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TÉCNICAS DE ALTERACIÓN DE LA EXPRESIÓN GÉNICA

Una fecha para recordar : 2001, la publicación (por duplicado) de un borrador del genoma humano

La comparación entre el genoma anotado humano y de otras especies animales ha permitido establecer correlaciones entre genética humana y animal, o entre genética de tejidos sanos y enfermos

Estos datos incluyen tanto proteínas conocidas como proteínas cuya función es totalmente desconocida, y aquí comienza el reto de la biología en la era postgenómica, asignar una información funcional a las proteínascodificadas (GENÓMICA FUNCIONAL O FUNCIONÓMICA)

Abordajes para asignar una función a un gen

Experimentos de “knockout”o silenciamiento génico

Experimentos de “knock-in”o sobreexpresión

Ratones KOSilenciamiento químicoSilenciamiento génico

WHAT IS AN ANTISENSE?

Antisense technology involves the use of synthetic segments of DNA or RNA called ON to stop the production of any protein of interest by interacting at the genetic level with target strands of mRNA.

Antisense binds with mRNA and inhibits translation of proteins.Traditional pharmaceuticals intervene after a disease-causing protein is formed, antisense

therapeutics block mRNA translation intervening before a protein is formed

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Ribosomebindingprevented

Ribosometranslocationstopped

Nascent protein

INHIBITION OF GENE EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES

Hybridization

1. Inhibition of protein synthesis by interfering translation

2. Destruction of the mRNA due to the activity of RNase H

Problems of DNA OAS:

Inespecific toxic effects

Requires repetitiveapliccation because mRNA iscontinuosly produced

Degradation of OAS

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WHAT IS THE PROBLEM WITH MY ANTISENSE?

1) ENDO- AND EXONUCLEASES

Design OAS resistant to nucleases (i.e. metilphosphonate)

1. Passive Diffusion2. Receptor Mediated Endocytosis3. Fluid Phase Pinocytosis4. Adsorptive Endocytosis

2) HOW CAN THE ANTISENSE REACH ITS TARGET?

One more problem:They are charged!

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DNA S-DNA PNAS’-Methoxyethyl

Morpholino

M-DNA

CH3

Non-specificeffects-+ + ++ + +

Directs RNAse H-+ + ++ + +

Hybridization strength (to RNA)++ + + + +

Resistant tonucleases+ + ++ +-

Charged-+ + ++ + +

Phosphodiester Phosphorothioate Methylphosphonate

ImprovingImproving stabilitystability, , efficiencyefficiency ofof deliverydelivery and and selectiveselective unionunion ofof OASOAS

phosphorothioatephosphorothioate MethilhosphonateMethilhosphonate

phosphorodiamidatephosphorodiamidate

Morpholinos

AdvantagesAdvantages::HigherHigher especificityespecificity thanthan ASOASOResistantResistant toto nucleasesnucleasesGoodGood solubilitysolubility

DisadvantagesDisadvantages::DesignDesign ofof controlscontrols ((theythey do do notnot degrade degrade mRNAmRNA))DesignDesign ofof morpholinosmorpholinos (5(5´́UTR)UTR)DeliveryDelivery (as (as anyany OAS)OAS)

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Secondary structure of a hammerhead ribozymeannealed to its target. The ribozyme is in uppercaseand the target is in lowercase. Cleavage occurs as indicated just downstream of the X in the NUX targetsite. The targeting arms of the ribozyme anneal to thetarget to form stems I and III while stem II is formedinternal to the ribozyme. Catalytically inactive ribozymes are produced by substituting the conservedG with a C as indicated.

RIBOZYMES

Advantages:Higher especificity than ASOAllelic discriminationHigher potency than ASO

Types:Hairpin ribozymes

(YNGUC)Hammerhead rybozymes

(NUX or NXX)

OH

3’

P

5’

+

product

CataliticCatalitic mRNAsmRNAs (intron auto-splicing, viral auto-hydrolisis, Rnase P, spliceosome, ribosome...)

RNA INTERFERENCE 1st description : Fire et al., 1998Antecedents:- Blocking effect of OS and OAS - Antiviral response in plants- Cossupresion in plants (PTGS)- Quelling in Neurospora

Different mechanismHigher potency

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WHAT DO WE KNOW ABOUT THE MECHANISM OF RNAi?

