Dr.Padmesh.

Basic Principles of
Application of Genetic Engineering
as a New Modality of Treatment of
Many Congenital Disorders and
Cancer

Prof. Dr. Mohamed Ali, M.Sc.. M.Phil., B.Ed., M.S (Sing)., Ph.D (Sing)
Department of Clinical Biochemistry
Faculty of Medicine
University of Tabuk
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GENETIC ENGINEERING
 Changing the genetic information in a cell.

 Specific trait of one organism may be isolated, cut, and

moved into the cell of another organism.

 Results of Gen. Eng. Are said to be “transgenic”.

 Genetic material in an organism has been altered

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m o d e l o r g a n i s m s
sizes
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v i r u s e s

 proteins involved in DNA,

RNA, protein synthesis
 gene regulation
 cancer and control of
cell proliferation
 transport of proteins and
organelles inside cells
 infection and immunity
 possible gene therapy
approaches
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b a c t e r i a

 proteins involved in DNA,

RNA, protein synthesis,
metabolism
 gene regulation
 targets for new antibiotics
 cell cycle
 signaling
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yeast
Saccharomyces cerevisiae
 control of cell cycle and cell
division
 protein secretion and
membrane biogenesis
 function of the cytoskeleton
 cell differentiation
 aging
 gene regulation and
chromosome structure
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r o u n d w o r m
Caenorhabditis elegans
 development of the body
plane
 cell lineage
 formation and function of the
nervous system
 control of programmed cell
death
 cell proliferation and cancer
genes
 aging
 behaviour
 gene regulation and
chromosome structure
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f r u i t f l y
Drosophila melanogaster
 development of the body plan
 generation of differentiated cell
lineages
 formation of the nervous system,
heart and musculature
 programmed cell death
 genetic control of behaviour
 cancer genes and control of
cell proliferation
 control of cell polarisation
 effect of drugs, alcohol and
pesticides
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f r u i t f l y
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zebrafish

 development of
vertebrate body
tissue
 formation and
function of brain and
nervous system
 birth defect
 cancer
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mice

 development of body
tissues
 function of mammalian
immune system
 formation and function of
brain and nervous system
 models of cancer and
other human diseases
 gene regulation and
inheritance
 infectious disease
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mouse with human ear

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APPLICATIONS OF
GENETIC ENGINEERING
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AGRICULTURE AND INDUSTRY

 Almost everything we eat and much of what we wear come from living
organisms.

 Researchers have used genetic engineering to try to improve the products

we get from plants and animals.

 Genetic modification could lead to better, less expensive, and more

nutritious food as well as less harmful manufacturing processes.

 Since their introduction in 1996, genetically modified (GM) plants have become
an important component of our food supply.

 One genetic modification uses bacterial genes that produce a protein known
as Bt toxin.

 This toxin is harmless to humans and most other animals, but enzymes in the
digestive systems of insects convert Bt to a form that kills the insects.

 Plants with the Bt gene do not have to be sprayed with pesticides.

 In addition, they produce higher yields of crops.

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GM ANIMALS
 Transgenic animals are becoming more important to our food
supply.

 About 30 percent of the milk in U.S. markets comes from cows that
have been injected with hormones made by recombinant-DNA
techniques to increase milk production.

 Pigs can be genetically modified to produce more lean meat or high

levels of healthy omega-3 acids.

 Using growth-hormone genes, scientists have developed transgenic

salmon that grow much more quickly than wild salmon.
 Scientists are working to combine a gene for lysozyme—an
antibacterial protein found in human tears and breast milk—into the
DNA of goats.

 Milk from these goats may help prevent infections in young children
who drink it.
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PREVENTING DISEASE

 Golden rice is a GM plant that contains increased amounts of

provitamin A, also known as beta-carotene—a nutrient that is essential
for human health.

 Two genes engineered into the rice genome help the grains produce
and accumulate beta-carotene.

Provitamin A deficiencies produce serious medical problems, including

infant blindness. There is hope that provitamin A–rich golden rice will
help prevent these problems.

 Other scientists are developing transgenic plants and animals that

produce human antibodies to fight disease.
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TREATING DISEASE
 Recombinant-DNA technology can be used to make
important proteins that could prolong and even save human
lives.

 For example, human growth hormone, which is used to treat

patients suffering from pituitary dwarfism, is now widely
available because it is mass-produced by recombinant
bacteria.

 Other products now made in genetically engineered bacteria

include insulin to treat diabetes, blood-clotting factors for
hemophiliacs, and potential cancer-fighting molecules.
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TREATING DISEASE

 Gene therapy is the process of changing a gene to treat a

medical disease or disorder.

 In gene therapy, an absent or faulty gene is replaced by a

normal, working gene.

 This process allows the body to make the protein or enzyme it

needs, which eliminates the cause of the disorder.
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TREATING DISEASE —
ONE EXAMPLE OF GENE THERAPY
 To deliver therapeutic genes to target cells researchers engineer a virus that cannot
reproduce or cause harm.

 The DNA containing the therapeutic gene is inserted into the modified virus.

