RNA  interference  (RNAi)    and  its  applica3ons  

Carlos  Camilleri  

Content  of  the  presenta3on  

•  Introduc1on  •  Argonaute  proteins  •  piRNAs:  biogenesis  and  gene  silencing  •  miRNA  and  siRNA  

–  Differences  –  Biogenesis  –  Gene  Silencing  –  Transla1onal  ac1va1on  

•  Applica1ons  of  RNA  interference  –  Poten1al  therapeu1c  applica1on  in  gene1c  diseases  –  Poten1al  an1viral  response:  HIV  

•  Current  drawbacks  of  using  RNAi  •  Conclusion  

Introduc3on  

•  RNA  interference  is  a  process  of  gene  silencing  

•  Three  different  types  of  RNA  involved:  – Micro  interference  RNA  (miRNA)  – Small  interference  RNA  (siRNA)  – Piwi-­‐interac1ng  RNA  (piRNA)  

 •  RNA-­‐induced  silencing  complex  (RISC)  

Argonaute  family  

•  Highly  specialized  small-­‐RNA-­‐binding  proteins  •  Key  of  RNA-­‐silencing  pathways  •  2  human  subfamilies  with  4  members  each:  

•  10-­‐fold  increase  in  finding  target  mRNA  

Subfamily   Expression   Associated  to  AGO   Ubiquitous   miRNAs  and  siRNAs  PIWI   Germline   piRNAs  

piRNAs  biogenesis  and  gene  silencing  

•  Derive  from  transposons  à  share  homology  

•  Transcribed  from  piRNA  clusters:  –  Long  single-­‐stranded  transcript  –  Sense  or  an1sense  transcrip1on  

•  Biogenesis  of  piRNAs  follows  2  different  pathways:  –  Primary  processing  pathway  –  Amplifica1on  pathway  (ping-­‐pong  cycle)  

•  Mature  piRNAs  are  25-­‐33  nt  in  length  

•  Mechanism  of  transposon  silencing  in  mammals  

piRNAs  biogenesis  and  gene  silencing  

anchors the 5′ end of the small RNA by providing a binding pocket in which the 5′ terminal base engages in stacking interactions with a conserved tyrosine. In addition, several hydrogen bonds coordinate correct 5′ end binding8. The N domain is required for small RNA loading and assists in unwinding the small RNA duplex10.

The PIWI domain is structurally similar to RNase H, and it has indeed been shown that Argonaute proteins can function as endonucleases and cleave target RNA that is fully complementary to the bound small RNA8. The PIWI domain contains a catalytic triad composed of DDX (where X is D or H). Recent structural work on an Argonaute protein from yeast revealed that a fourth residue is also essential turning the catalytic centre into a tetrad of DEDX11. Generally, only a subset of Argonaute proteins possesses cleavage activity. In mam-mals, for example, only AGO2 of the AGO subfamily is catalytically active and functions as an endonuclease12,13. Besides the PIWI domain, an unstructured loop in the N domain is also important for cleavage14.

Recently, crystal structures of human AGO2 have been reported15,16. Overall, their structure is strikingly similar to known bacterial and archaeal Argonaute pro-teins, underlining the high conservation of the basic principles of small RNA pathways.

Mechanisms of Argonaute loadingThe AGO subfamily and the PIWI subfamily use differ-ent mechanisms for small RNA loading, and they will be discussed separately with an emphasis on AGO clade proteins.

Loading AGO clade members. Dicer processing gener-ates a short double-stranded RNA of about 20–24 nt in length. However, only one strand associates with the AGO protein and becomes the guide strand. How is the correct strand selected, and how is it loaded into AGO proteins? One important determinant for strand selection lies in the small RNA duplex itself: both in the miRNA pathway and the siRNA pathway, the strand with the less stably paired 5′ end is preferentially loaded into AGO proteins. These thermodynamic differ-ences between the small RNA ends are known as the asymmetry rule17,18.

