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Targeting the Palm: A Leap ForwardToward Treatment of KeratinDisordersWera Roth1, Mechthild Hatzfeld2 and Thomas M. Magin1
Any rational therapy benefits from an understanding of basic biology and thesimplicity of its strategy. Among keratinopathies, epidermolytic palmoplantarkeratoderma stands out by virtue of hotspot mutations in the KRT9 gene,exclusively expressed in the palmoplantar epidermis. In this issue, Leslie Pedrioliet al. report on the successful application of KRT9-specific siRNAs in cultured cellsand in a mouse model. The study beautifully illustrates the potency of a thoroughexperimental approach and the challenges that remain, especially in its delivery.
Journal of Investigative Dermatology (2012) 132, 1541–1542. doi:10.1038/jid.2012.99
Efficacy, specificity, and potency of a drugrepresent the lynchpins of a successfultherapy. In the case of genetic disorders,onset of disease and the cell type of originmount additional hurdles to be overcome.Keratinopathies are caused mostly bydominant mutations in at least 23 of the54 human keratin genes expressed asthe ‘‘keratin pairs’’ that typify epithelialdifferentiation (Szeverenyi et al., 2008;http://www.interfil.org). Therefore, sites
of expression reveal the major site(s)of disease, despite the notion thatmost keratinocytes express 4–10 differentkeratin proteins. Further, there appears tobe reasonable genotype–phenotype corre-lation, indicating that mutations severelycompromising the cytoskeleton’s integritycause more severe disease phenotypesthan those that do not. Although patho-mechanisms of the keratinopathies aremore complex than originally thought
See related article on pg 1627
1Division of Cell and Developmental Biology, Translational Centre for Regenerative Medicine and Instituteof Biology, University of Leipzig, Leipzig, Germany and 2Institut für Molekulare Medizin, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany
Correspondence: Thomas M. Magin, Division of Cell and Developmental Biology, Translational Centre forRegenerative Medicine and Institute of Biology, University of Leipzig, Philipp-Rosenthal-Stra�e 55, LeipzigD-04103, Germany. E-mail: [email protected]
(Coulombe and Lee, 2012), one canreasonably argue that reducing theexpression of the mutant allele in domi-nant keratin disorders should restore amore functional cytoskeleton from theintact allele, leading to greater tissueintegrity. Proof of principle stemmedfrom mouse models in which the ratio ofmutant and normal keratin alleles hasbeen modified genetically (Cao et al.,2001; Hesse et al., 2007).
Among keratinopathies, epidermo-lytic palmoplantar keratoderma (EPPK)is unique for several reasons: the expres-sion of the culprit, KRT9, is restrictedto the upper strata of the palmo-plantar epidermis, forming a cytoskele-ton together with at least six additionalkeratins. The majority of EPPK patientssuffer from a missense mutation in oneof the three hotspot codons, giving riseto a focal epidermolytic keratoderma(http://www.interfil.org). This settinginvited Leslie Pedrioli et al. (this issue,2012) to develop an siRNA-based ther-apy approach, testing both generic andmutation-specific siRNAs directed againstKRT9. The team first scanned all possible19-mer siRNAs for the repression of KRT9,using transiently expressed luciferasereporters to monitor specificity andefficacy of siRNAs. Next, siRNAs thatefficiently inhibited the two prominentKRT9 missense mutations M157V andR163Q were identified using a similarstrategy. The best siRNAs were able torepress a mutant KRT9 allele in the 50 pMrange, without apparently affecting theexpression of other keratins. Ultimately,siRNAs must be delivered in situ. Unfortu-nately, no mouse model for KRT9 iscurrently available. As a first step, LesliePedrioli et al. coinjected the most potentsiRNA, siR163Q-13, together with amutant KRT9-luciferase reporter carryingthe same mutation, into mouse foot-pad epidermis. This delivery route hadbeen previously approved in a phase Ibclinical trial for pachyonychia congenita(Leachman et al., 2010). To test forspecificity, a wild-type KRT9-luciferasereporter was applied together with theabove siRNA in another footpad. Despitethe limitations imposed by the nature ofthe delivery, i.e., injection, the data sug-gest that the siRNA was more specific inrepressing the mutant compared with thenormal allele.
COMMENTARY
www.jidonline.org 1541
http://www.jidonline.orghollyText BoxThis is a commentary on the Generic and Personalized RNAi-Based Therapeutics for a Dominant Negative Epidermal Fragility Disorder. That article begins on page 3
As one finds with any good study,Leslie Pedrioli and colleagues’ data raisea number of issues that in the abovesetting relate to RNAi, keratin biology,and skin physiology. The specific andeffective repression of mutant KRT9alleles is supported by encouragingdata in model systems for the related,dominant keratinopathies pachyony-chia congenita, epidermolysis bullosasimplex, and Meesmann cornealdystrophy (Leachman et al., 2010; Liaoet al., 2011; Coulombe and Lee, 2012).These studies imply that specificallytargeting individual keratin mutations isfeasible, although current bioinforma-tics approaches are unable to deliverreliable predictions. Further, siRNAcomplexed into stable nucleic acidparticles appears to be stable for up to2 weeks (Davidson and McCray, 2011).Whether the use of repeated cycles ofsiRNA that are necessary to treat chronicdiseases will avoid immunological recog-nition through RIG and TLR receptorsexpressed in keratinocytes remains to bedetermined. The stability of the targetmRNA and encoded protein(s) representsanother challenge. The long half-life of keratin intermediate filament pro-teins and their mRNAs may indeed outlastmost siRNA formulations. Therefore,including data from well-establishedthree-dimensional keratinocyte culturemodels is of paramount importancein future studies. In combination withexperiments on animal models, thequestion of how efficient siRNA-mediatedrepression of mutant keratins must bein order to eliminate dominant-negativeeffects remains to be answered.