1)1) InducedInduced by by dsRNAdsRNA and and requiresrequires a a proteinprotein complexcomplex

2)2) TranslationTranslation ofof mRNAmRNA isis blockedblocked by by thethe complementarycomplementary RNA RNA ofof thethe dsRNAdsRNA

3)3) SeparationSeparation ofof dsRNAdsRNA requiresrequires anan helicasehelicase and ATPand ATP

4)4) AntisenseAntisense RNA RNA ofof dsRNAdsRNA recognizesrecognizes targettarget mRNAmRNA

5)5) IdentificationIdentification ofof genes genes involvedinvolved in in RNAiRNAi pointspoints toto helicaseshelicases, , RNaseRNase III III relatedrelated nucleasesnucleases, , argonauteargonaute proteinsproteins and and RdRPsRdRPs

6)6) In In allall cases, cases, dsRNAdsRNA isis breakbreak downdown by a by a specificspecific RNaseRNase calledcalledDicerDicer

PROPOSED MECHANISM OF RNAi AND OTHER RNA SILENCING PHENOMENA

•Induced by dsRNA and requires a protein complex

•dsRNA is cleaved by Dicer intosiRNAs

•siRNA associates with a ribonucleoprotein complex thatallows siRNA unwinding

•Unwinding of siRNA activatesRISC

•The as strand of siRNA binds totarget mRNA and guides itscleavage (by “slicer”?)

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Unc 22

Unc 52

AMPLIFICATION OF RNAi: The role of RdRP

First model: (plants, fungi, nematodes)

RdRP creates new dsRNA

RNAi does not require RISC*

RNAi requires a free OH in 3´

RNAi can affect differentaleles in heterozygotes

Second model: (flies and mammals)

No RdRP present

siRNA guides mRNA cleavage by RISC

Requires a P in 5´ (autentification)

First model: (plants, fungi, nematodes)

1 2

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In the last decade, few areas of biology have been transformed as thoroughly as RNA biology, due to discovery of small (20-30 nt) noncoding RNAs that regulates genes and genomes

The effects of these small RNAs are generally inhibitory

The small RNAs serve as specifical factors that bound effectorproteins to target nucleic acid moleules via base-pairing interactions

The core of effector machinery is a member of the Argonaute family

Chromatin structure

Chromosomal segregation

transcription

RNA processingRNA stability

translation

The three main classes of small regulatory RNAs

Martin Jinek & Jennifer A. Doudna (2009) Nature 457, 405-412

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Carthew RW and Sontheimer EJ. Cell 2009, 136(4):642-55.

Core features of miRNA and siRNA silencing

miRNA are mainly endogenousand siRNA exogenous. They alsodiffer in the type of match (complete versus incomplete compementarity).

ss forms asociates into de RISC complexes

Both are involved in post-transcriptional regulation

The similarities led to the searchof common mechanisms in theirprocessing, and in this way wefound:

Dicer proteinsAgo proteins21-23 ds RNA

Helicase PAZRNAse III

DSRMDICER

••DicerDicer firstfirst describeddescribed in in DmDm••OrthologousOrthologous in in mammalsmammals, , fungi, fungi, plantsplants, Ce..., Ce...••AllAll DicerDicer orthologousorthologous show show nuclear nuclear localizationlocalization signalssignals

PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their two-nucleotide 3' overhangs.

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Hs Dicer

Dm Dicer-1Dm Dicer-2Hs Drosha

Dm DroshaDm Pasha

Dm R2D2Dm Ago2Dm Ago1-4

Domain structures of proteins involved in the RNA silencing pathway

Filopowicz et al., Curr Op Struct Biol 15: 331-341 (2005)

Model of DICER function

Martin Jinek & Jennifer A. Doudna (2009) Nature 457, 405-412

DICER structure and function

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RNAse III proteins and their mechanism of action

Nuclear proteins

Kim, Han & Siomi, (2009) Nature Reviews Molecular Cell Biology 10, 126-139

Martin Jinek & Jennifer A. Doudna(2009) Nature 457, 405-412

Argonaute proteins structure

Farazi, T. A. et al. Development 2008;135:1201-1214

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YL Wang et al. Nature 456, 921-926 (2008)

Argonaute proteins structure

Catalytic site

3’ end ofguide strand

5’ end ofguide strand

Diversity of siRNA sources and genesis of siRNA

Pre-RISCAspectos a destacar:Aspectos a destacar:

Dependiendo de su origen, puede existir una fase nuclear

La selección de la hebra guía y la hebra pasajera no requiere la presencia del mRNA

Si el apareamiento no es perfecto, se produce inhibición de la traducción (off-target effect)

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Mechanisms of siRNA silencing

Carthew RW and Sontheimer EJ. Cell 2009, 136(4):642-55.

