branched macromolecules. Their surface
can be charged by various methods and the properties of the final structure
depends on the charged surface. The nucleic acid linkage is formed with the
cationic dendrimer which is transferred into cell by endocytosis.
Non-viral methods
A. Ormasil.
Ormasil is the modified silica or organic silicates and are used as a non-viral
method in genetic engineering. Commonly silica is used in combination with
the nanoparticles and amino silicones. This method is preferred due to less
toxicity and ease of operating the Ormasil. The reaction between the serum
proteins during the delivery is the limiting factor of this method.
B. Injection of naked DNA.
It is the simplest non-viral method. The gene expression with this
method is very low as compared to other methods. However, the clinical trials have
been successful. The naked DNA uptake by the cell is not so efficient. This limitation
has given rise to the certain physical methods such as electroporation and gene gun
etc.
Types of gene therapy.
 Somatic cell gene therapy.
The non reproductive cells of the body are called as the somatic cells i.e.,
the cells other than the sperms and egg. Any alteration in the somatic cells
don’t cause any inheritance. This gene therapy is carried out to treat
certain genetic disorders, acquired diseases such as leukemia, protein
deficiency that may cause serious disorders that are due to absence of a
gene etc. The dysfunctional gene is removed and a functional copy of a
cloned gene is introduced in an organism. The gene may be inserted by
using a viral vector or liposomes. Cancer and cystic fibrosis are the disease
treated.
 Germ cell gene therapy.
The reproductive cells of an organism are called as germ cells. When the
DNA is injected into the germ cells of an organism then it is passed over
generations and lead to the inheritance alteration. Any genetic ailment
inherited from the parents can be treated. Introducing genetic changes
into germ cells could have lasting effect. The genetic modifications are
performed on sperm or egg before fertilization. The modifications can be
carried out directly into cells or may be carried in vitro fertilization. It helps

6. in the prevention of genetic disorders, inherited diseases and
enhancement of the traits. Altering the human germline raises the ethical
issues.
Ex vivo gene therapy.
It can be applied to only selected tissues like bone marrow cells that can be
cultured in laboratory. It involves following steps:
 Isolate the cells from patient that are defected
 Growing the cells in culture
 Introducing the therapeutic gene to remove the defect
 Select the genetically modified cells and grow
 Transplant these cells without defect in the patient
The patient cells is extracted, cultured and after the genetic modification it is
returned to patient.
Ex vivo gene therapy can be applied to a wide range of cell types including
genetic disorders, cancer and immune related diseases.
In vivo gene therapy.
In vivo gene therapy can be defined as the direct delivery of the therapeutic gene
into the target cell. These include liver, muscle, skin, spleen, lung and brain cell.
The delivery of gene can be carried out by viral and non-viral vectors. The

7. expression capability of gene, uptake of gene and the intracellular degradation of
gene determine the efficiency of the process.
Ethical issues of gene therapy.
 Altering genes could have unforeseen and potentially harmful effects, not just for
the individual treated but also for the future generations as changes passed
down.
 Editing gene in embryo raising concern about passing modified traits and leading
to questions about “playing God”.
 Gene therapy high cost can divide between those who can afford and those who
can’t afford
 Altering the human genome and the desirable traits can lead to inequality and
societal bias.
 Developing effective regulations to ensure the safety and ethical use of gene
therapy while avoiding stifling innovation poses a challenge
 If gene edited organism are released into environment unintended ecological
consequences could occur.
Advantages of gene therapy.
 Germ line therapy offers a true cure for various genetic disorders.

8.  It can be tailored to an individual’s genetic makeup allowing for personalize
treatment
 Successful gene therapy can lead to long term benefits and provide permanent
solution to some conditions
 Compared to traditional treatment like chemotherapy, gene therapy may have
fewer effects.
 It opens the door to treating disease that were previously considered untreatable
 It can enhance the quality of life for individuals with chronic diseases
 Effective gene therapies could reduce the need for frequent and costly medical
interventions
Disadvantages of gene therapy.
 There is a risk of unintended genetic changes or adverse reactions, which could
have a long term or unpredictable consequences
 The body’s immune system might react against the modified genes or vectors
used in gene therapy
 Manipulating genes raises ethical questions about potential misuses, unintended
consequences
 Developing and administrating the gene therapies and can be expensive, limiting
access for certain regions or individuals
 The technology behind gene therapy requires specialized expertise and facilities
 In some cases, the therapeutic effect of the gene may disappear over a short
period of time, requiring repeated treatments
 Ensuring that the therapies reach the correct cell or tissue into the body can be
challenging
 Since gene interactions are complex so modifying a single gene might lead to
unexpected effects on other genes or systems