VIRAL AND NON VIRAL GENE TRANSFER

This Slide is prepared by JYOTIRMOY BHATTACHARYYA

1. PRESENTED BY
JYOTIRMOY BHATTACHARYYA
M.PHARM 2
ND
SEMESTER
ROLL NO. 180520011004

2. CONTENTS
What is gene therapy ?
Gene Transfer
Introduction
Gene Transfer Techniques
Non viral delivery systems
Physical Methods
Chemical Methods
Viral delivery systems
Conclusion
References

3. What is gene therapy ?
GenetherapyisanExperimentaltechniquethatusesgenes
totreatorpreventdisease.
Inthefuture,thistechniquemayallowdoctorstotreata
disorderbyinsertingageneintoapatient’scellsinsteadof
usingdrugsorsurgery.

4. Gene Transfer
Itisdefinedasatechniquetoefficientlyandstably
introduceforeignintothegenomeofthetargetcells.
Theinsertionofunrelated,therapeuticgeneticinformation
intheformofDNAintotargetcells.

5. Introduction
Therearedifferentreasonstodogenetransfer.Perhaps
foremostthesereasonsisthetreatmentofdiseasesusing
genetransfertosupplypatientswiththerapeuticgenes.
Therearedifferentwaystotransfergenes.Someof
methodsinvolvetheuseofavectorsuchasavirussoitcan
takethegenealongwithitwhenitentersthecell.
Itprovidesanovelapproachfortheinvestigationand
potentialtreatmentofavarietyofdisease.

6. Gene Transfer Techniques
Basedonthevectorsusedthegenetransfertechniquescan
bedividedas
•Nonviralmethods
•Viralmethods

7. Non viral delivery systems
Nonviralsystemscompriseallthephysicalandchemical
methods.
Generallyincludechemicalmethodseitherchemical
methods,suchascationicliposomeandpolymers,or
physicalmethods,suchasgenegun,electroporation,
particlebombardment,ultrasoundandmagnetofaction.

8. Physical Methods
DNAparticlebombardment
bygenegun
Goldortungstenspherical
particlesarecoatedwith
plasmidDNA andthen
acceleratedtohighspeedby
pressurizedgastopenetrateinto
targettissuecells.
Actuallyitisamodification
techniquecalled“biolistic”,
originallydevelopedforplant
transgenesis,butnowusedfor
invitroandinvivogenedelivery
intomammaliancellstoo.

9. Ultrasound
Ultrasoundcanmakesomenanomericporesinmembranetofacilitate
intracellulardeliveryofDNAparticlesintocellsofinternalorgansor
tumours.
Themostimportantlimitationsofthesystemislowefficiencyofit
especiallyinvivo.
Electroporation
Electroporationistemporarydestabilizationofthecellmembrane
targetedtissuebyinsertionofapairofelectrodesintoit.
DNAmoleculesinthesurroundingmediaofthedestabilizedmembrane
wouldabletopenetrateintocytoplasmandneoplasmofthecell.
Electroporationhasbeenusedinvivoformanytypesoftissues,suchas
skin,muscle,lung,HPRTgenedeliveryandtumourtreatment.
Someproblemsinthismethodare
Thedifficultyinsurgicalprocedureintheplacementofelectrodesintothe
internaltissues
Thehighvoltageappliedtotissuemightdamagetheorganandaffectgenomic
DNAstability.

10. Magnetofaction
Inthismethodthemagnetic
fieldsareusedtoconcentrate
particlescontainingnucleic
acidintotargetcells.
Inthisway,themagnetic
forceallowsaveryrapid
concentrationoftheentire
appliedvectordoseonto
cells.
Magnetofactionhasbeen
adaptedtoalltypesofnucleic
acids,nonviraltransfection
systemsandviruses.

11. Chemical methods
Cationicliposome
Cationicliposomesaremoreimportantcurrentnonviralpolycationic
systems,whichcompactnegativelychargednucleicacidsleadtothe
formationofnanomericcomplexes.
Cationicliposomeshaveuniquecharacteristics–
Capabilitytoincorporatehydrophilicandhydrophobicdrugs
Lowtoxicity
Noactivationofimmunesystem.
Targeteddeliveryofbioactivecompoundstothesiteofaction.
Cationicliposomesarebeingusedingenedeliveryintolung,skeletal
muscles,spleen,kidney,liver,testis,heartandskincells.

12. CationicPolymers
Cationicpolymershavealsobeenusedextensivelyforgenetransfer.
UponmixingwithDNA,thesepolymersformnanosizedcomplexes,
oftencalledpolyplexes.
Amongcationicpolymers,PEIisconsideredoneofthemosteffective
polymer-basedtransfectionagents.
PEIleadstoaninfluxofchloridecounterionswithinthecompartment
andabuildupofosmoticpressurethatcausestheswellingandrupture
oftheendosomalmembrane.
Recently,morepolymerswithimprovedbiocompatibilityand
biodegradabilityhavebeenreporteddemonstratingequalorsuperior
performancecomparingtonondegradablePEIs.

