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tRECOMBINANT DNA TECHNOLOGY AND DRUG DISCOVERY
PRESENTED BYDIVYA V1st MPHARMPHARMACEUTICAL CHEMISTRY
RECOMBINANT DNA TECHNOLOGY
INTRODUCTION
A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from different organism.
Also called as chimeric gene.
Achieved by cutting DNA(restriction enzymes)into suitable fragments and joining together the appropriate fragments(ligation).
Proteins expressed by rDNA called as recombinant proteins.
Cloning is the process to create rDNA
TOOLS OF GENETIC ENGINEERING
A)RESTRICTION
ENDONUCLEASES(RE)
Bacterial enzyme that can cut DNA at
specific sites
Recognition sequences; site in DNA
which is cut by RE.
Cleavage pattern; Form sticky ends
which can easily pair with other DNA
having complementary sticky ends.
Also called as molecular scissors
B)DNA LIGASE
Cutted DNA fragments are covalently
joined by this.
Join the fragments by forming
phosphodiester bond between
phosphate group of 5’carbon of one
deoxy ribose with hydroxyl group of
3’carbon of another deoxy ribose.
BASIC PRINCIPLES
STEPS IN GENE CLONING
1)Identification and isolation of desired
gene.
2)Insertion of isolated DNA into a suitable
vector
3)Introduction of this vector into suitable
organism
4)Selection of transformed host cells
5)Multiplication/Expression/Integration
followed by expression of the gene in
the host
1)ISOLATON OF DESIRED GENE
DNA fragment to be cloned is called as
DNA insert.
Desired fragments can be obtained
from,
a)Genomic libraries
Libraries are collection of DNA clones
in a certain vector.
Genomic - made from RE DNA
fragments of total genomic DNA
cDNA (complementary DNA) – made
from DNA synthesized from mRNA
b)Polymerase chain reaction
Allows the isolation of a specific
segment of DNA from a small DNA (or
cell sample) using DNA primers.
c)Chemical synthesis of gene
Base sequence of protein is identified,
a polynucleotide of same sequence
can be synthesized chemically or
enzymatically.
2)INSERTION OF THE GENE INTO
SUITABLE VECTORS
Can carry foreign DNA fragment to be
cloned and are self replicating in host
cells
vectors
BACS
YACS
expression
cosmid
Bacteriopha
ge
plasmid
PLASMID
They are extrachromosomal,circular,self
replicating DNA molecules.
E.g. pBR322
BACTERIOPHAGE
They are viruses that attack bacteria.
Can accept short fragments of foreign
DNA into their genome.
COSMID
It posses the characteristics of both
plasmid and bacteriophage.
ARTICIAL CHROMOSOME
VECTORS HUMAN ARTIFIAL CHROMOSOME(HAF)
Synthetically produced vector DNA
possesing characteristics of human
chromosome.
BACTERIAL ARTIFICIAL
CHROMOSOME
Bacterial artificial chromosomes
(BACS) are bacterial plasmids derived
from the F plasmid. They are capable
of carrying up to 300 kb of DNA.
YEAST ARTIFICIAL
CHROMOSOME(YAC)
Behaves like yeast chromosome and
can accept large pieces of foreign DNA.
PHAGEMID/PHASMID
Contain several copies of plasmid but
one copy of plasmid is retained in the
DNA.
3)INTRODUCTION OF rDNA INTO
SUITABLE HOST
rDNA is introduced in to suitable host.
Host are the living cells in which
carrier of rDNA/vector can be
propagated.
TYPES,• E.coli
• Bacillus subtilisProkaryotic
• Yeast
• Mammalian cellsEukaryotic
4)SELECTION OF
TRANSFORMED CELLS
rDNA containing cells can be identified
from non-transformed cells when a
marker gene is present in it.
Only the cells that posses such gene
will survive.
5)MULTIPLICATION/EXPRESSION
OF GENE
The multiplied copies of gene can be
used in number of ways,
Introduced to bacterium for production
of protein.
Introduced into eukaryotic host.
Expression of gene.
APPLICATIONS
Manufacture of proteins/hormones
Interferon, plasminogen activating factor,
blood clotting factors, insulin, growth
hormone,several enzymes etc.
Diagnosis of molecular diseases:
sickle cell anaemia, thalassaemia,
familial hypercholesterolaemia, cystic
fibrosis.
Prenatal diagnosis: DNA from cells
collected from amniotic fluid, chorionic
villi.
Gene Therapy:
This is achieved by cloning a gene into
a vector that will readily be taken up &
incorporated into genome of a host
cell.
ADA deficiency has been successfully
treated
Application in Agriculture:
Genetically engineered plants are
developed to resist draught &
diseases. Good quality of food &
increased yield of crops is also
possible.
HYBRIDOMA
TECHNOLOGY
INTRODUCTION
It is a hybridization technique which is
used to produce antibody producing
hybrid cell.
Antibodies produced are called as
Monoclonal antibodies.
APPLICATIONS
DIAGNOSTIC
A monoclonal antibody can be usedto detect pregnancy in only 14 daysafter conception.
Their selective binding property allow detection of low levels of human corionicgonadotropin (HCG) in urine andserum.
THERAPEUTIC
Earlier horses were inoculated with Coryne bacterium diphtheriae,the resulting crude horse antiserum was used totreat diphtheria.
Organ transplantation For the treatment of solid organ transplant rejection, several Mabs against T cell antigens have been evaluated.
Bone marrow transplantationMAbs are being evaluated for graft versus host disease in bone marrow transplantation.
CANCER TREATMENT
mAbs act directly when binding to
cancer specific antigens and induce
immunological response to cancer
cells. Such as inducing
cancer cell apoptosis, inhibiting growth
etc.
IMMUNOPURIFICATION
Monoclonal antibodies can also be
used to purify a substance with
techniques called affinity
chromatography.
NEW PHARMACEUTICALS DERIVED FROM BIOTECHNOLOGY
HORMONES
INSULIN
Used for treatment of diabetes.
VACCINES
Hepatitis B vaccine.
Myobloc vaccine.
Menveo vaccine.
Ixiaro vaccine.
MONOCLONAL ANTIBODIES
Used along with immunosuppressant's.
E.g. Infliximab,Basiliximab,rituximab
ENZYMES
Alteplase-Plasminogen activator.
Recombinant dornase alpha-Cystic
fibrosis
Idursulphase-Hunter syndrome.
GROWTH FACTORS
Recombinant erythropoietin-Anemia.
Palifermin-Oral mucositis in cancer
patients.
ANTIBIOTICS
Penicillin,Cephalosporin,Streptomycin
BLOOD FACTORS
Clotting factors 8,9-Hemophilia.
Anti-thrombin recombinant-Prevention
of thromboembolic events
OLIGONUCLEOTIDE
THERAPY
They are short DNA or RNA molecules
that has wide range of applications.
Antisense oligonuleotides(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.
MECHANISM OF ACTION
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 their target mRNA make these compounds useful as therapeutic agents against human cancer.
Suppresses malignant cells
ASO AS THERAPEUTIC AGENT
THALASSEMIA
Antisense 2'-O-methylribooligonucleotides were targeted against specific sequence elements in mutated human beta-globin and can repair thalassemia.
ARTHRITIS
Fibroblast-like cells obtained from RA synovium were stimulated with interleukin-1beta and treated with antisense or sense oligonucleotides targeting proliferating cell.
ASTHMA
ASOs directed against chemokine
receptor,granulocyte-macrophage
colony stimulating factor are designed
to inhibit allergic inflammation.
AMYLOIDOSIS
DIABETES
LIMITATIONS
High doses required.
Half life in plasma is short
Protected against nucleophilic attack.
GENE
THERAPY
INTRODUCTION
Gene therapy is a clinical procedure in
which a gene or other DNA sequence
used to treat a disease.
TYPES
EXVIVO
INVIVO
VECTORS
VIRAL VECTORS
Viral DNA has been removed and is
introduced into hosts.
E.g. Adenoviruses,Adeno associated
virus,retro virus,Lenti virus.
NON-VIRAL VECTORS
Pure DNA construct.
DNA molecular conjugates.
Lipoplexes.
Human artificial chromosome.
METHODS OF GENE DELIVERY
PHYSICAL METHODS
CHEMICAL METHODS
Using detergent mixtures
Lipofection
MICROINJECTION
RECENT ADVANCES &
APPLICATION OF GENE THERAPY
BLINDNESS
Cure blindness of inherited condition.
HOW IT WORKS;
used harmless viruses
enable access to the cells beneath the
retina of patients.
CANCER
Used to treat various types of cancer.
HOW IT WORKS;
Normal WBC taken from cancer
patients infected with retrovirus that
deliver genes to cells.
PARKINSON’S DISEASE
Improved the weakness of the
symptoms such as tremors, motor skill
problems,and rigidity.
HIV
Under clinical trials
CYSTIC FIBROSIS
Adenovirus vector was used to deliver
a
normal ion channel protein(CFTR) to
airway cells in a patient’s nose or
lungs.
SEVERE COMBINED
IMMUNODEFICIENCY
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 corrected by gene
therapy.
THALASSEMIA
It is an inherited autosomal recessive
blood disease.
Gene transfer of a regulated β-globin
gene in would reduce the imbalance
between a-and β-globin chains in
erythroid cells.
CLINICAL TRIALS
Alzheimers disease
Hepatitis-B
AIDS
CANCER-
Brain, Ovarian,Small cell lung,
Prostrate, Breast cancer.
Chronic granulomatous disease.
Important applications in PCR.
DIPHENYLAMINE METHOD
Diphenylamine + deoxy ribose
Blue coloured complex(absorbs at
595nm)
Concentration Vs Absorbance plotted.
SPECTROPHPTOMETRIC
METHOD Sample is exposed to wavelength at
260nm and photo detectors 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 seperated.
ANALYSIS WITH FLUORESCENT DYE
TAGGING
Sample is tagged with fluorescent dye.
Intensity of the dye that bind to nucleic
acids is measured.
REFERENCES T.A.Brown.Gene cloning and DNA
analysis.Blackwell publishers;2006(5);302-322.
B.D.Singn.Text book of biotechnology. Kalyani Publishers.;2006(1);11-104.
U.Satyanarayana and U.Chakrapani. Textbook of biochemistry.Allied Publishers.2006(3).578-618.
Hugo Almeida, Maria Helena Amaral, Paulo Lobão. Drugs obtained by biotechnology processing. Brazilian Journal of Pharmaceutical Sciences;vol. 47;2011.
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