 The patient’s cells are then infected with the genetically engineered virus.

 In theory the virus will insert the healthy gene into the target cell and correct the
defect.
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TREATING DISEASE
• Gene therapy can be risky.

• In 1999, 18-year-old Jesse Gelsinger volunteered for a

gene therapy experiment designed to treat a genetic
disorder of his liver.
• He suffered a massive reaction from the viruses used to
carry genes into his liver cells, and he died a few days
later.

• For gene therapy to become an accepted treatment,

we need more reliable ways to insert working genes
and to ensure that the DNA used in the therapy does
no harm.
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FORENSIC SCIENCE

 The precision and reliability of DNA

fingerprinting has revolutionized
forensics—the scientific study of
crime scene evidence.

 DNA fingerprinting has helped solve

crimes, convict criminals, and even
overturn wrongful convictions.

 To date, DNA evidence has saved

more than 110 wrongfully convicted
prisoners from death sentences.
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PERSONAL IDENTIFICATION
 In DNA fingerprinting, restriction enzymes first cut a small sample of human DNA into
fragments containing genes and repeats.

 Note that the repeat fragments from these two samples are of different lengths.

 Next, gel electrophoresis separates the restriction fragments by size.

 DNA samples can be obtained from blood, sperm, or tissue—even from a hair strand
if it has tissue at the root.
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WHAT IS GENE THERAPY ?

 Definiton: 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:
 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.
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What is Cell/Gene Therapy

 A branch of Regenerative Medicine, an emerging field that involves the
"process of replacing, engineering or regenerating human cells, tissues or
organs to restore or establish normal function”.

 Gene therapy is the the delivery of therapeutic gene into a patient's cells
to treat disease.

 Cell therapy is the delivery of intact, living cells into a patient to treat
disease.

 Combination Cell/Gene Therapy approaches that seek to insert genes

into a patients’ own cells to control or kill HIV are in clinical trials now.
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Some targets for gene therapy

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Researchers are studying gene therapy for a

number of diseases, such as

 Severe combined immuno-deficiencies (SCID)

 Hemophilia
 Parkinson's disease
 Cancer
 HIV
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Definition of gene therapy:

“ Novel approach to treat,cure or ultimately

prevent a disease by changing the expression of a
person’s genes”
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Alright,tell me
how you want to
change…!

Gene
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STEPS IN GENE THERAPY:

1. Identification of the defective gene.
2. Cloning of normal healthy gene.
3. Identification of target cell / tissue / organ.
4. Insertion of the normal functional gene into the host
DNA.

METHOD:
Introduction of FUNCTIONAL GENES into appropriate cells
Transferred gene (TRANSGENE) encodes & produces proteins
The Proteins encoded by Transgene corrects the disorder
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 Gene Therapy:

 Approaches:
Two ways to deliver genes:
1. Ex vivo approach
2. In vivo approach
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 1. Ex vivo approach:
-Target cells are removed from the body and grown in vitro.
-The gene is then introduced into the cultured cells.
-These cells are then re-introduced into the same individual
-Examples: Fibroblast cells, Hematopoietic cells.
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 2. In vivo approach: (Direct Gene Transfer)

-Cloned therapeutic gene is introduced directly into the
affected tissue, without removing cells from the body.
-Specially designed vehicles are needed.
-Examples are: Lungs, Brain
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 METHODS OF GENE DELIVERY:

1. PHYSICAL METHODS:
-Parenteral injection
-Microinjection
-Aerosol
-Gene gun

2. CHEMICAL METHODS:
-Calcium phosphate
-DEAE-Dextran
-Liposomes

3. BIOLOGICAL METHODS:
Viral Vectors like
-Retrovirus -Adenovirus -HSV
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 METHODS OF GENE DELIVERY: contd…

4. NEO-ORGAN IMPLANTS

5. TISSUE TRANSPLANTATION

6. HUMAN ARTIFICIAL CHROMOSOMES

7. OTHERS:
-Receptor mediated delivery
-Virally directed enzyme prodrug therapy
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 TYPES OF GENE THERAPY:

1. SOMATIC CELL THERAPY

2. GERM LINE THERAPY

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 1.SOMATIC CELL THERAPY:

 Insertion of therapeutic gene into somatic cells like

fibroblasts,myoblasts,epithelial cells, nervous
cells,glial cells etc.

 This can correct the genetic defect in the patient

 However,in somatic cell therapy, Transgene cannot

be passed on to the siblings etc.
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 2. GERMLINE THERAPY:

 Introduction of the foreign gene into germ cells like

sperm / ovum / fertilized egg.

 Results in expression of modified features in both

somatic as well as germ cells of the offspring.

 Considered unethical,and is not advocated in humans.

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 VARIOUS STRATEGIES FOR GENE

THERAPY:
 1. GENE AUGMENTATION THERAPY

 2. TARGETED MUTATION CORRECTION

 3. INHIBITION OF GENE EXPRESSION

 4. GENE THERAPY TO ACHIEVE

PHARMACOLOGICAL EFFECTS
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 1. GENE AUGMENTATION THERAPY:

 If a disease is caused by a mutation causing

loss of function,
introduction of a FUNCTIONAL COPY OF
THE GENE into the cell will restore the
normal function of the gene.