13. Some Polymers Commonly Used for Gene Transfer
Polymer Abbreviation Unique feature
Poly(ethylene)glyc
ol
PEG Inert
PolyethyleniminePEI cationic
Poly(L-lysine) PLL Cationic
Poly(N-
vinylpyrrolidone)
PVP Neutral
Poly(propylenimin
e)
PPI Dendromer
Poly(amidoamine)PAMAM Dendromer
Triethylenetetrami
ne
TETA Cationic
Poly(allylamine) Cationic
Poly(phosphazene
)s
PPZ Biodegradable

14. Viral delivery systems
Virusesarenaturallyevolvedvehiclesthatefficiently
transfertheirgenesintohostcells.
Choiceofviralvectorisdependentongenetransfer
efficiency,capacitytocarryforeigngenes,toxicity,stability,
immuneresponsestowardsviralantigensandpotential
viralrecombination.
ThereareawidevarietyofvectorsusedtodeliverDNAor
oligonucleotidesintomammaliancells,eitherinvitroorin
vivo.
Themostcommonvectorsystembasedonretroviruses,
adenoviruses,herpessimplexviruses,adenoassociated
viruses.

16. Thethreecommonlyusedviralgenetransfersystemsare-
•Retrovirus(RV)
•Adenovirus(AV)
•AdenoAssociatedVirus(AAV)

17. RetroVirusVector
Commonlyemployedvectors,derivedfromMurineLeukemia
Virus(MuLV).
VirusgenomehastwosinglecopyRNAmolecules,complexedwithviral
coreproteins,surroundedbylipidenvelope.
Applications
Ex-vivogenetherapy.
In-vivogenetransferusingretroviralvectorsforsuicidegenesusedin
braintumour.
TreatmentofT-lymphocytedeficiency(ADA),TumourInfiltrating
Lymphocytes(TIL),Bonemarrowcells(ADAdeficiency,Gauchers
disease),hepatocytes(LDLreceptordeficiency)andmelanoma.

18. AdenoVirusVectors
ThesearenonenvelopedDNAviruses,lineargenomeanddouble
strandedDNAmoleculeofabout36kb.
Adenoviralvectorshavebeenisolatedfromalargenumberofdifferent
speciesandmorethan100differentserotypeshavebeenreported.
Adenovirusestype2andtype5canbeutilizedfortransferringboth
dividingandnondividingcellsandhavelowhostspecificity.
Applications
Invivogenetherapy–transducenondividingandterminally
differentiatedcells.
Transfectcellsinvivointheintactorgan.
Genetherapyforcystictherapy.
GenetherapyofmuscleinliverandtherapyofdiseaseofCNS.

19. AdenoAssociatedVirusVector
MembersofParvovirusfamily.
Heatstableandresistanttovariouschemicals.
Dependonvirus–cannotreplicateitsown,anothervirusisnecessary
forreplicate,.
Majordisadvantagesofthesevectorsarecomplicatedprocessofvector
productionandthelimitedtransgenecapacityoftheparticles.
Applications
Usedinhaematopoieticstemcellsfortreatmentofß-thalassemiaand
sicklecellanaemia.
ß-thalassemiaerythrocytecontainsinsufficientß-globinchain
whereas,mutantß-globinchainsareproducedinsicklecell.

20. Conclusion
Althoughnumerousviralandnonviralgenedeliverysystemshavebeen
developedinthelast3decades,allofthemhavesomedisadvantages
thathavemadesomelimitationsintheirclinicalapplicationandyetno
deliverysystemhasbeendesignedthatcanbeappliedingenetherapy
ofallkindsofcelltypesinvitroandinvivowithnolimitationandside
effects.Soitseemsthattheprocessofdevelopingsuccessfuldelivery
systems,especiallynonviralsystems,foruseininvivoisstillinits
adolescenceandmoreeffortsareneeded.Totally,keystepseffectivein
improvingthecurrentlyavailablesystemsincludethefollowing:(1)
improvingextracellulartargetinganddelivery,(2)enhancing
intracellulardeliveryandlong-timeexpression,and(3)reducing
toxicityandsideeffectsonhumanbody.

21. References
1.NayerossadatNet.al,“Viralandnonviraldeliverysystemsforgene
delivery”,AdvancedBiomedicalResearch.2012;1(2):1-12.
2.StoneD.Novelviralvectorsystemsforgenetherapy.Viruses
2010;2:1002-7.
3.KatareDP,AeriV.Progressingenetherapy:Areview.I.J.T.P.R
2010;1:33.
4.ASIANJ.EXP.BIOL.SCI.Vol1(1)2010:208-218.
5.SmithKR.GeneTherapy:ThePotentialApplicabilityofGeneTransfer
TechnologytotheHumanGermline.IntJMedSci2004;1:76-91.

22. THANK YOU