In various organisms, Dicer proteins directly inter-act with AGO proteins and strand selection, and load-ing is achieved within such complexes19–21. In addition, Dicer proteins partner with various different dsRNA-binding proteins, which have been most extensively studied in D. melanogaster. In flies, two Dicer proteins exist: Dcr1 processes precursor miRNAs (pre-miRNAs) and loads Ago1 for miRNA-guided gene silencing, whereas Dcr2 cleaves perfectly paired long dsRNA and loads siRNAs into Ago2. Dcr1 interacts with loquacious (Loqs), which is essential for the miRNA pathway22. Dcr2 requires R2d2, which is important for the siRNA pathway. R2d2 functions as a sensor for the thermodynamic asymmetry within an siRNA duplex and positions Dcr2 for correct strand selection23,24. In mammals, the dsRNA-binding domain proteins TRBP

Nature Reviews | Genetics

Primaryprocessing

PrimarypiRNAs

PIWI protein

Sense transcript

Trimming

Secondaryprocessing(ping-pong cycle)

Mature sensepiRNA

Mature antisense piRNA

Antisense piRNASense piRNA

Antisense transcript

Trimming

piRNA cluster

Sense or antisensetranscript

Zuc

Box 2 | piRNA biogenesis and function

Mobile genetic elements such as transposons are a constant threat for the genome. PIWI-interacting RNAs (piRNAs) protect germline cells from transposons in organisms as diverse as flies, fish or mammals. piRNAs are 25 to 33 nt in length, depending on the PIWI clade protein that they bind to. piRNAs derive from distinct transposons that are referred to as piRNA clusters, but the piRNAs from each locus are characterized by a complex mixture of sequences spanning large portions of the transposon5. piRNA clusters are transcribed in the sense or antisense direction, and the long single-stranded RNA serves as the basis for piRNA production.

The biogenesis of piRNAs is independent of Dicer165 and requires other nucleases. Two biogenesis pathways are important for piRNA production. First, a primary processing pathway generates primary piRNAs, and these are then amplified by an amplification cycle referred to as the ping-pong loop5. In the primary biogenesis pathway, the long transposon transcript is initially cleaved by the nuclease zucchini (Zuc; see the figure)166–168, which probably generates the 5′ ends of primary piRNAs. The other steps of primary piRNA maturation are not understood5.

In the ping-pong cycle (see the lower right panel of the figure), mature sense primary piRNAs guide PIWI clade proteins to complementary sequences on antisense transcripts from the same piRNA cluster. PIWI proteins use their slicer activity to cleave the target antisense transcript to generate a new 5′ end. This 5′ end is bound by another PIWI protein. In subsequent steps, the 3′ end is trimmed to the length of the mature piRNA, leading to a mature antisense secondary piRNA, which can now target sense transcripts transcribed from the piRNA cluster. In Drosophila melanogaster, the two Piwi proteins Aubergine and Ago3 cooperate in secondary piRNA production to generate sense and antisense piRNAs. However, antisense piRNAs dominate, and a protein called Qin, which contains E3 ligase and Tudor domains, seems to modulate such a heterotypic ping-pong cycle169. In the mouse germ line, the PIWI proteins MILI and MIWI collaborate in piRNA generation. After trimming, piRNAs receive a methyl group at the 3′ end by the methyltransferase HEN1. Primary piRNAs carry such modifications as well5. In Caenorhabditis elegans, an additional piRNA production pathway exists. Short and capped RNA polymerase II transcripts are decapped, processed and directly loaded into PIWI proteins170.

piRNAs guide PIWI proteins to complementary RNAs derived from transposable elements. Similarly to in RNA interference, PIWI proteins cleave the transposon RNA, leading to silencing. In flies, mutations in piwi, aub and Ago3 (which encode the Piwi proteins in &�|OGNCPQICUVGT) are required for transposon silencing in the germ line5. Similar observations were made when the mouse PIWI proteins MILI and MIWI were genetically inactivated171. Here, long interspersed nuclear elements (LINE) and long terminal repeat (LTR) retrotransposons accumulated172.