1. Canonical siRNA

2. miRNA-like

3. H

eter

ocho

rmat

info

rmat

ion

4. Secondary dsRNAs

5. Secondary siRNAs

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MECHANISMS OF miRNA SILENCING

Carthew RW and Sontheimer EJ. Cell 2009, 136(4):642-55.

Krützfeldt and Stoffel, Cell Metab. 2006 Jul;4(1):9-12.

microRNAs FUNCTIONS

MicroRNAs constitute as many as 2%–3% of all genes in the genome

Many miRNAs are clearly involved in important cellular processes such as differentiation and development andmetabolism

Up to one-third of all human genes may be miRNA targets

These studies point to novel therapeutic opportunities based ontargeting of miRNAs.

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J Krützfeldt, MN Poy & M Stoffel Nature Genetics 38, S14 - S19 (2006)

Strategies to determine the biological function of microRNAs

miRNA identification

Forward genetics (mutants)Directional cloningbioinformatics

miRNA microarrays

Target identification

Strategies to determine the biological function of microRNAs

Krutzfeldt, J. et al. Nucl. Acids Res. 2007 35:2885-2892;

miRNA silencing

Dicer KO

miRNA genes KO

miRNA target mutations

Antisense targeting of miRNA:ANTAGOMIRS

miRNAsmiRNAs silencingsilencing isis dosedose--dependentdependent, , Fast Fast developmentdevelopment (24 h)(24 h)Long Long lastinglasting (> 3 (> 3 weeksweeks))SpecificSpecific ofof miRNAmiRNA, , allowsallows thethe studystudy ofof single single miRNAsmiRNAs fromfrom a a commoncommon precursor. precursor.

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ENDOGENOUS siRNAs and microRNAs: SILENCE FROM WITHIN

Three main categories (depending on precursors)

RNA pol

miRNAEndogenous

siRNArasiRNA (repeat-associated siRNA)Y piRNAS

RNA polRNA pol

RdRP RdRP

RNA pol RNA pol

Dicer Dicer-like

Dicer

miRISC EndogenoussiRISC

RITS (RNAi-induced transcriptional silencing)

traslation mRNAstability

heterochromatin

Drosha

In Dm, rasiRNAs and piRNAs are producedby a DICER-independent pathway

They function through the PIWI subfamilyto ensure silencing of retrotransposons

Sontheimer and Carthew, Cell 122, 9-12 July 2005

PIWI is a member of the Dm Argonautefamily, which is part of theribonucleoprotein complex forming RISC

There are 5 members of the Dm Argonautefamily,:•AGO1 and AGO2, that bind miRNA•Aub, Piwi and Ago3 (the PIWI subfamily), that are enriched in germline

Colin D. Malone and Gregory J. Hannon. Cell 136: 656-668 (2009)

Ras-siRNAs and piRNAs-Different size (24-26 nt)-Not dsRNA precursor-Different Argonaute proteins (Piwi clade)

Ping-pong cycle amplifies small RNA population against target molecules

PrimaryPrimary piRNAspiRNAs (as)(as)

GenesisGenesis ofof sensesensepiRNAspiRNAs + + AGo3AGo3((secondarysecondary piRNApiRNA) )

RdRP can amplify the ping-pong cyclegenerating more piRNAs (not in humans)

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•Primary piRNAs are AS toexpressed transposons

•Primary piRNAs cleave theytargets, creating secondaryS piRNAs (bound to AGO3 orMIWI2)

•The AGO3-bound S piRNAcleaves transposons AS sequences, creatingsecondary AS piRNAs(bound to Aub or MILI)

•The ping-pong cycle uses primary piRNAs againstactive transposons, optimizing the inhibition ofmobile elements

PING-PONG CYCLE

miRNAs, your RNAs....

Roles of RNAi

Processing of mRNAhairpin precursors

Control timing ofdevelopment

Generation of miRNA (stRNA)

Arrest translation

Degradation ofexo- dsRNA

Generation of siRNA

mRNA degradation

Combating viral infections

Degradation ofendo- dsRNA

TransposonsimmobilizationChromatin

remodelling

Degradation ofmobile elements

Generation of piRNA

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PTGS IN PLANTS

PTGS IN PLANTS: The mechanisms

Systemic propagation:Sequence-specificSpreaded to distant parts(involves RNA)

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PTGS IN PLANTS: the mechanisms

Model for dsRNA-dependent dimerization and activation of PKR. Binding to dsRNA is required to induce a conformational change toexpose dimerization sites within intact PKR. Activated PKR (PKR*) leads to eIF-2 α phosphorylation and global arrest of protein synthesis.

