rDNA Technology and gene Therapy & it's applications

1. Recombinant DNA Technology
And Drug Discovery
Presented By: Panchal Ashitosh
M. Pharm. Sem-I Roll No.546
Guided By: Dr. Revan Karodi
Dr. D. Y. Patil College Of PharmacyAkurdi, Pune.

2. Contents
• R-DNA Technology
• Hybridoma Technology
• New Pharmaceuticals derived from biotechnology
• Oligonucleotide Therapy
• Gene Therapy
• Clinical Applications
• Recent Advances in Gene Therapy
• Principles of RNA & DNA Estimation
• References

3. • What is DNA?
DNA = DeoxyriboNucleic Acid
• DNA is a very large molecule, made up
of smaller units called nucleotides.
• Each nucleotide consist of three parts; a
sugar (ribose), a phosphate group and a
nitrogenous base.
• The nitrogenous base is the part of the
nucleotide that carries genetic
information.
• The bases found in DNA are; Adenine,
Cytosine, Guanine and Thymine (ATP,
CTP, GTP and TTP).

4. Recombinant DNA Technology
• Definition:- Recombinant DNA (rDNA) technology is the
technique used in genetic engineering that involves the
identification, isolation and insertion of desired gene into
a vector such as a plasmid or bacteriophage to form a
recombinant DNA molecule and production of large
quantities of that gene fragment or product encoded by
that gene.

5. Discovery of Recombinant DNATechnology

6. Basic steps involved in the process
1. Isolating genomic DNA of a 'donor’.
2. Fragmenting this DNA using “molecular scissors” (enzymes).
3. Screening the fragments for a ‘desired gene.’
4. Inserting the fragments with desired gene into a ‘cloning vector.’
5. Introducing the recombinant vector into a competent host cell.
6. Culturing these cells to obtain multiple copies or clones of desired
fragments of DNA.
7. Using these copies to 'Transform’ suitable host cells so as to express
the desired gene.

7. Fig. Steps involvedin rDNATechnologyfor the productionof human insulin

8. Tools Used in rDNA Technology
• Enzymes
• 1. Nucleases
• 2. Ligases
• Vectors
• Host
• DNA to be
cloned

9. Tools Used in rDNA Technology
A. Restriction Endonucleases
• Bacterial enzyme that can cut DNA at specific sites.
• Recognition sequences; site in DNA which is cut by
restriction endonuclease enzyme.
• Cleavage pattern; Form sticky ends which can easily pair
with other DNA having complementary sticky ends.
• Also called as molecular scissors.

12. B. DNA Ligase
• Cutted DNA fragments are
covalently joined by DNA
ligase.
• Join the fragments by forming
phosphodiesterase bond
between phosphate group of 5’
carbon of one deoxyribose
with hydroxyl group of 3’
carbon of another deoxyribose.

15. Vectors used in rDNATechnology
• In molecular cloning a vector is a
DNA molecule used as a vehicle
to artificially carry foreign
genetic material into another cell,
where it can be replicated and/ or
expressed (eg. Plasmids,
Cosmids, Lambda Phages).
• A vector containing foreign DNA
is termed as recombinant DNA.

16. Plasmid Vector
• Plasmids are small, circular
DNA molecules that are
separate from the rest of the
chromosome.
• They replicate independently
of the bacterial chromosome.
• Useful for cloning DNA
inserts less than 20 kb ( kilo
base pairs ).

17. Lambda Phage
• Lambda Phage vector are
recombinant vectors, containing
the phage chromosome.
• Useful for cloning DNA inserts
from 8-25 kb ( kilo base pairs ).
• In all phage vectors can convey
bigger DNA groupings than
plasmid vectors .

18. Cosmid Vectors
• Cosmid Vectors ( 5-7 kb ) are
hybrid between plasmids and
phages. They contain antibiotic
genes and replication origin from
plasmids and 'cos’ sites from
phages which are necessary for
packaging of DNA .
• These vectors can clone larger
fragments of size ranging from 35 to
50 kb.

19. Expression Vectors
• Expression Vectors are
vectors that carry host
signals that facilitate the
transcription and
translation of an inserted
gene.
• They are very useful for
expressing eukaryotic
genes in bacteria.

20. Yeast Artificial Chromosome ( YACS )
• Yeast Artificial Chromosomes
( YACS ) are yeast vectors that
have been engineered to
contain a centromere,
telomere, origin of replication
and a selectable marker.
• They can carry upto 1000 kb
of DNA.
• They are useful for cloning
eukaryotic genes.

21. Bacterial Artificial Chromosomes
• Bacterial Artificial Chromosomes
( BAC ) vectors are plasmids
constructed with the replication
origin of E.coli F factor and so
can be maintained in a single
copy per cell.
• These vectors can hold DNA
fragments of upto 300 kb.
• Recombinant BACs are
introduced into E.coli by
electroporation.

22. Host Cells
• rDNa introduced into suitable host.
• The host are the living cells in which the carrier of rDNA
molecule or vectors can be propagated.
• Host cells can be prokaryotic or eukaryotic.

23. Applications of rDNA Technology
• Various therapeutic products are made by using rDNA
technology such as proteins, hormones, immune modulators,
plasminogen activator factor, blood clotting factors, insulin,
growth hormones and several enzymes etc.
• Diagnosis of molecular diseases:
• Sickle cell anemia, thalassemia, familial
hypercholesterolemia, cystic fibrosis.
• Prenatal diagnosis:
• DNA from cells collected from amniotic fluid, chorionic villi.

24. • Gene Therapy:
• This is achieved by cloning a gene into a vector that will
readily be taken up and incorporated into genome of a host
cell. ADA ( Adenosine deaminase ) deficiency has been
successfully treated.
• Applications in Agriculture:
• Genetically engineered plants are high yielding and pest
resistant. Good quality of food and increased yield of crops is
also possible.

25. Hybridoma Technology
• In 1975, these technogy
developed by Georges J. F.
Kohler and Cesar Milstein.
• In 1984, they shared a Nobel
Prize for this discovery.
• They make a hybrid cell that
will make a numbers of
monoclonal antibodies
against antigen.

26. • The hybrid cell has the capacity of antibody production
derived from B-cells ( spleen cell ).
• At the same time it can divide continuously by the quality
derived from myeloma cell.
• By combining the desired qualities of both the cells, the
technogy ensures large antibody production of single
specificity.
• Specific hybridomas ( spleen cell and myeloma cell ) obtain
monoclonal antibodies in artificial media, this technology
called as Hybridoma Technology.
Principle

28. Monoclonal Antibody
• Monoclonal antibodies ( mAb) are antibodies that are
identical because they are produced by one type of immune
cell, all clones of a single parent cell.
• Basically produced by white blood cell which is called as
plasma cell.
• It is used for treatment of cancerous cells and as anti-
venom (anti-snake venom).

29. Procedure:
1. Immunization of specific animal which generate
Hybridoma cell with spleen cell.
2. Isolation of myeloma cell.
3. Fusion between spleen cell and myeloma cell.
4. Selection of HAT medium.
5. Isolation of Hybridoma Cell.
6. Screening of Hybridoma Cell.

30. Applications of Hybridoma Technology:
1. Serological:
• Identification of ABO blood group.
2. Diagnosis:
• Detection of pregnancy by assaying of hormones with monoclonal
antibodies.
• Separation of one substance from a mixture of very similar molecules.
3.Immunopurification:
• Purification of individual interferons using monoclonal antibodies.
• Inactivation of T-lymphocytes responsible for rejection of organ
transplant.

31. 4. Therapy:
• Removal of tumor cells from bone marrow.
• Treatment of acute renal failure.
• Treatment of malignant leukemia cells, B cell lymphoma and
a variety of allograft rejections after transplantation.

33. 1. Hormones
•Insulin:
Used for treatment of diabetes.
2. Vaccines
• Hepatitis B vaccine
• Myobloc vaccine
• Menveo vaccine

34. Monoclonal antibodies:
• Used along with immunosuppressants.
Eg. Infliximab, Basiliximab, Rituximab.
Enzymes:
• Alteplase-plasminogen activator
• Recombinant dornase alpha-cystic fibrosis
• Idursulphase-Hunter syndrome.

35. Antibiotics:
• Penicillin, Cephalosporins, Streptomycin.
Blood Clotting Factors:
• Clotting factors 8,9 – Haemophilia
• Anti-thrombin recombinant- prevention of thromboembolic
events.

36. Oligonucleotide Therapy
• They are short DNA or RNA molecules that has wide
range of applications.
• Antisense oligonucleotides ( ASO) are single strand
of DNA or RNA that are complementary to a chosen
sequence.
• They are chemically synthesized from protected
phosphoramides or chemically modified nucleosides.

37. • Mechanism of Action

38. ASO As Therapeutic Agent
• Muscular Dystrophy
• Group of diseases that cause weakening and breakdown of muscles.
ASO therapy used to remove mutated Exon.
• Cancer
• The high specificity of binding of ASO to target mRNA make these
compounds useful as therapeutic agents against human cancer.
• Suppresses malignant cells.
• Thalassemia
• Antisense 2’-O-methylribooligonucleotides were targeted against
specific sequence elements in mutated human beta-globin and can
repair thalassemia.

39. • Arthritis
• Fibroblast-like cells obtained from synovium were stimulated
with interleukin-1beta and treated with antisense
oligonucleotides targeting proliferating cells.
• Asthma
• ASO directed against chemikine receptor, granulocyte-
macrophage colony stimulating factor are designed to inhibit
allergic inflammation.

40. Gene
Therapy

41. Gene Therapy
• Definition: Gene therapy may be defined as the introduction
of a normal functional gene into cell of patient which
contains the defective allele of concerned gene with the
objective of correcting a genetic disorder or an acquired
disorder.
Types:

43. Vectors
• Viral vectors:
• Viral DNA has been removed and is introduced into hosts.
Eg. Adenoviruses, retrovirus, Lenti viruses.
• Non-viral vectors:
• DNA molecular conjugates
• Lipoplexes
• Human artificial chromosomes.

44. Methods of gene delivery
• Physical Methods:
Gene Gun:
Employs a high pressure delivery system
to shoot tissue with gold or tungsten
particles that are coated with DNA.
Microinjection:
In this process glass micropipette is used to
insert microscopic substances into a single
living cell.
Normally performed under a specialized optical
microscope set-up called a micromanipulator.

45. • Chemical Methods
• Using detergent mixtures
• Lipofection.
Some times chemicals are used to make easy delivery of DNA into
recipient cell. Using detergent mixture. Certain charged chemical
compounds e.g. calcium phosphate, dextran or lipids are
mixed with functional cDNA of desired function. The mixture is
introduced near the vicinity of recipient cells.

46. Recent Advances & Applications of Gene
Therapy:
• Cancer
Miltiple gene therapy strategies have been developed
to treat a wide variety of cancers, including suicide
gene therapy, anti-angiogenesis and therapeutic gene
vaccines.

48. •Severe Combined Immunodeficiency (ADA-SCID):
• Also known as 'Alimphocytosis’ and Bubble-boy disease.
• SCID caused due to defect of gene coding Adenosine deaminase.
• Gene of ADA is introduced for its treatment.
•Ornithine Transcarboxylase (OTC) deficiency:
• Leads to accumulation of ammonia and can be treated by gene therapy.
•Cystic Fibrosis:
• Adenovirus vectors was used to deliver a normal ion channel protein
to airway cells in a patient’s lungs.

49. • Thalassemia:
• It is an inherited autosomal recessive blood disease.
• Gene transfer of a regulated beta-globin gene reduce the
imbalance between alpha and beta-globin chains in erythroid
cells.
• Blindness:
• Cure blindness of inherited condition.

50. Principles of RNA and DNA Estimation:
• Important application in PCR.
Diphenylamine Method
Diphenylamine + deoxy ribose
Blue coloured complex (absorbs at 595 nm)
ConcentrationVs Absorbance plotted.

51. Spectrophotometric Method:
• Sample is exposed to wavelength at 260 nm and
photodetectors measures the light that passes through the
sample.
Agarose Gel Electrophoresis:
• Used to separate nucleic acid based on their size under the
influence of electric field. Nucleic acids are negatively
charged, on applying electric field they move to anode based
on size and separated.

52. Analysis with Fluorescent Dye Tagging:
• Sample is tagged with Fluorescent Dye.
• Intensity of the dye that bind to nucleic acid is measured.

53. References:
• Molecular Biotechnology, principles and applications of recombinant
DNA, 4th edition.
• Elements of Biotechnology by P.K. Gupta.
• https://www.researchgate.net/publication/327111668_Recombinant
_DNA_Technology_and_its_Applications_A_Review
• Brazilian Journal of Pharmaceutical Sciences vol. 47, n. 2, apr./jun.,
2011_Drugs obtained by biotechnology processing
• https://www.google.com_rdna&genetherapy_google images