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miRNA  and  siRNA  

•  O[en  referred  to  as  the  ‘true’  interference  RNA  •  Similar  biogenesis  but  s1ll  key  differences  

miRNA   siRNA  

Mature  structure   dsRNA  of  19-­‐25nt  with  a  2nt  3'overhang  

dsRNA  of  21-­‐23nt  with  a  2nt  3'overhang  

TargeBng   Mul1ple  targets,  par1ally  complementary  

One  highly  specific  target  with  fully  complementariety  

Mechanisms  of  gene  regula3on  

 1.  Transla1onal  repression    2.  mRNA  degrada1on  

3.  Endonucleotydic  cleavage  1.  Endonucleotydic  cleavage  

Biogenesis  of  miRNA  and  siRNA  

•  Both  form  RNA  duplexes  (dsRNA)  a[er  transcrip1on  

•  miRNA  requires  an  extra  processing  in  the  nucleus  

•  Both  are  exported  to  the  cytoplasm  by  Expor1n-­‐5  

•  In  the  cytoplasm,  their  processing  is  the  same  

www.moleculartherapy.org/mtna

siRNA Versus miRNA TherapeuticsLam et al.

3

backbone of the mRNA between bases 10 and 11 relative to the 5!end of the guide strand.30 The mRNA fragments gener-ated are subsequently degraded by different exonucleases.31 By contrast, the target recognition of miRNA is more complex, as different binding sites and different degree of complemen-tarity between the miRNA and the target RNA exist. This is a consequence of imperfect base pairing; miRNA only needs

to be partially complementary to its target mRNA. The com-plementary pairing between mRNA and the mature miRNA typically occurs at the 3! untranslated region (UTR) of the for-mer and the seed region (nucleotides 2–7 from the 5! end) of the latter (Figure 2).32,33 Other miRNA binding sites, such as the centered sites, 3! supplementary sites and bulged sites, are considered to be atypical.32,34,35 Since miRNA-mRNA

Figure 1 Gene silencing mechanisms of siRNA and miRNA. siRNA: dsRNA (either transcribed or artificially introduced) is processed by Dicer into siRNA which is loaded into the RISC. AGO2, which is a component of RISC, cleaves the passenger strand of siRNA. The guide strand then guides the active RISC to the target mRNA. The full complementary binding between the guide strand of siRNA and the target mRNA leads to the cleavage of mRNA. miRNA: Transcription of miRNA gene is carried out by RNA polymerase II in the nucleus to give pri-miRNA, which is then cleaved by Drosha to form pre-miRNA. The pre-miRNA is transported by Exportin 5 to the cytoplasm where it is processed by Dicer into miRNA. The miRNA is loaded into the RISC where the passenger strand is discarded, and the miRISC is guided by the remaining guide strand to the target mRNA through partially complementary binding. The target mRNA is inhibited via translational repression, degradation or cleavage.

miRNA gene

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Complementary binding of theguide strand to target mRNA

Incomplete complementarybinding of the guide strand to

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3!UTR

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mRNA cleavage

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mRNA mRNA

miRISC

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miRNA  and  siRNA  gene  silencing  

Endonucleotydic  cleavage  of  target  mRNA  

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•  Needs  perfect  complementarity  

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Moreover, miRNA duplexes often contain sufficient bulges to prevent slicing of miR* strands even by com-petent enzymes. Therefore, it has been proposed that miR* strands dissociate in a cleavage-independent man-ner by unwinding — a process that is facilitated by the presence of mismatches in the loaded duplexes113,118,132. Biochemical evidence supports unwinding as a passive, ATP-independent process, with degradation of the miR* strand on its release. It is unclear how plant Argonautes remove the miR* or passenger strand during RISC mat-uration. MiR* and passenger strands could be cleaved through the slicer activity of AGOs (similar to fly AGO2) or unwound passively (like fly AGO1)12–14,132.

The impact of sorting on target regulationThe ultimate result of accurate strand selection and sort-ing is that an active RISC is formed, which is imbued with the ability to regulate a target gene or process. Argonaute family members differ in their biochemical properties, subcellular localization and expression pat-terns, and matching the right small RNA with the correct partner is key to proper biological function.