RNAi IN MAMMALIAN CELLS

PKRPKR

PKR PKR

PKR* PKR*

ATPADP elF-2α

elF-2α∗P

INHIBITION OF PROTEIN SYNTHESIS

APOPTOSIS

dsRNA

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Synthetic 21-nt siRNA duplex prepared by chemical synthesis (23) aligned to a target mRNA. Target regions are selected such that siRNAsequences may contain uridine residues in the 2-ntoverhangs. Uridine residues in the 2-nt 3'-overhangcan be replaced by 2'-deoxythymidine without loss ofactivity, which significantly reduces costs of RNA synthesis and enhance nuclease resistance of siRNAduplexes

Methods for the delivery of siRNAs to somatic mammalian cells

Plasmid-based expression of short hairpin loops. The polymerase III promoter of H1 RNA (human RNase P RNA) drives the transcription of a nineteen-base-pair/nine-nucleotide-loop RNA hairpin. Thetranscription is terminated by the encounter of a polythymidine tract (T5) after the incorporation of twoto three uridine residues encoded by the T5 element.

Brummelkamp et al., Science 296, 550-53 (2002)

Elbashir et al., Nature 411, 494-8 (2001)

Brummelkamp et al., Science 296, 550-53 (2002)

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ATransient

applicationLong dsRNA

Interferon responseApoptosis

Transientapplication siRNA

RNAiKnock down

B

Transient orpermanent

endogenousexpression of siRNA Production

of siRNA

RNAiKnock down

C

RNAiKnock out

Vector forendogenousproductionof dsRNA

DPermanent

endogenousexpression oflong dsRNA

Which RNAiapproach to use?

H1-promoter

Which RNAiapproach to use?

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siRNA DESIGN

A guide for siRNA selection:

1) Target mRNA analysis: • select region: coding vs. non-coding, regions free of

interactions with mRNA binding proteins…• Aditional constrains: analysis of orthologous, splice variants…

2) Database search for published and validated siRNA

3) Selected algorithms and siRNA sequence selection tools:• intinsic sequence and stability features• accesibility (secondary structure)• specificity (genome-wide search for unintentionated matches)

4) Prevalidation of siRNAs

PREVALIDATON OF siRNAs

Plasmids that carry the targetsequence fused to a reportergene are coexpressed with thetarget-specific siRNA

The prevalidated siRNA can thenbe used to validate the depletionof endogenus mRNA

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THE ART OF SELECTING siRNAs

•Sequence asymmetry but structural symmetry

•Differences in thermodynamic stability of the 2 base-paired ends: The strand with theless stable 5’ end is favorably loaded into RISC (i.e. the guide strand): Selection ofduplexes with the less stable (A+U)-enriched 5’ on the guide strand

•Low to medium C+G content (30-50%): Enough to stabilize it but not too much tointerfere with RISC loading

• The center of the duplex may havelow internal stability (pos. 9-14)

• 5’ U-specific interaction contributesto effective RISC assembly (in theguide strand)

•Avoid internal repeats orpalindromes that can formintrastrand secondary structures

Pei and Tuschl, Nature Methods 3, 670-75, September 2006

UU

SEQUENCE CHARACTERISTICS OF siRNAs

AACCAA

A + U enriched region

Pei and Tuschl, Nature Methods 3, 670-75, September 2006

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YesNo Alternative splicing

YesNo Transcriptional regulation

Physiological rangeOften not physiological

Dependent on natural promoterDependent on artificial promoterExpression level

High Bac coverage of themouse/human genome

Limited number of full-lengthORF cDNAs

Availability of rescueconstructs

Use of transgenic BACsModification of full-lengthcDNA clone inserts

Method

CONTROL OF RNAi

TheThe ultimateultimate control control ofof thethe specificityspecificity ofof lossloss ofof functionfunction by by RNAiRNAiisis thethe rescuerescue experimentexperiment..

-- CreatingCreating silentsilent pointpoint mutationsmutations in in thethe targettarget sitesite-- OverexpressingOverexpressing thethe cDNAcDNA ¿¿??-- TargetingTargeting a a sequencesequence in in thethe 33’’ UTR UTR ofof thethe cDNAcDNA ¿¿??-- UsingUsing scramblescramble siRNAsiRNA-- ExpressingExpressing anan orthologousorthologous gen in a BAC (gen in a BAC (KittlerKittler et al)et al)

RNAi RESCUE BY BAC TRANSGENESIS

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THERAPEUTIC POTENTIAL OF RNAi

Davidson and Boudreau, Neuron 53, 781-88 March 2007

Protecting against viral infection (VIH)

Gene therapy of inherited diseases

Antitumoral therapy

Generation of transgenic knockouts

Gene-specific drug development

METHODS OF GENE DELIVERY

Non- toxic

Biodegradable

Specific for a given target cell

Non inflamatory or immunogenic

Stable for storage

With high capacity for genetic material

Provided by an efficient transfection method

Able to be produced at large ammounts and low cost

The ideal system for gene trasfer with biomedical aplications should be:

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METHODS OF GENE DELIVERY

FactorsFactors toto considerconsider

MethodMethod:: how how toto deliverdeliver genes genes totospecificspecific cellscells oror tissuestissues

Time and dosis:Time and dosis: how how muchmuch and how and how long long thethe gene gene willwill be be expressedexpressed

SiteSite:: ofof gene gene deliverydelivery

AdversedAdversed consequencesconsequences:: ToxicToxiceffectseffects, , immunologicalimmunological reactionsreactions......

1) Viral 1) Viral vectorsvectorsAdenovirusAdenovirusRetrovirusRetrovirusLentivirusLentivirusAdenoAdeno--AssocitedAssocited virus (AAV)virus (AAV)Herpes Herpes simplexsimplex virus (HSV)virus (HSV)AlphavirusAlphavirusVacciniaVaccinia virusvirus

2) Non2) Non--viral viral vectorsvectorsChemicalChemical methodsmethodsElectricalElectrical methodsmethodsPhysicalPhysical methodsmethods

aa

bb

TRANSFECTION VERSUS INFECTIONTRANSFECTION VERSUS INFECTION

WhyWhy wewe willwill use nonuse non--viral viral vectorsvectors??

AdvantagesAdvantages::CheapCheap productionproductionNo No biosafetybiosafety isuesisuesSizeSize ofof thethe constructconstruct isis notnot a a limitinglimiting factorfactor

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CALCIUM PHOSPHATE TRANSFECTION

CHEMICAL METHODSCHEMICAL METHODS

DNADNA

--

--

ClCl22CaCa solutionsolution

PhosphatePhosphate bufferbuffer

CaPCaP--DNA mixDNA mix

-----

---

CaPCaP precipitateprecipitate

AddAdd toto cellscells

AsessAsess transfectiontransfection

DNA

-

-- - -

--

--

-

+++

+ +

Cationic liposomes

- ---

--

--

-

+

+

+

++

+

+

Cationic lipid – DNA complexEndocytosis

Release and transport of DNA

to nucleus

LIPOFECTION

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DNA

Transferrin Poly-L-Lysisne

MODIFIED LIPOFECTION

Poly-L-Lysisne – Transferrin – DNA complex

DNADNA--lipidlipid complexcomplex cross cross bloodblood--brainbrain barrierbarrier and and

entersenters thethe cellcell by receptorby receptor--mediatedmediated transporttransport

DNA DNA isis encapsidatedencapsidated in ain apegilatedpegilated liposomeliposome, ,

((conjugatedconjugated withwith PEG) and PEG) and attachedattached toto mAbmAb againstagainst

insulininsulin receptorreceptor

TheThe heterologous DNA heterologous DNA goesgoes toto thethe nucleusnucleus and and

thethe desireddesired proteinprotein isisproducedproduced

proteinprotein

IMMUNOLIPOSOMES

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MICROINJECTIONPHYSICAL METHODSPHYSICAL METHODS

Pressure gaugeDisk with DNA-coated particles

Stop plate

Sample goes here

Vacuum lineGas line

Direct transformation

The most common technique for direct transformation is microprojectile or particle bombardment. This technique is based on the use of a “particle gun” or “gene gun”. The expression vector with YFG is precipitated onto tungsten or gold particles which are then shot into the plant tissue. In most cases we will see only transient expression (i.e. the DNA does not integrate into the genome but is transcribed until it degrades). In a small percentage of the cells the DNA will integrate and we will see stable expression. The main drawback of this technique is that often multiple copies of the transgene insert. It is also necessary to have an in vitro regeneration system in place.

Rupture disk

Vacuum chamber

BIOLISTIC

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MAGNETOFECTION

cDNA

GFP

In vitrotranscription

In vitropoli(A)

RNA

Neurons grown on coverslip

microporation

2-4 h for GFP traslation

Microporator device

ELECTROPORATIONELECTRICAL METHODSELECTRICAL METHODS

Most efficentMost versatile

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SINGLE-CELL ELECTROPORATION•• AproximaciAproximacióón de la n de la

micropipetamicropipeta cargada con el cargada con el DNA a la membrana de la DNA a la membrana de la ccéélulalula

•• AplicaciAplicacióón del pulso de voltajen del pulso de voltaje

•• Apertura de poros en la Apertura de poros en la membranamembrana

•• Entrada de las Entrada de las moleculasmoleculas de de DNA a favor de gradiente DNA a favor de gradiente electroquelectroquíímicomico

•• Seguimiento del marcaje Seguimiento del marcaje fluorescente del DNA fluorescente del DNA mediante sistema de imagenmediante sistema de imagen

NUCLEOFECTIONhttp://www.amaxa.com/products/technology/how-it-works/

VIRAL VECTORS

Virus DNA

Host DNA

Viral DNA integratedinto host

Virus mRNA

Viral proteins

Infection

Integration

Transcription

Translation

WhyWhy use viral use viral vectorsvectors

Virus are Virus are obligatoryobligatory intracellularintracellularparasitesparasites

VeryVery efficientefficient at at transferringtransferring DNADNAintointo hosthost cellscells

TheyThey can can targettarget cellscells specificallyspecifically((dependingdepending onon viral viral attachmentattachmentproteinsproteins))

ExogenousExogenous genes can be genes can be insertedinsertedin place in place ofof nonnon--esentialesential viral genesviral genesthatthat are are deleteddeleted

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GENERATION OF VIRAL VECTORS FOR GENE DELIVERY

ReplicationReplication--competentcompetent virusvirus

ReplicationReplication--defectivedefective virus: AMPLICONvirus: AMPLICONDoesDoes notnot encodeencode structuralstructural proteinsproteinsCan Can notnot replicatereplicate beyondbeyond thethe firstfirst cyclecycle ofof infectioninfection

ElementsElements neededneeded toto generategenerateampliconsamplicons::

Transfer vectorTransfer vector: : plasmidplasmid ((containingcontainingpromoterpromoter, GOI, , GOI, oriori, , packagingpackaging signalsignal, , ΨΨ))

PackagingPackaging vectorvector ((cosmidcosmid oror cellcell lineslines) ) providingproviding thethe viral viral structuralstructural proteinsproteins

HelperHelper virusvirus: : packagingpackaging oror transfer vector transfer vector withwith deleteddeleted packagingpackaging signalsignal sequencesequence

Pac (Ψ) ori

Cotransfection with HSV-1 lackingpackaging sequences (helper virus)

Plasmid is replicated into concatemers,cut into genome-unit length molecules andpackaged into viral particles by the helpergenome

Helper-free amplicon

AMPLICON VECTOR SYSTEMAMPLICON VECTOR SYSTEM

ADENOVIRAL VECTORSand the mechanims of replication

NonNon--enevelopedeneveloped dsds DNA, 36 DNA, 36 KbsKbs

Express Express earlyearly (E1(E1--E4) and late (L1E4) and late (L1--L5) L5) proteinsproteins

Causes Causes benignbenign respiratory respiratory infectionsinfections in in humanshumans

SerotypesSerotypes 2 and 5 are 2 and 5 are commonlycommonlyusedused as as vectorsvectors

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Recombination in HEK cells

Cre-mediated excision

CharacteristicsCharacteristics

AdvantagesAdvantages::HighHigh titerstitersDividingDividing and nonand non--dividingdividing cellscellsWide Wide tissuetissue tropismtropismEasilyEasily modifymodify tissuetissue tropismtropism

DisadvantagesDisadvantagesTransientTransient expressionexpression ((notnot goodgood forfor

geneticgenetic diseasesdiseases))HighlyHighly immunogenicimmunogenicHighHigh titerstiters can be can be toxictoxicMore More suitablesuitable forfor cancercancer

immunotherapyimmunotherapy

GutlessGutless adenoviraladenoviral vectorsvectors ((ampliconamplicon))

+

ITR ψ CMV RSV pA loxP

VgEcR RXR+ Ponasterone A

ITR ψ pA loxP

MCSIRESEcd Promoter

ModifiedEcdysone receptor

Retinoid X receptor

EGFP

GOI

AdVgRXR(Receptor virus)

AdEGI

AdEGI-GOI

Ecdysone-inducible adenovirus constructs

InducibleInducible adenoviraladenoviral vectorsvectors ((ampliconamplicon))

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LinearizeLinearize withwith PmeIPmeI

LinearizeLinearize withwith PacIPacI

Transfect HEK

FollowFollowtransfectiontransfectionwithwith GFPGFP Harvest virus

AdEasyAdEasy adenoviraladenoviral vectorsvectors

CotransfectCotransfect intointo bacteriabacteriaSelectSelect withwith KanamycinKanamycin

ADENO-ASSOCIATED VIRUS VECTORS (AAV)

Ψ

A A plasmidplasmid containingcontaining onlyonlythethe essentialessential genesgenes

TheThe wholewhole AdVAdV

NonNon--pathogenicpathogenic hymanhymanparvovirusparvovirus, non, non--immunogenicimmunogenic

Wide Wide tissuetissue tropismtropism

NonNon--envelopedenveloped ssss DNA, 4,6 DNA, 4,6 KbsKbs

DependentDependent onon a a helperhelper virus virus (adenovirus (adenovirus oror HSV) HSV) forfor replicationreplication

AAVAAV-- 2 2 commonlycommonly usedused as vectoras vector

+

+

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CharacteristicsCharacteristics ofof AAV AAV vectorsvectors

AdvantagesAdvantages::IntegrationIntegration: : permanentpermanent expressionexpression in in fixedfixed positionposition ((ChrChr. 19). 19)No No insertionalinsertional mutagenesismutagenesisInfectingInfecting dividingdividing and nonand non--dividingdividing cellscellsSafeSafe

DisadvantagesDisadvantagesSizeSize limitationslimitations (4,9 (4,9 KbKb))Low Low titertiter ofof virus, virus, lowlow levellevel ofof gene gene expressionexpression

AAV can AAV can dimerizedimerize and and formform concatemersconcatemers

TheThe GOI GOI isis expressedexpressed in in twotwo halfshalfs

ITR are ITR are eliminatedeliminated by by includingincluding splicingsplicing sequencessequences

IncreassingIncreassing thethe packagingpackaging sizesize ofof AAVAAV

RETROVIRUS

MoloneyMoloney murinemurine lekemialekemia virus virus ((muLVmuLV))

EnvelopedEnveloped ssRNAssRNA vector, vector, withwith twotwoidenticalidentical ssRNAssRNA strandsstrands

ContainingContaining specificspecific enzimesenzimes, , includingincluding reverse reverse transcriptasetranscriptase

CapableCapable ofof integratingintegrating in in hosthostgenomegenome (DNA (DNA endonucleaseendonuclease))

Express Express alsoalso proteinsproteins forfor thethe coatcoat(gag) and (gag) and thethe envelopeenvelope ((envenv))

ContainsContains specificspecific cellularcellular tRNAtRNAOncogenicOncogenic

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RETROVIRAL VECTORS

GenerationGeneration ofof replicationreplication--defectivedefective retroviral retroviral vectorsvectors

Transfer Transfer plasmidplasmid vector:vector:TransgeneTransgeneLTR: LTR: promoterpromoter, , poly(Apoly(A), ), integrationintegration, , replicationreplication, RT, RTPrimer Primer bindingbinding sitesite (PBS = (PBS = oriori))RNA RNA packagingpackaging signalsignal ((ΨΨ))PolipurinePolipurine tracttract (PPT)(PPT)

PackagingPackaging vector:vector:••CellCell lineline stablystably transfected transfected withwithplasmidplasmid constructsconstructs expressingexpressinggag/gag/polpol and and envenv

oror••TransientTransient transfectiontransfection ofofplasmidplasmid constructsconstructs gag/gag/polpol and and envenv

Transient transfection oftransgene, gag/pol and env

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CharacteristicsCharacteristics ofof retroviral retroviral vectorsvectors

AdvantagesAdvantages::IntegrationIntegration: : permanentpermanent expressionexpressionPseudotypedPseudotyped virusvirus

DisadvantagesDisadvantagesOnlyOnly infectinginfecting dividingdividing cellscellsInsertionalInsertional mutagenesismutagenesis

CellCell deathdeathactivateactivate oncogenesoncogenesinhibitinhibit suppressorsuppressor genesgenes

NotNot packagedpackaged at at highhigh titerstiters

Pseudotyped packaging vectors

LENTIVIRAL VECTORS

PPTStop signalDNA-

DNA+Secondary DNA+

Triple DNA helix

Signal for nuclear entry

pro

pro

Retroviral Retroviral vectorsvectorsInfectionInfection ofof dividingdividing and nonand non--

dividingdividing cellscellsVIHVIH

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HERPESVIRUS VECTORS

Herpes Herpes simplexsimplex virus 1 (HSV1), virus 1 (HSV1), mildmild disseasedissease in in humanshumans

EnvelopedEnveloped virus, linear virus, linear dsDNAdsDNA152Kb (152Kb (halfhalf ofof thethe genes nongenes non--esentialesential forfor replicationreplication))

4040--50 50 KbKb ofof foreignforeign DNA can be DNA can be accommodatedaccommodated

NeurotrophicNeurotrophic virus, virus, targetedtargeted totonervousnervous systemsystem

RemainsRemains episomalepisomal

HSV parental DNA

mRNA

Inmediate earlyproteins

Delayed early

HSV progeny DNA

Late

HSV progeny

Life cycle of HSV

1) Fusion of viral envelope with cellmembrane and transport of capsidto nuclear membrane

2) DNA entry, circularisation andsequential transcription

3) Replication of genome, translation ofviral proteins and packaging

4) Fusion with cell membrane and cell exit

1 2

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transfectiontransfection infectioninfection

helper

Plasmidvector

Packaging Procedure

Permissive cells

helper

packaged vector

wt HSV revertants

lac Z

pHSVlacAmpr

IE 4/ 5prom

IE 4/5promoter

oris

Col E1or i

poliASV40

HSV-1packaging site

AmpliconAmplicon systemsystem

- Cloning of therapeutic gene into plasmid (amplicon) containing viral origin of replication and packaging signal

- Transfection of target cell

- Infection of a complementing cell line with a defective helper virus

- Production of packaged amplicon vector and helper virus

COMPARISON OF DIFFERENT VIRAL VECTORS

yeslowNot so good107AAV

yeslowNot so good107Herpesvirus

Some (lentivirus)Low good107Retrovirus

yesVery highterrific1011Adenovirus

Infection of non-diving cellsImmunogenicity

Manipulationof tropismTitersViral vector

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VACCINIA VIRUS VECTORS (POXVIRUS)

““CultivedCultived”” virusvirusEnvelopedEnveloped dsDNAdsDNA virus virus withwith ITR , ITR ,

180Kb180KbReplicationReplication cyclecycle in in cytoplasmcytoplasm, , thethe

virus virus containscontains allall neededneeded enzimesenzimesInsertionInsertion ofof transgenetransgene by by

homologoushomologous recombinationrecombination

RecombinantRecombinant vacciniavaccinia virus virus vectorsvectors

In In thethe presencepresence ofofTkTk, , BrdUBrdU isis

metabolizemetabolize toto a a toxictoxiccompoundcompound thatthat killskillsthethe cellscells. . CellsCells withwith

thethe recombinantrecombinantvirus are virus are TkTk--

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ALPHAVIRUS VECTORS

TogavirusTogavirus, (, (SindibisSindibis, , SemlikySemlikyForestForest))

ssRNAssRNA in a simple in a simple nucleocapsidnucleocapsid((onlyonly 1 viral 1 viral proteinprotein))

EnvelopedEnveloped virus, virus, formedformed by 2 viral by 2 viral proteinsproteins

TwoTwo ORF, ORF, oneone forfor replicases and replicases and oneoneforfor structuralstructural proteinsproteins

EnterEnter intointo thethe cellcell by receptor by receptor mediatedmediated endocytosisendocytosis

ReplicationReplication cyclecycle in in cytoplasmcytoplasm, , thethevirus virus containscontains allall neededneeded enzimesenzimes

OrfOrf rerplicasererplicase

PΨ

VIRAL VECTOR TARGETING

TraslationalTraslational targetingtargeting : : DirectingDirecting thethe infectivityinfectivity towarstowars a a cellcell typetype

TrascriptionalTrascriptional targetingtargeting : : TheThe virus virus infectinfect manymany cellcell typestypes, , butbut thetherecombinantrecombinant proteinprotein isis onlyonly transcribe in transcribe in oneone cellcell typetype

PseudotypingPseudotypingMonoclonal Monoclonal antibodiesantibodiesFusionFusion proteinsproteins

PromotersPromoters ((tissuetissue oror cellcell specificspecific))Locus control Locus control regionsregions (LCR) (LCR)

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MIXED VIRAL VECTORS

PlasmidPlasmid vectorvector AdVAdV helperhelper withwith retroviral retroviral proteinsproteins