PTT 104:
Biotechnology
and Medicine
Department of Chemical
Engineering Technology,
UniMAP
Semester 1, 2013/2014
khadijahhanim@unimap.edu.
my
Course outcome:

Able to differentiate scopes and importance
of various biotechnological streams.

TOPIC COVERS:
Illustrate scopes of medical biotechnology
and examine gene transfer methods, gene
therapy and Human Genome Project (HGP)
and applications.

Lesson Contents
1. Detecting and Diagnosing
Human
Disease Conditions
2. Medical Products and
Applications of Biotechnology
3. Gene Therapy
4. The Human Genome Project
Detecting and diagnosing
human disease conditions
 Molecular
biology has advanced
tremendously- providing scientists
molecular techniques in combating
human diseases.
 Using model organism: mice, rats, worm
and flies.
 We not really unique at genetic level.
Share some similarities (genes) with these
organisms.
A
no. of human genetic diseases also
occur in model organisms.
 Therefore, can use model organisms to
identify disease genes and test gene
therapy and drug based therapeutic
approaches to check their safety and
effectiveness prior to clinical trials in
human.
 Why model organism?
- Because we cannot manipulate human
genetics for experimental purposes.
- Illegal or unethical to force human to
breed/ to take out their genes to learn
how they function. Therefore, use model
organisms.
 Many
important genes are highly
conserved between species.
 If a gene found in model organisms, we
can make hypotheses/predictions about
how the gene might function in humans.
 Related genes are called homologs.
 For eg: a gene thought to play a role in
human illness can be eliminated/knock
out. Then, the effects can be studied to
learn about the function of the gene.
Biomarkers for disease
detection
 In
theory, right diagnostic tools, possible to
detect every disease at early stage.
 For many diseases, early detection is
critical for providing the best treatment
and improving the odds of survival.
 Detection approach- BIOMARKERS as
indicator of disease.
What is biomarkers?


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Protein produced proteins produced by diseased
tissue or proteins whose production is increased
when a tissue is diseased
Many biomarkers are released into body fluids ie:
blood and urine as a product of cell damage
For eg: protein called prostate-specific antigen
(PSA) released into bloodstream when prostate
gland is inflamed and elevated levels can be
marker for prostate inflammation/prostate cancer.
Many companies are actively searching for better
biomarkers that can be used in early detection
and diagnosis.
Detecting genetic diseases
 Testing
-
-
for chromosome abnormalities
and defective genes:
Genetic testing occurred on fetus: identify
sex/genetic diseases (occur because of
alteration of chromosome no).
Eg: Down syndrome: most contain 3
copies of chromosome 21.
Symptoms: mental retardation, short
stature and broadened facial features.
Methods of detection:
Amniocentesis
•When the developing fetus is around
16 weeks.
•A needle is inserted thru the mother’s
abdomen into the pocket of amniotic
fluid surrounding and cushioning the
fetus
• fluid contain fetus’s cells. Isolated and
cultured for a few days to increase
the no.
•Chromosomes are put onto glass slide
and stained with diff. dyes.
•The chromosomes are counted
•Technique also called as karyotype:
use to determine the sex of a child.
Amniocentesis
Chorionic
villus
sampling
• A suction tube is used to
remove small portion of a
layer of cells called
chorionic villi- tissue that
help forms the placenta.
• Advantage: enough cells
obtained for immediate
karyotyping
• Can be done earlier: 8-10
weeks.
• Can cause miscarriage.
Detecting genetic diseases

Testing for chromosome abnormalities
 Fluorescence
in situ hybridization (FISH) – new
technique for karyotyping
 Blood withdrawn from adult- white blood cells
used for karyotyping
 Chromosome spread is prepared on a slide.
 Fluorescent probe are hybridized to each
chromosome.
 Each probe is specific for certain marker
sequences on each chromosome.


Useful for identifying missing chromosomes and extra
chromosomes, but much easier to detect defective
chromosomes
For eg: in leukimia: DNA is exchanged between
chromosomes 9 and 22 so genes from 9 are swapped
onto 22 and vice versa. This exchange can be detected
by FISH.
Definition of Karyotypes
Karyotypes describe the number of chromosomes,
and what they look like under a light microscope.
A karyotype is an organized profile of a
person's chromosomes. In a karyotype,
chromosomes are arranged and
numbered by size, from largest to
smallest. This arrangement helps
scientists quickly identify chromosomal
alterations that may result in a genetic
disorder.
Other methods of detection
 Restriction
fragment length polymorphism
(RFLP) analysis: defective gene
sequences may be cut differently by RE
than their normal genes because of
nucleotides changes in mutant genes
can affect RE cutting sites to create more
or fewer fragments.
Medical products and
applications of Biotechnology

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Major areas in medical biotechnology:
identifying novel drugs and developing new
ways to treat disease.
Eg: Oncogenes: genes involved in the growth
of cancer.
Oncogenes: produced proteins that function
as transcription factors and receptors for
hormones to change growth properties of
cells causing cancer.
Researchers are focusing on the protein
produced by the oncogenes as a target for
inhibitors: drugs that can bind to the protein
and block their function.
Pharmacogenomics:
personalized medicine


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Pharmacogenetics is referred to the study
of genetics influence on individual respond
to drugs.
Designing the most effective drug therapy
and treatment strategies based on the
specific genetic profile of a patient
Individuals can react differently to the same
drugs

Different degrees of effectiveness and side
effects because of genetic polymorphisms
Improving techniques for drug
delivery

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In addition to developing new drugs, companies
are working to develop innovative ways to deliver
drugs to max their effectiveness.
Getting drugs to where it need to function.
Eg: drug to treat arthritis taken orally. Small amount
is absorbed by body and transported to knee joint.
Other factors that influence drug effectiveness:
Drug solubility (ability to dissolve in body fluids)
Drug breakdown by body organs
Drug elimination by liver/kidneys
 Microspheres:
tiny particles that can be
filled or coated with drugs.
 Made by materials that closely resemble
the lipids in cell membranes.
 Delivery of microspheres as a mist sprayed
into airways thru nose/mouth has been
successfully in treating lung
cancer/asthma/tuberculosis/flu.
Nanotechnology and
nanomedicine

Nanotechnology – involved in designing,
building, and manipulating structures at the
nanometer scale


nm is 1 billionth of a meter
Nanomedicine – applications of
nanotechnology to improve human health
Nanodevices to monitor blood pressure, blood
oxygen levels, hormone concentrations
 Nanoparticles that can unclog arteries, detect
and eliminate cancer cells; smart drugs that
could seek out and target specific cells


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Scientists have developed
smart
drugs
using
viruses/nanoparticles that are
introduced into the body to
seek out or target specific cells
to treat or destroy the cells.
Figure
shows
a
plastic
nanoparticle covered with
antibodies against cancer cell
proteins that allow the particle
to bind to the cancer cells.
Inside the agent are contrast
agents- can be used for MRI or
X-ray
to
detect
tumors,
chemotherapy drugs that can
diffuse out of the cell to kill
tumor.
Vaccines and therapeutic
antibodies

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Vaccines: can be used to stimulate body’s
immune system to produce antibodies and
provide a protection against infectious
microbes.
Vaccines against cancer is still being
developed.
Cancer vaccines: not preventive but
designed to treat a person who already has
cancer.
A person is injected with cancer cell antigens to
stimulate immune system to attack cancer cells
Vaccine for Alzheimer’s disease
 Primary
purpose of vaccination: to
stimulate antibodies production by
immune system to ward off foreign
materials.
 Antibodies can also be used to treat
existing condition as opposed to
preventing infectious microbes from
causing a disease.
Monoclonal antibodies used
to treat
 Cancers
 Cardiovascular
disease
 Allergies
 Other
conditions
Gene therapy
 Gene
therapy is the delivery of
therapeutic genes into the human body
to correct disease conditions created by
a faulty gene or genes


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How are genes delivered?
How can genes be sent to the proper
tissues and organs?
Can it be effective and safe?
Gene therapy: How is it done?
Ex vivo gene therapy
In vivo gene therapy
Ex = out of
Introducing genes directly into
tissues and organs in the body
without removing the cells
Vivo = something alive
Cells from a diseased person
are removed, treated in the
lab using techniques similar to
bacterial transformation and
reintroduce into the patient.
Challenges: delivering genes
only to intended tissues not
tissues throughout the body.
Eg: using virus
Vectors for gene delivery
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Require a safe and effective delivery of
therapeutic genes.
Both ex-vivo and in-vivo rely on viruses as vectors
to introduce genes into cells.
A viral vector would use a viral genome to carry
therapeutic gene/s to infect human cells
thereby introducing the gene/s.
Some of the potential vectors:
Adenovirus (causing common cold)
Influenza (flu)
Herpes virus (cold sore or STD)
Must make sure the virus has been genetically
engineered and inactivated so that it can neither
produce disease nor spread throughout the body
Viral infection to human body
cells

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By binding to and entering cells and then
releasing their genetic material into
nucleus/cytoplasm of human cell.
DNA/ RNA
Infected human cell serves as a host for
reproducing the viral genome and producing
viral RNA and proteins.
Viral protein ultimately assemble to create
more viral particles that break out of host cells
so they are free to infect other cells and
repeat the cycle.
Viral life cycle
Viruses are effective in
infecting human cells
They can make good vectors: efficiently
infecting human cells
 Adenovirus: can infect many types of body cells
efficiently
 HIV virus: on entering a host cell they copy their
RNA genome into DNA and then randomly
insert their DNA into the genome of the host cell
where it remains parmanently- Integration.
- Integrate therapeutic genes into DNA of human
host cells, allowing permanent insertion of genes
into chromosome as a way to provide lasting
therapy.

 Some
virus only infect certain body cells.
Allow for targeted gene therapy.
 For eg: herpes virus (infects cells of the
central nervous system) may effectively
treat genetic disease of the brain,
Alzheimer and Parkinson disease.
Other delivery options:


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Naked DNA
DNA by itself without viral vector, that is
injected directly into body tissues.
Transfect cells by mixing them with naked
DNA in a tube.
Disadvantages: relatively small no of cells
uptake. May not be enough cells expressing
the therapeutic genes for gene therapy to
have any effect on the tissue.
Other delivery options:
 Liposomes
-
small-diameter hollow microspheres
made of lipid molecules
-
Packaged with gene and injected or sprayed into
tissues
A similar technique involves coating a tiny gold
particles into cells using a DNA gun

Antisense RNA and RNA
interference for gene therapy
 Antisense

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RNA Technology
A way to block translation of mRNA
molecules to silence gene expression
Called RNA or gene silencing
Promising way to turn off disease genes
 Used
successfully in cell culture, but has yet to
live up to its promise as a treatment for
disease
RNA interference (RNAi)

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Double-stranded RNA molecules are
delivered into cells where the enzyme Dicer
chops them into 21-nt-long pieces called
small interfering RNAs (siRNAs)
siRNAs join with an enzyme complex called
the RNA-induced silencing complex (RISC)
RISC shuttles the siRNAs to their target
mRNA where they bind
siRNA-bound mRNAs are degraded so they
cannot be translated into a protein
Curing genetic diseases:
Targets for gene therapy
 Focusing
on single gene mutations or
deficiencies.
 For eg: sickle cell anemia
 Easier to cure compare to multiple genes
The first human gene therapy
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In 1990, a patient with severe combined
immunodeficiency (SCID).
SCID- lack a functioning immune system
caused by a defect in a gene called
adenosine deaminase (ADA)
ADA produce enzyme involved in metabolism
of nucleotide dATP.
Lack of ADA causes accumulation of dATP. and
is toxic to T cells
 Without T cells, B cells cannot recognize antigen
and make antibodies
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To treat SCID:
Normal gene for ADA was cloned into a
vector and then introduced into an
inactivated retrovirus.
Ex vivo gene therapy was used. Small
amounts of T-cells were isolated from patient’s
body and cultured in the lab.
Her T cells, were infected with ADAadenovirus and the infected adenovirus were
further cultured.
Retrovirus integrate their genome (containing
ADA genes) into the patient’s chromosome
during culturing.
These ADA-containing T cells were
reintroduced back to the patient’s body.
Treating cystic fibrosis
 Cystic
Fibrosis – two defective copies of a
gene encoding a protein called cystic
fibrosis transmembrane conductance
regulator (CFTR).
 Normal protein serves as a pump to
remove chloride ions from cell
 The CFTR important for maintaining the
proper balance of Cl inside cells.
 Absence/altered of CFTR causes CF.


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Produced by many cells in the body – skin,
pancreas, liver, digestive tract, male
reproductive tract, and respiratory tract.
Abnormality of this gene in trachea can
cause accumulation of Cl ions lead to
extremely thick mucus that clog airways.
Normally, mucus in trachea helps sweep dust
and particles out of the airways to keep this
materials out of the lungs. But when water enter
trachea- mucus become thick.

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Thick mucus-provide ideal environment for
microbes to grow- leading to infections.
Can lead to pneumonia and respiratory
failure.
Others: can cause infertility and salty sweat.
Treatment of CF
 Normal
CFTR gene into liposomes and
spraying the liposomes into the nose and
mouth of CF patients as an aerosol or into
airways via hose.
 But not reliable cure because DNA
delivered via liposomes does not
integrate into chromosomes.
Challenges facing gene
therapy

Potential risks of viruses as vectors
 Death
of Jesse Gelsinger in 1999 due to
complications related to adenovirus vector
 Death of 2 children in France in 2002
 Temporary cessation of a large number of
gene therapy trials and FDA stopped most
retroviral studies
 Greater patient monitoring
Challenges facing gene
therapy
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Can gene expression be controlled?
Can we safely and efficiently target only
the cells that require the gene?
How can gene therapy be targeted to
specific regions of the genome?
How long will therapy last?
Will immune system reject?
How many cells need to be corrected?
Human genome project
 Many
of the disease genes were
discovered thru HGP.
 Scientists have been developing complex
map showing the locations of normal and
diseased genes on human chromosomes.