Although AGO proteins evolved as ribonucleases, animal miRNAs affect their targets without the need for this activity. miRNAs generally interact with their tar-gets through limited base-pairing interactions that are insufficient to place the scissile phosphate of the target in the enzyme active site where cleavage can occur. The prevalence of cleavage-independent repression modes is also reflected in the diversity of the Argonaute family. In mammals, three of the four AGO proteins have lost catalytic potential, and AGO1, the D. melanogaster AGO protein into which most miRNAs are sorted, is a poor enzyme compared with its siRNA-binding cousin113.

miRNA-directed target cleavage has only been reported in a few cases144,145. However, this is assumed to be the principal regulatory mode for endo-siRNAs and for piRNA-mediated repression of transposons. Here again, the choice of a particular AGO partner is crucial. Piwi family members all retain catalytic compe-tence and D. melanogaster AGO2, the main partner for endogenous and viral siRNAs, is tuned for highly active slicing (BOX 4).

AGO1-associated plant miRNAs usually share exten-sive sequence complementary with their mRNA targets and these interactions often result in target cleavage146. However, recent studies have indicated that cleavage-independent translational repression is widespread in plants, even for highly complementary target sites147. Nevertheless, miRNA-mediated cleavage is of key importance for some processes like the biogenesis of ta-siRNAs, for which the initial slicing event is key to RdRP recruitment and dsRNA synthesis88.

Notably, small RNAs that direct cleavage, for exam-ple, plant miRNAs, piRNAs and fly endo-siRNAs, often have a 2 -O-methyl modification on their 3 termini. Although the purpose of this modification was initially mysterious, it is now clear that this functions as a pro-tective group to prevent small RNA destruction64,142,143. In flies and mammals, small RNAs that have extensive complementarity to their targets can be recognized by terminal uridyl transferases, which mark small RNAs for degradation64. The uridylation event is blocked by the 2 -O-methyl modification, preserving these small RNAs, which have evolved to function through cleav-age64. The balance between protection and targeted destruction has been proposed as a quality control on small RNA sorting and as an evolutionary mechanism to drive animal miRNAs toward a cleavage-independent repression mode64.

hc-siRNAs are thought to function by different mechanisms148,149. They must be sorted into a particu-lar Argonaute, AGO4, which they guide to target DNA loci by base pairing with nascent non-coding transcripts synthesized by RNAPV. Effector proteins, such as the chromatin-remodelling factor DRD1, the de novo meth-yltransferase DRM2 and other factors, are then recruited, resulting in DNA methylation at cytosine residues150,151. As this regulation functions by repressing RNA synthe-sis, it was termed transcriptional gene silencing to distin-guish it from post-transcriptional gene-silencing modes. Some piRNAs in flies and mammals must associate with particular Piwi-family proteins — that is, PIWI and

Box 4 | Mechanisms of target regulation in Drosophila melanogaster

Individual Argonaute (AGO) proteins differ in their expression patterns, subcellular localization and enzymatic properties. Thus, distinct AGOs can function through many different effector modes that may involve slicing of target transcripts, cleavage-inde-pendent regulation and chromatin modification (reviewed in REFS 15–17). Another layer of complexity is added by the degree of sequence complementarity between the AGO-bound small RNA and target transcripts, which determines the mechanism of regulation. a | In flies, AGO1-associated microRNAs (miRNAs) typically target mRNAs in their 3 UTRs to reduce protein synthesis. Owing to limited sequence complementarity between the small RNA (seed region) and the mRNA, such interactions usually do not result in direct cleavage of the targeted transcript. Instead, AGO1 and its partner protein GW182 are likely to disrupt crucial interactions between the polyA tail and the cap of the transcript, leading to a reduction in translational initiation and an induction of mRNA decay173. In mammals, it was recently shown that reduced protein output is predominantly owing to destabilization of the target transcript174. b | Small RNAs bound to Drosophila melanogaster AGO2 do not exhibit a bias towards binding their targets in the 3 UTR. AGO2 primed with a small RNA sharing extensive complementarity with its target typically directs endonucleolytic cleavage of the mRNAs through AGO2 slicer activity. The 2-O-methyl modification of AGO2-bound small RNAs prevents their degradation when targeting perfectly complementary transcripts64,142,143. However, other modes are possible: AGO2 can also regulate targets with limited sequence complementarity through a block in translation initiation (not shown)173. PABP, poly(A)-binding protein

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28 | JANUARY 2011 | VOLUME 12 www.nature.com/reviews/genetics

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miRNA-­‐only  gene  silencing  

Transla3onal  repression  

ANRV413-BI79-13 ARI 27 April 2010 19:36

A(n)

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Ribosomedrop-o!