Examples:
1. Deficiency of ADA
2. Haemophilia
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 2.TARGETED MUTATION CORRECTION:

 Correction of mutation, by changing the mutated

nucleotide sequence to normal.

 Practically difficult, but in principle can be done by

homologous recombination.
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3. INHIBITION OF GENE EXPRESSION:

In case of mutations that have a negative dominant effect,

the expression of the mutated gene can be blocked at the
DNA / RNA / protein level.

Examples:
This strategy is useful in Cancers caused by inappropriate
expression of a gene.
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4. GENE THERAPY TO ACHIEVE PHARMACOLOGICAL

EFFECTS:

 Examples:
1. Introduction of a gene that makes cancer cells
susceptible to anticancer drugs.
2. Introduction of a toxic gene whose expression kills
cancer cells.
3. Genes of cytokines can be introduced into cells of
immune system to enhance their potential to kill
diseased cells.
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COMMON VECTORS
USED FOR GENE
THERAPY:

 1.Retro viruses

 2. Adeno viruses

 3. Liposomes
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1. RETRO VIRUSES:
 Retroviruses used in gene therapy are made incapable of
independent replication,to prevent side effects associated
with infectivity.
 Retroviruses are used ONLY in EX VIVO THERAPY.

 Advantages:
 Chromosomal integration & stable modification of target
cells.

 Disadvantages:
 Uncontrolled integration; May be oncogenic.
 Cannot infect non-dividing cells.
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 2. ADENO VIRUSES:
 Second most commonly used delivery system in gene
therapy.
 Adenoviruses can be produced at high titres in cultures.
Advantages:
 Can infect non-dividing cells,thus suitable for gene therapy
of Cystic fibrosis, DMD.
 Non-integration to chromosome. Avoids the risks of
uncontrolled integration.
 Efficient gene transfer.
 Disadvantages:
 Transient expression of gene due to episomal integration.
 Provokes immune response.
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 3.LIPOSOMES:
 These are lipid bilayers surrounding an aqueous vesicle.
 Can be used to introduce foreign DNA into a target cell.

 Advantages:
 Safer when compared to Viral vectors.
 Can carry large DNA molecules.

 Disadvantages:
 Inefficient transfer.
 Transient expression.
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SUCCESS CASES OF GENE

THERAPHY

BLINDNESS

PARKINSON’S DISEASE
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GENE THERAPY CURES

BLINDNESS

 Cure blindness of inherited condition

 Leber’s conginetal amaurosis
 - inherited disease caused by an abnormality in
a gene called RPE65.
 - The condition appears at birth or in the first few
months of life and causes progressive worse and
loss of vision.
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HOW IT WORKS??
 used harmless viruses
 enable access to the cells beneath the
retinas of patients
 By using a very fine needle
 -safe in an extremely fragile tissue and can
improve vision in a condition previously
considered wholly untreatable
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GENE THERAPY REDUCES PARKINSON’S

DISEASE SYMPTOMS
 it significantly improved the weakness of the
symptoms such as tremors, motor skill
problems, and rigidity
 Main- overactive brain region: the subthalamic
nucleus should be introduced with gene
 that would produce GABA—an inhibitory
chemical—then they could potentially quiet
that brain region and alleviate tremors.
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HOW IT WORKS??
 Done with local anesthesia, used a harmless,
inactive virus [AAV-2 GAD].

 Deliver the GAD gene into patient’s subthalamic

nucleus.

 The gene instructs cells to begin making GABA

neurotransmitters to re-establish the normal
chemical balance that becomes dysfunctional as the
disease progresses
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 Some results of gene therapy:

1. Adenosine Deaminase deficiency:
-First attempt at gene therapy.
2. Severe Combined Immuno Deficiency:
-SCID-X1 successfully treated with gene therapy.
3. Hemophilia A & Hemophilia B:
-Ex vivo method using fibroblasts
-Clinical improvement was present.
4. DMD:
-Successful in mice,but human trials not yet.
5. Cystic Fibrosis:
-In vivo trials with Transmembrane Conductance Regulator
CFTR.
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 RISKS OF GENE THERAPY:

 1.Adverse reactions due to the virus or new
genes.

 2.Activation of proto-oncogene leading to

formation of oncogene.

 3.Introduction of a mutation to the next

generation.
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Clinical trials—blood cancer patients

 Many trials recruit lymphoma or leukemia patients who need
a transplant.

 Undergo conditioning to eliminate current immune system

to create space for a new system.

 The HSCs used in these trials are autologous, meaning that

they are taken from the patients not from a donor.

 Their HSCs are gene modified to resist HIV, and are then
transplanted back into the participant in a mix of modified
and unmodified cells.
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Clinical trials-other patient populations

 Other trials propose going into healthier

patients—currently, either immunologic non-
responders or patients who have quit taking
ART (treatment fatigue) as participants.

 Some of these trials include conditioning

regimens which present toxicity issues
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