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.