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40S

60S

m7G

A(n)m7G

a Initiation block

b Postinitiation block

AGO

GW182?? ?

PABP

miRISC

Open reading frameAUG

Figure 2Schematic diagram of miRNA-mediated translational repression. (a) Initiation block: The miRISC inhibits translation initiation byinterfering with eIF4F-cap recognition and 40S small ribosomal subunit recruitment or by antagonizing 60S subunit joining andpreventing 80S ribosomal complex formation. The reported interaction of the GW182 protein with the poly(A)-binding protein(PABP) (106, 156) might interfere with the closed-loop formation mediated by the eIF4G-PABP interaction and thus contribute to therepression of translation initiation. (b) Postinitiation block: The miRISC might inhibit translation at postinitiation steps by inhibitingribosome elongation, inducing ribosome drop-off, or facilitating proteolysis of nascent polypeptides. There is no mechanistic insight toany of these proposed “postinitiation” models. The 40S and 60S ribosomal subunits are represented by small and large gray spheres,respectively. Ovals with question marks represent potential additional uncharacterized miRISC proteins that might facilitatetranslational inhibition. Abbreviations: AGO, Argonaute; A(n), poly(A) tail; m7G, the 5!-terminal cap.

358 Fabian · Sonenberg · Filipowicz

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Transla3onal  repression  

ANRV413-BI79-13 ARI 27 April 2010 19:36

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GW182?? ?

PABP

miRISC

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eIF4E

Elongationblock Proteolysis

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40S

60S

m7G

A(n)m7G

a Initiation block

b Postinitiation block

AGO

GW182?? ?

PABP

miRISC

Open reading frameAUG

Figure 2Schematic diagram of miRNA-mediated translational repression. (a) Initiation block: The miRISC inhibits translation initiation byinterfering with eIF4F-cap recognition and 40S small ribosomal subunit recruitment or by antagonizing 60S subunit joining andpreventing 80S ribosomal complex formation. The reported interaction of the GW182 protein with the poly(A)-binding protein(PABP) (106, 156) might interfere with the closed-loop formation mediated by the eIF4G-PABP interaction and thus contribute to therepression of translation initiation. (b) Postinitiation block: The miRISC might inhibit translation at postinitiation steps by inhibitingribosome elongation, inducing ribosome drop-off, or facilitating proteolysis of nascent polypeptides. There is no mechanistic insight toany of these proposed “postinitiation” models. The 40S and 60S ribosomal subunits are represented by small and large gray spheres,respectively. Ovals with question marks represent potential additional uncharacterized miRISC proteins that might facilitatetranslational inhibition. Abbreviations: AGO, Argonaute; A(n), poly(A) tail; m7G, the 5!-terminal cap.

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mRNA  degrada3on  

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DCP2

DeadenylationDecapping(and subsequent decay)

12

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Figure 3Schematic diagram of miRNA-mediated mRNA decay. The miRISC interacts with the CCR4-NOT1deadenylase complex to facilitate deadenylation of the poly(A) tail [denoted by A(n)]. Deadenylation requiresthe direct interaction of the GW182 protein with the poly(A)-binding protein (PABP) (see previous figures).Following deadenylation, the 5!-terminal cap (m7G) is removed by the decapping DCP1-DCP2 complex.The open reading frame is denoted by a black rectangle. Abbreviations: AGO, Argonaute; CAF1,CCR4-associated factor; CCR4, carbon catabolite repression 4 protein; NOT1, negative on TATA-less.

The conclusions from the cell culture stud-ies are strongly supported by in vitro exper-iments using cell-free systems that faithfullyrecapitulate the action of miRNAs in cells.In cell-free extracts from mouse Krebs II as-cites cells (referred to as Krebs extracts) (97),Drosophila embryos (96), and HEK293 cells(95), inhibition of mRNA translation or dead-enylation was dependent on the ability of the“seed sequence” of the miRNA to base-pairto the target sequence in the mRNA. Addi-tion of oligonucleotides, which are comple-mentary to miRNAs (antimiRs), to the ex-tract prevented miRNA function. The threesystems mentioned above made use of an en-dogenous miRNA targeting for in vitro syn-thesized mRNAs. All these aforementionedstudies concluded that the miRNA-mediatedtranslation inhibition occurs at the initiationstep and is due to the interference with thecap recognition process. This is further sup-ported by the findings that miRNAs failed toinhibit IRES-dependent translation or trans-lation from ApppG-capped mRNAs (95–97).More detailed analyses revealed that miRNAsinhibited ribosome initiation complex forma-tion; miR-2 inhibited both 40S ribosomalsubunit recruitment and 80S initiation com-

plex formation in fly embryo extract (96),and 80S initiation complex formation was im-paired in mouse Krebs extract (97). A study byZdanowicz et al. (98) points to the 5!-cap structure itself as being a direct targetof miRNA-mediated translational repression.miR-2-targeted mRNAs bearing modificationsto the triphosphate bridge of the 5! cap demon-strated increased translational repression inboth Drosophila embryonic extracts and S2cells (98). Strong evidence for the notion thatthe cap recognition machinery is indeed thetarget for miRNA-mediated translational re-pression was the demonstration that addinga purified eIF4F complex to the Krebs ex-tract alleviated translational repression of let-7-targeted mRNAs (97). In contrast to theseresults, Wang et al. (99) showed that in arabbit reticulocyte lysate the CXCR4 artificialmiRNA impairs translation by inhibiting thejoining of the 60S subunit, even though the 5!

cap was required for the inhibition. It is pos-sible that this inconsistence with former re-sults stems from nuclease-treated rabbit retic-ulocyte lysate not displaying cap-poly(A) tailtranslational synergy (100, 101), which may, inturn, alter the outcome of miRNA-mediatedrepression.

www.annualreviews.org • Regulation of mRNAs by microRNAs 359

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Transla3onal  ac3va3on  by  miRNA  

•  Mechanism  s1ll  under  study  •  Only  found  in  quiescent  cells  arrested  in  G0/G1  •  Involves  recruitment  of  FXR1  (fragile  X-­‐related  protein  1)  

Applica3ons  of  RNA  interference  

•  Poten1al  therapeu1cs  for  gene1c  diseases  – Dominant  gene1c  disorders  caused  by  a  mutant  allele  in  the  presence  of  a  second,  normal  allele  

–  Currently  no  cures:  difficult  to  treat  the  origin,  in  some  cases  the  muta1on  is  a  SNP  

Demonstrated  in  vitro  that  a  SNP  is  enough  for  RNAi  to  select  the  mutant  allele  over  the  normal  one  

S1ll  under  development  

Applica3ons  of  RNA  interference  

•  Poten1al  an1viral  responses:  HIV  –  Successful  targe1ng  of  HIV-­‐encoded  RNAs  –  Successful  downregula1on  of  cellular  cofactors  needed  for  the  HIV  replica1on  

–  Inhibi1on  of  HIV  replica1on  achieved  in  several  human  cell  lines  

Big  challenge  going  from  in  vitro  to  in  vivo  –  High  viral  muta1on  rate  à  escaping  mutants  –  Challenging  delivery  of  the  RNAi  to  the  infected  cells  

S1ll  under  development  

Current  major  drawbacks  of  using  iRNA  

•  Off-­‐target  effects  – Gene  silencing  of  unwanted  genes  

•  Delivery  methods  – Large  size  of  iRNAs  (around  14  kDa)  – Nega1ve  charge  of  RNA  – Generally  unstable  in  vivo  – Problems  targe1ng  specific  cells  or  1ssues  

Conclusion  Clinical  trials  with  RNAi  are  currently  underway,  but  major  obstacles,  such  as  off-­‐target  effects,  toxicity  and  unsafe  delivery  methods,  have  to  be  overcome  before  RNAi  can  

be  considered  a  conven1onal  drug  

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