In many epidermal keratinocytes, ker-atins represent some of the most abun-dant proteins. Current studies on treatingkeratinopathies with siRNAs assume thatrepressing a single keratin isotype, i.e.,KRT9 as in the Leslie Pedrioli et al. (thisissue, 2012) paper, is of little conse-quence for skin integrity and physiologyin view of keratin’s abundance and theircomplexity of expression. This may notbe the case and is not well supported by
in vivo data. The only supporting mousestudies are those of the functionalreplacement of KRT18 by KRT 19 andthe partial replacement of KR14 byKRT18 (Hutton et al., 1998; Maginet al., 1998). More recent data, rather,point to non-overlapping keratin func-tions, spear-headed by KRT17’s role incontrol of protein biosynthesis andinflammation (Coulombe and Lee,2012). With these and additional datain mind, future studies should includemore comprehensive assays to evaluatetreatment success in the context of skinphysiology.
Delivery in vivo is themajor limitation inapplying siRNAtechnology to skindiseases.
In addition, the greatest hurdle forsiRNA-based treatment of skin disordersremains delivery to the cell of origin.Recent advances in lipid-mediatednucleic acid delivery to the skin haveconsidered trans- and intracellular, aswell as transfollicular and transappenda-geal, routes to treat a range of geneticand non-genetic conditions (Geusenset al., 2011). The truth is that the under-lying mechanisms for successful deli-very of nucleic acids (the basis of anyex vivo therapy into live keratino-cytes, including stem cells) are not wellknown. As odd as it seems, there is noother way than back from bedside to thebench: are all keratinocytes equal in theirability to take up, transport, and processsiRNAs? Which of the aforementionedroutes are actually being taken by siRNAthat is delivered to cells? How manystem cells are targeted, and does resto-ring their phenotype confer a selectiveadvantage over their neighbors? Theseare some of the questions that must beanswered before the exciting stridestaken by Leslie Pedrioli et al. (this issue,2012) find their way to the clinic.
CONFLICT OF INTERESTThe authors state no conflict of interest.
ACKNOWLEDGMENTSWork in the Thomas M. Magin lab is supportedby the DFG (MA-1316), the BMBF (network EB),and the Translational Center for RegenerativeMedicine, TRM, Leipzig, PtJ-Bio, 0315883). Workin the Mechthild Hatzfeld lab is supported by theDFG (Ha1791/7-1 and 8/1, SFB 610, GRK 1591),the BMBF (ProNET T3), and Sachsen-Anhalt.
REFERENCES
Cao T, Longley MA, Wang XJ et al. (2001) Aninducible mouse model for epidermolysisbullosa simplex: implications for gene ther-apy. J Cell Biol 152:651–6
Coulombe PA, Lee CH (2012) Defining keratinprotein function in skin epithelia: epidermo-lysis bullosa simplex and its aftermath. J InvestDermatol 132(Part 2):763–75
Davidson BL, McCray PB Jr (2011) Current pro-spects for RNA interference-based therapies.Nat Rev Genet 12:329–40
Geusens B, Strobbe T, Bracke S et al. (2011) Lipid-mediated gene delivery to the skin. Eur JPharm Sci 43:199–211
Hesse M, Grund C, Herrmann H et al. (2007) Amutation of keratin 18 within the coil 1Aconsensus motif causes widespread keratinaggregation but cell type-restricted lethalityin mice. Exp Cell Res 313:3127–40
Hutton E, Paladini RD, Yu QC et al. (1998)Functional differences between keratins ofstratified and simple epithelia. J Cell Biol143:487–99
Leachman SA, Hickerson RP, Schwartz ME et al.(2010) First-in-human mutation-targetedsiRNA phase Ib trial of an inherited skindisorder. Mol Ther 18:442–6
Leslie Pedrioli DM, Fu DJ, Gonzalez-Gonzalez Eet al. (2012) Generic and personalized RNAi-based therapeutics for a dominant-negativeepidermal fragility disorder. J Invest Dermatol132:1627–35
Liao H, Irvine AD, Macewen CJ et al. (2011)Development of allele-specific therapeuticsiRNA in Meesmann epithelial corneal dys-trophy. PLoS One 6:e28582
Magin TM, Schroder R, Leitgeb S et al. (1998)Lessons from keratin 18 knockout mice: for-mation of novel keratin filaments, secondaryloss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates. J CellBiol 140:1441–51
Szeverenyi I, Cassidy AJ, Chung CW et al. (2008)The Human Intermediate Filament Database:comprehensive information on a gene familyinvolved in many human diseases. HumMutat 29:351–60
COMMENTARY
1542 The Journal of Investigative Dermatology (2012), Volume 132
Generic and Personalized RNAi-Based Therapeuticsfor a Dominant-Negative Epidermal FragilityDisorderDeena M. Leslie Pedrioli1, Dun Jack Fu1, Emilio Gonzalez-Gonzalez2,3, Christopher H. Contag2,3,Roger L. Kaspar3,4, Frances J.D. Smith1 and W.H. Irwin McLean1
Epidermolytic palmoplantar keratoderma (EPPK) is one of 430 autosomal-dominant human keratinizingdisorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in thekeratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerablekeratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations,makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific,short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeuticpackage for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations.Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using aninnovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potentgeneric K9 inhibitors, eliciting 485% simultaneous knockdown of wild-type and mutant K9 protein synthesis atpicomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibitedstrong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The mostpromising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentiallyinhibit mutant allele expression in vivo.
Journal of Investigative Dermatology (2012) 132, 1627–1635; doi:10.1038/jid.2012.28; published online 8 March 2012
INTRODUCTIONRNA interference (RNAi) was first reported in plants just overtwo decades ago (Napoli et al., 1990), and its subsequentcharacterization in eukaryotic cells (Fire et al., 1998)revolutionized the fields of molecular, cellular, and devel-opmental biology, as well as molecular medicine. Thedemonstration that small interfering RNAs (siRNAs) couldpotently and specifically control the gene activity viahomology-dependent mRNA degradation suggested thatpersonalized, or allele-specific, therapeutics were theoreti-cally attainable (Davidson and McCray, 2011). Monogenicdominant-negative interference or gain-of-function disease
pathologies, where pleiotropic phenotypes are not observed,lend themselves best to proof-of-principle RNAi-based thera-peutics (Lane and McLean, 2008). Keratin disorders, whichprimarily affect the epidermis, a highly accessible tissue, areideal model diseases for developing this type of therapeutic(Lane and McLean, 2008; McLean and Moore, 2011).
Keratins form cytoplasmic intermediate filaments inepithelial cells, which primarily function to protect thesecells from mechanical stress (Omary et al., 2004). Accordingto the Human Intermediate Filament Database (www.interfi-l.org; Szeverenyi et al., 2008), 23 of the 54 human epithelialkeratin genes are linked to epithelial fragility disorders (Smith,2003; McLean and Irvine, 2007; Lane and McLean, 2008).Most causative variants are heterogeneous missense or smallin-frame insertion/deletion mutations that inhibit cytoskeletalfunction via dominant-negative interference. Thus, treatingkeratinizing disorders will require silencing, or limitingthe activity, of these mutant alleles. To date, a handful ofpotentially therapeutic siRNAs have been identified for threeof these disorders: pachyonychia congenita (Hickerson et al.,2008; Smith et al., 2008), epidermolysis bullosa simplex(Atkinson et al., 2011), and Meesmann epithelial cornealdystrophy (Liao et al., 2011). Indeed, one of these inhibitorselicited therapeutic benefits in a phase I clinical trial(Leachman et al., 2010). These ground-breaking studies haveclearly demonstrated that RNAi-based therapeutics are well
See related commentary on pg 1541
& 2012 The Society for Investigative Dermatology www.jidonline.org 1627
ORIGINAL ARTICLE
Received 6 September 2011; revised 14 December 2011; accepted 22December 2011; published online 8 March 2012
1Dermatology and Genetic Medicine, Division of Molecular Medicine,Colleges of Life Sciences and Medicine, Dentistry & Nursing, University ofDundee, Dundee, UK; 2Molecular Imaging Program at Stanford (MIPS),Stanford University School of Medicine, Stanford, California, USA;3Department of Pediatrics, Radiology, Microbiology & Immunology, StanfordUniversity School of Medicine, Stanford, California, USA and 4TransDerm,Santa Cruz, California, USA
Correspondence: Deena M. Leslie Pedrioli, Dermatology and GeneticMedicine, Division of Molecular Medicine, College of Life Sciences,University of Dundee, Dundee DD1 5EH, UK.E-mail: [email protected]
Abbreviations: EPPK, epidermolytic palmoplantar keratoderma;KRT9, keratin 9; RNAi, RNA interference; siRNA, short interfering RNA
http://dx.doi.org/10.1038/jid.2012.28www.interfil.orgwww.interfil.orghttp://www.jidonline.orgmailto:[email protected]
suited for keratinizing disorders; unfortunately, each is linkedto a large number of different mutations in at least twoindependent keratin genes. Therefore, standardizing down-stream preclinical studies and subsequent clinical trials forsiRNAs targeting all of pathogenic mutations becomesprohibitive.
To circumvent these downstream hurdles, we havefocused our attention on developing an RNAi-based thera-peutic package for epidermolytic palmoplantar keratoderma(EPPK). EPPK is unique because it is an autosomal-dominantdisorder caused only by mutations in human keratin K9(KRT9; Smith, 2003; McLean and Irvine, 2007), which isexclusively expressed in the suprabasal cells of palm and soleepidermis (Langbein et al., 1993). EPPK presents as a well-circumscribed epidermolytic keratoderma, and B90% ofcases carry a missense mutation in one of three hotspotcodons (www.interfil.org; Smith, 2003; McLean and Irvine,2007).
The palmoplantar epidermis must withstand the greatestmechanical stress in the body; therefore it is believed to haveadapted to express the greatest number of keratin genes (atleast 10; Swensson et al., 1998), which provide these cellswith the mechanical resilience to survive these arduousconditions. The abundance of keratins here, and to a lesserextent in other epidermal tissues, suggests that a degree offunctional redundancy occurs between coexpressed keratins.This hypothesis is supported by several keratin knockout andreplacement studies that demonstrate keratin redundancies inseveral epithelial tissues (Magin et al., 1998; Porter and Lane,2003; Coulombe et al., 2004; Wong et al., 2005; Lu et al.,2006). No recessive and/or loss-of-function mutations havebeen identified in KRT9 (www.interfil.org; Szeverenyi et al.,2008). Therefore, it is possible that complete ablation ofKRT9 expression may be tolerable.
Here, we present our initial studies developing a broadpreclinical RNAi-based therapeutic package for EPPK, whichcontains generic KRT9 siRNA inhibitors and four individualpatient (allele)-specific siRNA inhibitors. We used a KRT9-luciferase reporter assay to systematically identify the mostpotent and specific lead inhibitors. These potencies andspecificities were independently verified in vitro underdisease modeling conditions using an innovative dual-tagquantitative immunoblot assay. Finally, the efficacy of one ofour patient (allele)-specific siRNAs was confirmed in vivo inan intact mouse epidermis using a mouse model.
RESULTSRNAi-based inhibition of human KRT9 expression
Our initial objective was to develop siRNA inhibitors thatindiscriminately and specifically downregulate the expres-sion of human KRT9. Six independent KRT9-targeting siRNAs(siKRT9-1–6) were designed based on two criteria: theytarget KRT9 transcripts outside of the mutation hotspot 1Aand 2B a-helical subdomain coding regions, and they displaystrong sequence specificity for KRT9 transcripts comparedwith all other type I and II keratins expressed in the humanepidermis (Figure 1a; see Supplementary Materials andMethods online). A dual-luciferase reporter assay (Atkinson
et al., 2011) was used to assay the inhibitory potential of eachof these siRNAs. To this end, a full-length KRT9 complemen-tary DNA firefly luciferase reporter construct (pfLUC-flKRT9/WT) was co-transfected into AD293 cells with a Renillaluciferase construct (for normalization) and each siRNA.Optimal expression levels for pfLUC-flKRT9/WT and knock-down potentials were defined using a positive control siRNAtargeting firefly luciferase (siLUC) and a nonspecific controlsiRNA (NSC4). NSC4 did not affect fLUC-flKRT9/WT expres-sion, whereas siLUC potently inhibited KRT9-luciferase
aKRT9 mRNA
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siKRT9–1 siKRT9–3
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siKRT9–5
Figure 1. Inhibition of wild-type K9 in AD293 cells. (a) Schematic diagram
of human keratin 9 (KRT9) mRNA and the positions where each short
interfering RNA (siRNA) targets KRT9. Black: protein-coding sequence; gray:
50 and 30 untranslated regions (UTRs). (b) AD293 cells were co-transfected
with a wild-type K9-luciferase reporter construct (pfLUC-flKRT9/WT) and
each of the KRT9-targeting siRNAs (siKRT9-1-6), a negative control siRNA
(NSC4), or a positive control siRNA (siLUC) over a concentration range of
0–6.25 nM. Luciferase activities were measured using the dual-luciferase
reporter assay 24 hours after transfection. Renilla luciferase activities were
used for normalization. Normalized firefly luciferase activities at each siRNA
concentration are expressed as percentages of firefly luciferase activity at
0 nM siRNA concentration. Error bars indicate SD of the mean from three
biological replicate experiments.
1628 Journal of Investigative Dermatology (2012), Volume 132
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
www.interfil.orgwww.interfil.org
activity in a dose-dependent manner, displaying IC50 ofB70 pM (Figure 1b). Although all of the KRT9 siRNAs wereactive and relatively potent inhibitors of fLUC-flKRT9/WTreporter gene expression, displaying IC50 values rangingbetween 100 and 250 pM (data not shown), siKRT9-1 andsiKRT9-3 induced the strongest inhibitions (Figure 1b). OursiRNA design criteria (see Supplementary Materials andMethods online) required several mismatches betweenthese KRT9 siRNAs and other potential keratin transcripts.Nevertheless, the off-target effects of one of the inhibitors(siKRT9-3) were investigated in a human keratinocyte cellline (HACATs) that expresses several keratins also found inthe palmoplantar epidermis. siKRT9-3-mediated knockdownof endogenous keratins was not observed in cytoskeletalextracts (Supplementary Figure S1 online), whereas strikingand specific reduction in endogenous keratin 6a (K6a) wasobserved with a validated K6a inhibitor, siK6a-2 (Smith et al.,2008).
Simultaneous inhibition of wild-type and mutant K9protein synthesisUnfortunately, the dual-luciferase assay only tested theinhibitory capacities of siKRT9-1 and siKRT9-3 for wild-typeKRT9 in isolation. As EPPK patients express both wild-typeand dominant-negative mutant alleles of KRT9, it waspossible that allelic discrepancies could occur when attempt-ing to simultaneously repress both in vivo. To test this,KRT9 Flag-HA (K9-WT) and four dominant-negative mutantKRT9 Strep-HA epitope-tagged (p.Arg163Trp (K9-R163W),p.Arg163Gln (K9-R163Q), p.Met157Val (K9-M157V), andp.Met157Thr (K9-M157T)) expression plasmids were gener-ated. AD293 cells were co-transfected with no siRNA, 0.25or 0.5 nM of siKRT9-1, -3, -5, or NSC4 siRNAs, and equalamounts of pKRT9-WT/FlagHA and either pKRT9-R163W/StrepHA, pKRT9-R163Q/StrepHA, pKRT9-M157V/StrepHA,or pKRT9-M157T/StrepHA. A dual-tag quantitative immuno-blotting assay was developed, using anti-Flag monoclonaland anti-Strep-TagII polyclonal antibodies, to confirm inhibi-tion of K9 protein translation. Fusion protein levels werequantified using state-of-the-art infrared fluorochrome-coupled secondary antibodies and direct infrared fluores-cence detection with the LI-COR Odyssey Infrared ImagingSystem (Fogarty et al., 2007). Although NSC4 had no effecton wild-type or mutant K9 protein production, we confirmedthat siKRT9-1 and siKRT9-3 were potent inhibitors ofKRT9 and demonstrated equal inhibition of all KRT9 alleles(Figure 2). Both siKRT9-1 and siKRT9-3 elicited 485%knockdown at 0.25 nM (Figure 2) and complete inhibitionof expression at 0.5 nM (data not shown). Unexpectedly,siKRT9-5, which showed the least potent inhibitory potentialin the luciferase assay, also proved to be an effectiverepressor under these conditions (Figure 2).
The half-lives and abundances of these transientlytransfected K9 fusion proteins in AD293 cells are likely lessthan those observed in vivo. Nevertheless, these data suggestthat they should effectively suppress endogenous KRT9expression. To assess their long-term knockdown efficacy,AD293 cells were co-transfected with pKRT9-WT/FlagHA,
pKRT9-R163W/StrepHA, and siKRT9-3 as described above.Dual-tag immunoblotting confirmed near-complete knock-down of both K9 fusion proteins 48 hours after transfection(Supplementary Figure S2a online) and maintained inhibitionat 72 hours (Supplementary Figure S2b online). Surprisingly,120 hours post-transfection production of K9-R163W andK9-WT remained inhibited by 60% and 80%, respectively(Supplementary Figure S2c online). Taken together, thesefindings suggest that our generic KRT9 inhibitors shouldsustainably inhibit de novo K9 protein synthesis in vivo.
In vitro screening for potent, allele-specific siRNA inhibitorsfor EPPK patients
As studies detailing the phenotypic consequences associatedwith homozygous and heterozygous loss of wild-type KRT9are currently lacking, it is unclear whether ubiquitousknockdown of KRT9 in EPPK patients will prove therapeutic.
a No siRNA NSC4 siKRT9–1 siKRT9–3 siKRT9–51
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Figure 2. Simultaneous inhibition of wild-type and mutant K9 protein
synthesis. AD293 cells were transiently co-transfected with a FlagHA-tagged
wild-type K9 reporter construct (K9-WT), either no siRNA, 0.25 nM of a
nonspecific control (NSC4), or 0.25 nM siKRT9-1, -3, or -5, and (a) the
StrepHA-tagged mutant K9 reporter construct K9-R163W, (b) the StrepHA-
tagged mutant K9 reporter construct K9-R163Q, (c) the StrepHA-tagged
mutant K9 reporter construct K9-M157V, or (d) the StrepHA-tagged mutant K9
reporter construct K9-M157T. Immunoblotting of whole-cell lysates with
mouse a-Flag and rabbit a-StrepTagII antibodies and differential fluorescencevisualization using a LI-COR Odyssey scanner demonstrated simultaneous
reduction of both wild-type and mutant K9 protein synthesis following
siKRT9-1, -3, and -5 transfection. As a loading control, each membrane
was also probed with an a-b-actin antibody.
www.jidonline.org 1629
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
http://www.jidonline.org
We, therefore, reasoned that allele-specific siRNA inhibitorsspecifically targeting EPPK-associated single-nucleotide poly-morphisms could function as patient-specific therapeuticsfor EPPK. To date, 45 of the 74 reported EPPK mutationsoccur within codons 163 (R163) and 157 (M157) of KRT9(www.interfil.org; Szeverenyi et al., 2008). To developthese alternative personalized RNAi-based therapies, siRNAsequence walks (Hickerson et al., 2008; Atkinson et al.,2011) were performed on four dominant-negative points:K9-R163W and K9-R163Q, and K9-M157V and K9-M157T(Reis et al., 1994; Covello et al., 1998). Arrays of 19individual siRNA inhibitors were designed for each mutationto define the optimal mutation-targeting nucleotide positionfacilitating selective and potent silencing of each EPPK-associated single-nucleotide polymorphism (SupplementaryFigures S3a–S6a online). Each siRNA within the array wasnamed according to the position of the mutation-specifictargeting nucleotide (Atkinson et al., 2011). Dual-luciferasereporter assays were developed, where exon 1 of wild-typeKRT9 and the four mutations were cloned into the 30
untranslated region of firefly luciferase (see SupplementaryMaterials and Methods online). We then screened all of thesiRNAs for their ability to selectively and potently inhibitmutant, but not wild-type, KRT9 expression. AD293 cellswere co-transfected in quadruplicate with the KRT9 exon 1constructs, Renilla luciferase (for normalization) and eachsiRNA, NSC4, or siLUC (Supplementary Figures S3b–S6bonline). Sequence walks were repeated three times and theaverage mutant and wild-type inhibitions, relative to 0 nM
siRNA, determined for the individual siRNAs (SupplementaryFigures S3c–S6c online).
siRNAs were considered allele specific if mutant alleleinhibition was X50%, wild-type allele inhibition was p50%,the mutant and wild-type inhibition difference was X30%,and the SD at the significant inhibition concentration wasp20%. Eight allele-specific siRNAs were identified thatspecifically target the EPPK-associated mutations queried.For K9-R163W, two inhibitors, siR163W-3 and siR163W-6,strongly inhibited K9-R163W reporter allele expression andsuccessfully discriminated between K9-WT and K9-R163W(Figure 3a). K9-R163Q reporter allele expression was speci-fically repressed by two inhibitors, siR163Q-3 and siR163Q-13, compared to K9-WT (Figure 3b). Inhibitors siM157V-11and siM157V-16 specifically and potently inhibited K9-M157V-luciferase expression, whereas a third inhibitor(siM157V-6) moderately inhibited K9-M157V (Figure 3c).Finally for M157T a single inhibitor, siM157T-16, showedstrong repressing characteristics but only moderate specificityfor K9-M157T (Figure 3d). The specificities of each allele-specific siRNA were further confirmed by their inabilityto silence the alternative codon mutation (SupplementaryFigure S7 online).
Preferential inhibition of mutant K9 protein synthesis
KRT9 Flag-HA and four KRT9 dominant-negative mutantStrep-HA epitope-tagged alleles were used to further validatethe allele specificity and potency of these siRNAs underpseudo-disease conditions. AD293 cells were transiently
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Figure 3. RNA interference (RNAi)-mediated independent mutant-specific inhibition of multiple K9 dominant-negative alleles. AD293 cells were co-
transfected with 0–6.25 nM of each of the 19 mutation-specific short interfering RNAs (siRNAs), or either of the control siRNAs (siLUC or NSC4), a Renilla
luciferase construct, and either wild-type K9 (K9-WT) or mutant K9-R163W (a), K9-R163Q (b), K9-M157V (c), or K9-M157T (d) firefly luciferase constructs.
Luciferase activities were measured 24 hours after transfection and normalized using Renilla luciferase activities. Normalized firefly luciferase activities at
each siRNA concentration are expressed as percentages of activity at 0 nM siRNA. Error bars indicate SD of the mean for biological replicate experiments.
The most promising mutant-specific inhibitors for each dominant-negative allele are shown here; the complete siRNA walk data sets are shown in
Supplementary Figures S3–S6 online.
1630 Journal of Investigative Dermatology (2012), Volume 132
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co-transfected with equal amounts of pKRT9-WT/FlagHAand either pKRT9-R163W/StrepHA, pKRT9-R163Q/StrepHA,pKRT9-M157V/StrepHA, or pKRT9-M157T/StrepHA aloneor with 0.5 nM each of their respective lead inhibitors andthe dual-tag immunoblotting assays performed as describedabove. In general, the potencies and allele specificities ofthese siRNAs mirrored those observed in our initial dual-luciferase siRNA sequence walks (Figure 4 and Supplemen-tary Figures S8–S10 online). The exceptions to this weresiR163Q-3 (Supplementary Figure S9 online) and siM157V-6(Supplementary Figure S10 online), whose inhibitory activ-ities were not confirmed in this independent assay. When
transfection concentrations of siR163Q-3 and siM157V-6were increased, similar knockdown of both the wild-type andmutant alleles was observed (data not shown).
To quantitatively define the specificity and potency of theremaining allele-specific siRNAs, the Flag-tag, Strep-TagII,and b-actin band intensities were defined using GelEval(www.frogdance.dundee.ac.uk) for all technical replicates ineach of the three biological replicate experiments (n¼ 9).Both siR163W-3 and siR163W-6 potently inhibited K9-R163W by 90% and 85%, respectively (Figure 4a andSupplementary Figure S8 online). Nevertheless, siR163W-3was the most allele discriminating of the two inhibitors,
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Figure 4. Validation of mutant-specific inhibition of K9 protein synthesis. AD293 cells were transiently co-transfected with pKRT9-WT/FlagHA (K9-WT)
and pKRT9-R163W/StrepHA (K9-R163W) (a), pKRT9-R163Q/StrepHA (K9-R163Q) (b), pKRT9-M157V/StrepHA (K9-M157V) (c), or pKRT9-M157T/StrepHA
(K9-M157T) (d) and 0.5 nM of the indicated short interfering RNA (siRNA). Immunoblotting of whole-cell lysates with a-Flag and a-StrepTagII antibodiesand differential fluorescence visualization using a LI-COR Odyssey scanner revealed preferential inhibition of mutant K9 protein synthesis by each of
the mutant-specific siRNAs (a–d; left panels). Quantitative analyses of a-Flag (wild-type) and a-StrepTagII (mutant) immunoblot signal intensities, relativeto b-actin, were used to quantify siRNA inhibition specificities and potencies (a–d; right panels). Mean relative abundances (n¼ 9) are shown, and error barsindicate signal intensity SD. *Pp0.01; **Pp0.001; ***Pp0.0001.
www.jidonline.org 1631
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
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knocking down K9-WT by o35% (Figure 4a), while450% knockdown was observed with siR163W-6 (Supple-mentary Figure S8 online). siR163Q-13 specifically andpotently elicited 480% knockdown of K9-R163Q whileminimally affecting K9-WT expression (Figure 4b). siM157V-11 (Figure 4c and Supplementary Figure S10 online) andsiM157T-16 (Figures 4d) proved to be the most promisingallele-specific inhibitors of K9-M157V and K9-M157T,respectively. Finally, the off-target effects of these allele-specific siRNAs were investigated in HACATs. The endo-genous keratin profiles were not affected following 0.5 nMtransfection with siR163W-3, siR163Q-13, siM157V-11, orsiM157T-16, whereas transfection with 0.5 nM siK6a-2resulted in near-complete knockdown of K6a (SupplementaryFigure S11 online).
Validation of mutant-specific siRNAs in mouse footpadepidermis
Previous studies have independently demonstrated, usinghigh-pressure intradermal injections into the footpads ofmice (Gonzalez-Gonzalez et al., 2010), that gene- andallele-specific siRNAs effectively repress the expression oftheir respective keratin reporter genes in vivo (Hickersonet al., 2008; Smith et al., 2008). Using this technique, we
assayed the inhibitory potential of our most discriminatingand potent allele-specific siRNA, siR163Q-13, in vivo(Figure 5). The footpads of the mice were co-injected witheither the K9-R163Q (pfLUC-ex1KRT9/R163Q; n¼45 mice)or the K9-WT (pfLUC-ex1KRT9/WT; n¼45 mice) luciferasereporter constructs (Figure 5a; top and bottom panels,respectively) and NSC4 siRNA (left paw; all animals) andeither siLUC (right paw; n¼ 22 mice) or siR163Q-13 (rightpaw; n¼23 mice). Using our positive control siRNA (siLUC),we confirmed that luciferase activities could be used as anin vivo readout of siRNA-mediated knockdown (images notshown). Moreover, we were able to confirm that siR163Q-13targets and represses the expression of K9-R163Q morefrequently and efficiently than K9-WT (Figure 5a).
Although every precaution was taken to deliver equalamounts of the reporter plasmids and siRNAs to each mousepaw, it is extremely difficult to ensure consistent luciferaseactivity in each animal. For this reason, large cohorts ofanimals were used in each treatment group to obtain astatistically robust data set. siLUC/NSC4 and siR163Q-13/NSC4 luciferase activity ratios for K9-R163Q or K9-WT werecalculated, loge transformed to fit a normal distribution, and abox-and-whiskers plot generated using R. Relatively equiva-lent in vivo knockdown was observed when either K9-R163Q
10
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aR/L= 3.14 R/L= 0.69 R/L= 0.08 R/L= 1.23 R/L= 0.16 R/L= 0.08
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*
*
(Pho
tons
sec
onds
–1 c
m–2
sr–
1 (×1
05))
Figure 5. Inhibition of K9-R163Q/fLuc reporter gene expression by R163Q-13 short interfering RNA (siRNA) in vivo. (a) CD1 female mouse footpads were
co-injected with 15 mg pfLUC-ex1KRT9/R163Q (top panel) or pfLUC-ex1KRT9/WT (bottom panel) expression plasmids and 15mg NSC4 siRNA (left paw) orsiR163Q-13 (right paw). Footpad luciferase expression was determined 24 hours after injection by whole-animal imaging using the Xenogen IVIS200 in vivo imaging
system. R/L values were calculated by dividing right paw luciferase light emissions (photons seconds–1 cm–2 sr–1) by left paw luciferase light emissions
(photons seconds–1 cm–2 sr–1); colored heat map depicts light emission intensity. (b) Box-and-whiskers plot of the loge-transformed in vivo K9-WT and K9-R163Q siLUC/
NSC4 and siR163Q-13/NSC4 luciferase light emission ratios. The Welch t-test was used to calculate P-values (P) in R. Asterisks (*) denote outliers within the data set.
1632 Journal of Investigative Dermatology (2012), Volume 132
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
or K9-WT was co-injected with siLUC (Figure 5b). In contrast,co-injection with siR163Q-13 more frequently and potentlyinhibited K9-R163Q compared to K9-WT (Figure 5b).A two-sample Welch’s t-test was used to determine whetherthis preferential inhibition of K9-R163Q compared toK9-WT was statistically significant. Although differences inmutant and wild-type K9 fusion protein knockdown withsiLUC were not statistically significant (P¼0.8734), thefavored inhibition of K9-R163Q compared to K9-WTobserved in vivo with siR163Q-13 approaches statisticalsignificance (P¼ 0.0783). Thus, although inherently variable,our pilot study with siR163Q-13 suggests that each of ourlead inhibitors should function as effective KRT9 repressors inan intact epidermis and that their defined specificities shouldbe maintained in vivo.
DISCUSSIONThe ‘‘holy grail’’ of translational research is to developbespoke, disease-tailored therapeutics for human diseases.The discovery that exogenous siRNAs can be used to harnessthe therapeutic potential of the endogenous RNAi pathway tospecifically and potently regulate and fine-tune gene expres-sion provided a theoretically ideal path to personalizedtherapeutics for some human diseases (Davidson andMcCray, 2011; Ketting, 2011). Although RNAi-based thera-peutics are not ideal for all diseases, they are well suitedfor inherited or acquired dominant-negative interference orgain-of-function disease pathologies.
Our group and our collaborators have made great stridestoward the development of siRNA inhibitors for the keratindisorders pachyonychia congenita (Hickerson et al., 2008;Smith et al., 2008; Leachman et al., 2010), epidermolysisbullosa simplex (Atkinson et al., 2011), and Meesmannepithelial corneal dystrophy ((Liao et al., 2011), clearlydemonstrating that RNAi-based therapeutics are promisingtreatment avenues. Unfortunately, pachyonychia congenita,epidermolysis bullosa simplex, and Meesmann epithelialcorneal dystrophy are all linked to a large number ofmutations in at least two independent keratin genes. Thiscomplicates downstream in vivo siRNA validation andsignificantly narrows patient cohorts who would benefit fromthe therapy. EPPK has advantages over other keratin disordersbecause (1) it involves only one keratin gene; (2) the affectedarea of the skin is limited and well circumscribed; (3) thereare many other keratins expressed in the affected tissue,allowing for a generic, or gene-specific, approach; and(4) there are important hotspot mutations, allowing an allele-specific approach. Thus, we have developed a two-prongedRNAi-based therapeutic package for EPPK. We have identi-fied potent and highly specific generic (gene-specific) siRNAsaimed at the KRT9 gene, as well as allele-specific inhibitorstargeting common hotspot mutations that exhibit no off-targeteffects on endogenous epithelial cell keratin profiles.Unfortunately, we have not yet been able to validate theefficacies and specificities of our lead inhibitors under endo-genous conditions. As a result, although these proof-of-principle preclinical studies clearly identify effective KRT9siRNA inhibitors, additional in vitro studies with primary
palmoplantar keratinocytes, as well as in vivo animal studies,are required to determine therapeutically beneficial dosingregimes for EPPK patients. Finally, EPPK is one of 430recognized phenotypes produced by mutations in 23 keratingenes (McLean and Moore, 2011), all of which share acommon pathomechanism. Therefore, one can reasonablyexpect that if a therapeutic strategy is successful for oneof these diseases, it could be extrapolated to other skindisorders, as well as many other dominant-negative geneticdisorders affecting other organ systems.
This study and others (Hickerson et al., 2008; Atkinsonet al., 2011) have described the development of personalizedsiRNA therapies targeting a particular mutation using siRNAsequence walks, which test all possible 19-mer siRNAstargeting the point mutation of interest. So far, we have notbeen able to predict the positions that will give allelicdiscrimination based on primary target sequence alone. Asummary of the data from all published siRNA walks againstkeratin mutations and the data presented here is shown inSupplementary Table S1 online. These siRNA walks haveyielded allele-specific siRNAs, approximately two per muta-tion. For each mutation, the pattern is highly reproducible; forexample, the K9 data presented here were consistent acrossthree biological replicate experiments, each containingfour technical replicates per data point. Thus, although thepattern is sequence dependent, the rules appear extremelycomplex. Understanding the rules that govern successfulallele-specific silencing would be advantageous as thistherapeutic strategy evolves; unfortunately, we have notyet accumulated a sufficient number of data sets tofacilitate bioinformatics approaches to define these para-meters. The only obvious rule that can be inferred from thecurrent combined data set is that the positions close to theends of the siRNA are ineffective, as positions 1 and 2 orpositions 17–19 have not produced mutation-specificinhibitors (Supplementary Table S1 online). As even ashort stretch of 19 nucleotides has 42.7� 1011 possiblesequence permutations, a systematic approach to deter-mining these rules may prove difficult. Therefore, thesequence walk methodology may be the best way to designfuture reagents of this type.
Overall, this study has further expanded the repertoireof potential siRNA therapeutic molecules for keratin disordersto include gene-specific and allele-specific inhibitors forthe KRT9 gene in EPPK, which exhibits many unique andattractive features as a model disease for application of siRNAtherapy within genodermatology. The potency, specificity,and in vivo efficacy data presented here provide a preclinicalpackage for future development of a clinically applicabletherapy for EPPK.
MATERIALS AND METHODSCell culture
Human AD293 embryonic kidney cells (Invitrogen, Paisley, UK)
and HACAT human keratinocytes were maintained in DMEM
(Invitrogen) supplemented with 10% fetal calf serum (Invitrogen).
Cells were incubated at 37 1C with 5% CO2 supplement andpassaged following standard laboratory procedures.
www.jidonline.org 1633
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
http://www.jidonline.org
DNA constructsFull-length wild-type KRT9-untagged (pKRT9-WT) and Flag-HA
(pKRT9-WT/FlagHA), and full-length p.Arg163Trp (pKRT9-R163W/
StrepHA), p.Arg163Gln (pKRT9-R163Q/StrepHA), p.Met157Val
(pKRT9-M157V/StrepHA), and p.Met157Thr (pKRT9-M157T/
StrepHA) mutant Strep-HA complementary DNAs, including the
50 and 30 untranslated regions, were synthesized and cloned into a
cytomegalovirus promoter–driven expression plasmid by DNA2.0
(Menlo Park, CA). pfLUC-ex1KRT9/WT, pfLUC-ex1KRT9/R163W,
pfLUC-ex1KRT9/R163Q, pfLUC-ex1KRT9/M157V, and pfLUC-
ex1KRT9/M157T firefly luciferase reporter constructs were generated
by PCR amplification and molecular cloning into the psiTEST-
Luc-target reporter plasmid (Yorkshire Bioscience, York, UK). See
Supplementary Materials and Methods online for further details.
siRNA design
The siDESIGN Center from Dharmacon RNAi technologies (Thermo
Scientific, http://www.dharmacon.com/designcenter/designcenterpage.
aspx) was used to identify candidate siRNAs targeting the coding
sequence and 30 untranslated region of KRT9 mRNA. The six siRNAs
(siKRT9-1–6) with the greatest number of mismatches between KRT9
and the other keratins queried were chosen for this study and
synthesized as previously described (Atkinson et al., 2011). A posi-
tive control siRNA (siLuc) targeting the firefly luciferase gene
(Atkinson et al., 2011) and a nonspecific control siRNA (NSC4)
(Hickerson et al., 2008) were also synthesized. To screen all possible
positions for the p.Arg163Trp (R163W), p.Arg163Gln (R163Q),
p.Met157Val (M157V), and p.Met157Thr (M157T) point mutations
within the 19-mer siRNA, mutation-specific sequence walk packages
containing 19 individual siRNAs were synthesized as previously
described (Hickerson et al., 2008; Atkinson et al., 2011).
Luciferase reporter assay
All siRNA screening studies were conducted as previously described
(Atkinson et al., 2011). