A general account of applications of Biotechnology in medicine

1. APPLICATIONS OF BIOTECHNOLOGY IN
MEDICINE
Dr. Saji Mariam George
Associate Professor
Assumption College Autonomous
Changanacherry

2. APPLICATIONS OF BIOTECHNOLOGY
IN MEDICINE
1. Production of human insulin
 Insulin – the hormone produced by the beta
cells of the Islets of Langerhans of Pancreas
 A protein – 2 polypeptides
A chain -21 amino acids
B chain - 30 amino acids
 Used for the treatment of Diabetes -
Hyperglycemia

3. Human Insulin
• Humulin – first therapeutic product produced by rDNA
technology (Eli Lilly & Co. , USA)
• Recosulin (Shreya Life Science, Pune)
• Humanzinsulin(Sarabhai - Ahmedabad )
• Lentard 40 (Ahmedabad)
• Human Insulatard(Novo Nordisk, Mumbai, Maharashtra)
• Huminsulin –R (Ranbaxy Ltd. Haryana)

4. • The gene for human insulin is inserted into
E.coli by rDNA technology.
• The gene for A chain and B chain are
introduced into separate plasmids (pBR type
vector) – the recombinant plasmids are
introduced into E. coli by transformation.
• Culture of transformed E. coli having human
insulin gene in bioreactors and harvesting.

6. 2. Production of human growth hormone (hGH)
 hGH – Somatotropin – the second genetically engineered
pharmaceutical approved for human use (1985, U.S Food
and Drug Administration)
 A single polypeptide chain of 191 amino acids.
 The hGH coding sequence was inserted into a plasmid
carrying the lac regulatory signals and introduced into
E.coli by transformation. The hGH synthesized in E.coli was
found to be active in humans.
 Somatotropin is required for normal growth
 Earlier source – human cadavers

7. Production of human growth hormone (hGH)

8. 3. Production of Vaccines
Vaccination
An effective, safest and most powerful tool
of medicine to prevent suffering, disability,
and death from various infectious diseases.
Reduce the mortality and morbidity due to
several infectious diseases – Tetanus,
Measles, Mumps, Poliomyelitis etc.

9. Vaccines – (L. vacca = cow) may consists of
i. Live attenuated microbes(Attenuate – to reduce
the virulence of a bacterium or virus to make a
vaccine
ii. Killed, inactivated microbes
iii. Purified microbial components
iv. Polysaccharide – carrier protein conjugates
v. Recombinant proteins

10. • Edward Jenner (1796) –
developed first vaccine -
used cow pox virus to
vaccinate against human
small pox (caused by
Variola virus) .
2. Small pox
1. Cow pox

11. • Louis Pasteur(1885)-
coined the term vaccine
• Developed vaccine against
rabies
• Genetically engineered
Vaccinia virus (Cow pox
virus) capable of
synthesizing anti rabies
vaccine was developed
in 1984 (Wistar
Institute, Philadelphia)

12. Hepatitis B vaccine – the first recombinant
vaccine for human use(1984), against
Hepatitis B virus (HBV).
• Genetically engineered Tobacco plants can
be used for the production of recombinant
vaccines against Hepatitis B virus – HBsAg
gene(Hepatitis B surface antigen gene) may
be integrated into the plant genome and the
recombinant protein produced by the plant is
purified for use as a vaccine.

13. VLP – Virus Like Particles Parenteral vaccine - administered or occurring
elsewhere in the body than the mouth and alimentary canal.

14. Production of Hepatitis B vaccine
• Produced by cloning
HBsAg gene (Hepatitis
B surface antigen
gene)of the virus in
yeast cells.

15. 4. Gene therapy.
 Potential tool for treating inherited human diseases -
it allows the transfer of genetic information into
patient tissues and organs.
 Gene therapy is still highly experimental, but has the
potential to become an important treatment
regimen.
 Involves adding a normal (wild –type) copy of a gene
to the genome of an individual carrying defective
copies of the gene.
 If successful, the transgene (for transferred gene) will
synthesize the missing gene product and restore the
normal phenotype.

16. Gene therapy - Types.
1. Somatic – cell gene therapy(Non heritable
gene therapy)
 Treat the disease symptoms of the
individual , but will not cure the disease.
 The defective gene/s will still be present in
the germ - line cells of the patient after
somatic – cell gene therapy and may be
transmitted to the children.
 All gene therapy treatments for human
diseases are somatic - cell gene therapies.

17. Add functional copies of the gene that is
defective – do not replace the defective gene.
Wild type genes are introduced into the cells
homozygous or hemizygous for a mutant
allele using retro viruses ( RNA viruses that
replicates using reverse transcriptase ) or
Adenoviruses and get the gene expressed.

18. Adenoviruses – the viruses that
attack the mucosa of the
upper respiratory tract and
lymph nodes.
• Most commonly
employed vector for
cancer gene therapy- can be
replication-defective; some
essential viral genes are
removed which in turn are
replaced by a gene cassette
- a type of mobile genetic
element - can move
between different
constructs - contain a gene
and a recombination site –
expresses a foreign
therapeutic gene.
Image:https://www.slideteam.net/0914-

19. Gene therapy using an adenovirus vector
Image: https://courses.lumenlearning.com/

20. • Somatic – cell gene therapy - Pioneering studies
on the correction of adenosine deaminase
deficiency, a lymphocyte-associated severe
combined immunodeficiency (SCID) .
• Successfully carried out in the treatment of
Adenosinedeaminase-deficient severe combined
immunodeficiency disease (ADA¯SCID) , a rare
autosomal disease of the immune system , in
1990. The enzyme adenosine deaminase is
encoded by a gene on chromosome 20. Children
with SCID were often known as 'bubble babies'.

21. SCID
“David Vetter from Texas had
SCID and had to live in a
sterile environment for
most of his life during the
1970/80s. He was known
to the media as 'the boy in
the plastic bubble'
and wore a special
'spacesuit' to protect him
from infections.”
https://www.yourgenome.org
/
Image credit: NASA Johnson
Space Center

22. Somatic cell gene therapy for SCID
STEPS
1) Isolation of White blood corpuscles (WBCs )from the patient.
2) Introduction of functional copies of the ADA gene into these
cells.
3) Demonstration of transgene expression in these cells.
4) Infusion of the transgenic cells back into the patient.
Effect short lived because of the limited life span of the WBCs
– repeated infusions of WBCs carrying functional ADA genes are
necessary.

23. Somatic cell gene therapy for SCID

24. Stem cells as vehicles for gene therapy
First used to treat two infants with Adenosine
deaminase-deficient severe combined
immunodeficiency disease (ADA¯SCID , 1993).
• Bone marrow stem cells that gives rise to WBCs could
be used to treat ADA¯SCID which could provide a
permanent or long - term treatment of the disease.
• In 2000, British and French Physicians used Somatic
cell gene therapy for the treatment of X- linked SCID
in boys. (caused by the mutations in a gene on X
chromosome– a fatal inherited disease – individuals
have no functional immune system).

25. 2. Germ- line gene therapy (Heritable gene
therapy) - modifying the genes in egg or
sperm cells – pass the genetic changes to
future generations .
Performed on mice and other animals , not on
humans – moral and ethical considerations are
involved in any decision to perform germ – line
modifications of humans.

26. Gene replacements ( Targeted gene
replacements)
• The ideal gene therapy – involves the
replacement of the defective gene with a
functional gene.
• Researchers are trying for such gene
replacements (targeted gene replacements).

27. 5. Production of Monoclonal antibodies
 Antibodies (Immunoglobulins) – proteins
synthesized in blood against specific antigens.
 Heterogeneous – contain a mixture of
antibodies (polyclonal antibodies) – do not
have specificity.
 A specific lymphocyte, after isolation and
culture in vitro , becomes capable of producing
a single type of antibody which has specificity
against a specific antigen – monoclonal
antibodies – used in the diagnosis of diseases.

28. Hybridoma technique for the continuous
production of antibodies- (George Kohler and
Caeser Milstein, 1974 – Nobel Prize 1984 in
Physiology & Medicine).
Hybridoma- formed by the fusion of a myeloma
cell (bone marrow tumour cell) and an antibody
producing lymphocyte.
Kohler and Milstein – isolated clones of cells from
the fusion of two parental cell lines –
lymphocytes from spleen of mice immunized
with sheep RBCs and myeloma and maintained
in vitro – produced antibodies which immunized
myeloma cells .

29. Hybridoma - maintain the character of
lymphocytes to secrete antibodies and
of myeloma cells to multiply in culture –
capable of continuous production of
antibodies.

30. Hybridoma technique for the continuous
production of antibodies
https://www.biomol.com/

31. Hybridoma cells grown in tissue culture
Image:https://commons.wikimedia.org/

32. Applications of Monoclonal Antibodies
 Useful in the diagnosis of diseases – Cancer,
Allergy, Viral diseases- e.g. For AIDS detection –
ELISA (Enzyme Linked Immuno sorbent Assay).
For classification of ABO blood groups, Rh etc.
For the treatment of Cancer- antibodies specific to
a cancer cell can be linked with a toxin
polypeptide to yield a conjugate molecule ,
immunotoxin – the antibody will ensure its
binding specifically and only to the target cells and
the attached toxin will kill such cells.
Useful in the purification of antigens specific to
pathogens , which can be used as vaccines.
ABZYMES – ANTIBODIES THAT CAN FUNCTION AS
ENZYMES

33. 6. Biopharming (Molecular farming or Molecular
pharming)
• Genes that encode the biosynthesis of certain
proteins or metabolic products (Secondary
metabolites) which are pharmaceutically
important are inserted into suitable hosts by
recombinant DNA technology (rDNA technology)
– get them expressed and the products are
harvested and used to produce pharmaceuticals.
• Transgenic proteins are most commonly
produced by transgenic bacteria or yeast in a
bioreactor.

34. i) Biopharming in plants
 Transgenic plants or animals are
used as bioreactors to produce
therapeutic proteins, drugs,
vaccines etc. – Biologics.
E.g. Transgenic tobacco, Potato,
Duck weed(Lemna minor)
 Genetically modifying plants so
that they can produce certain
proteins and other components
like secondary metabolites
which are therapeutically
important.
Duck weed
(Lemna minor)

35.  The first recombinant plant – derived protein
(PDP) was human serum albumin produced in
transgenic tobacco and potato plants.
 Transgenic banana, tomato, potato - to
produce edible vaccines against certain
diseases (e.g. Hepatitis B)
 Transgenic tobacco – to produce Cholera
vaccine (Chlorogen Inc.)

36. Plant Made Pharmaceuticals(PMPs)
Advantages
 Low cost of production
 Large production capacity
 Safer than recombinant proteins from
microorganisms
 Do not carry potentially harmful human or animal
viruses into the drug.
 The seeds and fruits of plants provide sterile
packaging containers for the valuable therapeutics
and guarantee a certain storage life.

37. Plant Made Pharmaceuticals(PMPs)-
Disadvantages
• Concern about the safety of GM foods –
edible vaccines – for human consumption.
• GM plants may cross pollinate with non-GM
crops.

38. Biopharming in animals
 Creation of transgenic mammals(Cow, Sheep, Goat )
that can produce the biopharmaceutical in its milk.
 The first drug produced in genetically modified
livestock is ATryn- Antithrombin protein (brand name of
the anticoagulant antithrombin manufactured by the
Massachusetts-based U.S. company rEVO Biologics)
purified from the milk of genetically modified goats.
Advantages
 Milk is produced in large quantity.
 Purification of the biopharmaceutical from milk is easy.

39. THANK YOU