Recombinant DNA Technology & Gene Therapy

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 Pharmacy Akurdi, 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
•DNAisaverylargemolecule,madeup
ofsmallerunitscallednucleotides.
•Eachnucleotideconsistofthreeparts;a
sugar(ribose),aphosphategroupanda
nitrogenousbase.
•Thenitrogenousbaseisthepartofthe
nucleotidethatcarriesgenetic
information.
•ThebasesfoundinDNAare;Adenine,
Cytosine,GuanineandThymine(ATP,
CTP,GTPandTTP).

4. Recombinant DNA Technology
•Definition:-RecombinantDNA(rDNA)technologyisthe
techniqueusedingeneticengineeringthatinvolvesthe
identification,isolationandinsertionofdesiredgeneinto
avectorsuchasaplasmidorbacteriophagetoforma
recombinantDNAmoleculeandproductionoflarge
quantitiesofthatgenefragmentorproductencodedby
thatgene.

5. Discovery of Recombinant DNA Technology

6. Basic steps involved in the process
1.IsolatinggenomicDNAofa'donor’.
2.FragmentingthisDNAusing“molecularscissors”(enzymes).
3.Screeningthefragmentsfora‘desiredgene.’
4.Insertingthefragmentswithdesiredgeneintoa‘cloningvector.’
5.Introducingtherecombinantvectorintoacompetenthostcell.
6.Culturingthesecellstoobtainmultiplecopiesorclonesofdesired
fragmentsofDNA.
7.Usingthesecopiesto'Transform’suitablehostcellssoastoexpress
thedesiredgene.

7. Fig. Steps involved in rDNA Technology for the production of 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
•BacterialenzymethatcancutDNAatspecificsites.
•Recognitionsequences;siteinDNAwhichiscutby
restrictionendonucleaseenzyme.
•Cleavagepattern;Formstickyendswhichcaneasilypair
withotherDNAhavingcomplementarystickyends.
•Alsocalledasmolecularscissors.

12. B. DNA Ligase
•CuttedDNAfragmentsare
covalentlyjoinedbyDNA
ligase.
•Jointhefragmentsbyforming
phosphodiesterasebond
betweenphosphategroupof5’
carbonofonedeoxyribose
withhydroxylgroupof3’
carbonofanotherdeoxyribose.

15. Vectors used in rDNA Technology
•Inmolecularcloningavectorisa
DNAmoleculeusedasavehicle
toartificiallycarryforeign
geneticmaterialintoanothercell,
whereitcanbereplicatedand/or
expressed(eg.Plasmids,
Cosmids,LambdaPhages).
•AvectorcontainingforeignDNA
istermedasrecombinantDNA.

16. Plasmid Vector
•Plasmidsaresmall,circular
DNAmoleculesthatare
separatefromtherestofthe
chromosome.
•Theyreplicateindependently
ofthebacterialchromosome.
•UsefulforcloningDNA
insertslessthan20kb(kilo
basepairs).

17. Lambda Phage
•LambdaPhagevectorare
recombinantvectors,containing
thephagechromosome.
•UsefulforcloningDNAinserts
from8-25kb(kilobasepairs).
•Inallphagevectorscanconvey
biggerDNAgroupingsthan
plasmidvectors.

18. Cosmid Vectors
•CosmidVectors(5-7kb)are
hybridbetweenplasmidsand
phages.Theycontainantibiotic
genesandreplicationoriginfrom
plasmidsand'cos’sitesfrom
phageswhicharenecessaryfor
packagingofDNA.
•Thesevectorscanclonelarger
fragmentsofsizerangingfrom35to
50kb.

19. Expression Vectors
•ExpressionVectorsare
vectorsthatcarryhost
signalsthatfacilitatethe
transcription and
translationofaninserted
gene.
•Theyareveryusefulfor
expressingeukaryotic
genesinbacteria.

20. Yeast Artificial Chromosome ( YACS )
•YeastArtificialChromosomes
(YACS)areyeastvectorsthat
havebeenengineeredto
containacentromere,
telomere,originofreplication
andaselectablemarker.
•Theycancarryupto1000kb
ofDNA.
•Theyareusefulforcloning
eukaryoticgenes.

21. Bacterial Artificial Chromosomes
•BacterialArtificialChromosomes
(BAC)vectorsareplasmids
constructedwiththereplication
originofE.coliFfactorandso
canbemaintainedinasingle
copypercell.
•ThesevectorscanholdDNA
fragmentsofupto300kb.
•Recombinant BACs are
introducedintoE.coliby
electroporation.

22. Host Cells
•rDNaintroducedintosuitablehost.
•ThehostarethelivingcellsinwhichthecarrierofrDNA
moleculeorvectorscanbepropagated.
•Hostcellscanbeprokaryoticoreukaryotic.

23. Applications of rDNA Technology
•VarioustherapeuticproductsaremadebyusingrDNA
technologysuchasproteins,hormones,immunemodulators,
plasminogenactivatorfactor,bloodclottingfactors,insulin,
growthhormonesandseveralenzymesetc.
•Diagnosisofmoleculardiseases:
•Sicklecellanemia, thalassemia,familial
hypercholesterolemia,cysticfibrosis.
•Prenataldiagnosis:
•DNAfromcellscollectedfromamnioticfluid,chorionicvilli.

24. •GeneTherapy:
•Thisisachievedbycloningageneintoavectorthatwill
readilybetakenupandincorporatedintogenomeofahost
cell.ADA(Adenosinedeaminase)deficiencyhasbeen
successfullytreated.
•ApplicationsinAgriculture:
•Geneticallyengineeredplantsarehighyieldingandpest
resistant.Goodqualityoffoodandincreasedyieldofcropsis
alsopossible.

25. Hybridoma Technology
•In1975,thesetechnogy
developedbyGeorgesJ.F.
KohlerandCesarMilstein.
•In1984,theysharedaNobel
Prizeforthisdiscovery.
•Theymakeahybridcellthat
willmakeanumbersof
monoclonal antibodies
againstantigen.

26. •Thehybridcellhasthecapacityofantibodyproduction
derivedfromB-cells(spleencell).
•Atthesametimeitcandividecontinuouslybythequality
derivedfrommyelomacell.
•Bycombiningthedesiredqualitiesofboththecells,the
technogyensureslargeantibodyproductionofsingle
specificity.
•Specifichybridomas(spleencellandmyelomacell)obtain
monoclonalantibodiesinartificialmedia,thistechnology
calledasHybridomaTechnology.
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.Immunizationofspecificanimalwhichgenerate
Hybridomacellwithspleencell.
2.Isolationofmyelomacell.
3.Fusionbetweenspleencellandmyelomacell.
4.SelectionofHATmedium.
5.IsolationofHybridomaCell.
6.ScreeningofHybridomaCell.

30. Applications of Hybridoma Technology:
1.Serological:
•IdentificationofABObloodgroup.
2.Diagnosis:
•Detectionofpregnancybyassayingofhormoneswithmonoclonal
antibodies.
•Separationofonesubstancefromamixtureofverysimilarmolecules.
3.Immunopurification:
•Purificationofindividualinterferonsusingmonoclonalantibodies.
•InactivationofT-lymphocytesresponsibleforrejectionoforgan
transplant.

31. 4.Therapy:
•Removaloftumorcellsfrombonemarrow.
•Treatmentofacuterenalfailure.
•Treatmentofmalignantleukemiacells,Bcelllymphomaand
avarietyofallograftrejectionsaftertransplantation.

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
•TheyareshortDNAorRNAmoleculesthathaswide
rangeofapplications.
•Antisenseoligonucleotides(ASO)aresinglestrand
ofDNAorRNAthatarecomplementarytoachosen
sequence.
•Theyarechemicallysynthesizedfromprotected
phosphoramidesorchemicallymodifiednucleosides.

37. •Mechanism of Action

38. ASO As Therapeutic Agent
•MuscularDystrophy
•Groupofdiseasesthatcauseweakeningandbreakdownofmuscles.
ASOtherapyusedtoremovemutatedExon.
•Cancer
•ThehighspecificityofbindingofASOtotargetmRNAmakethese
compoundsusefulastherapeuticagentsagainsthumancancer.
•Suppressesmalignantcells.
•Thalassemia
•Antisense2’-O-methylribooligonucleotidesweretargetedagainst
specificsequenceelementsinmutatedhumanbeta-globinandcan
repairthalassemia.

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

40. Gene
Therapy

41. Gene Therapy
•Definition:Genetherapymaybedefinedastheintroduction
ofanormalfunctionalgeneintocellofpatientwhich
containsthedefectivealleleofconcernedgenewiththe
objectiveofcorrectingageneticdisorderoranacquired
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 micropipetteis 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.
SometimeschemicalsareusedtomakeeasydeliveryofDNAinto
recipientcell.Usingdetergentmixture.Certainchargedchemical
compoundse.g.calciumphosphate,dextranorlipidsare
mixedwithfunctionalcDNAofdesiredfunction.Themixtureis
introducednearthevicinityofrecipientcells.

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. •SevereCombinedImmunodeficiency(ADA-SCID):
•Alsoknownas'Alimphocytosis’andBubble-boydisease.
•SCIDcausedduetodefectofgenecodingAdenosinedeaminase.
•GeneofADAisintroducedforitstreatment.
•OrnithineTranscarboxylase(OTC)deficiency:
•Leadstoaccumulationofammoniaandcanbetreatedbygenetherapy.
•CysticFibrosis:
•Adenovirusvectorswasusedtodeliveranormalionchannelprotein
toairwaycellsinapatient’slungs.

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)
Concentration Vs 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, 4
th
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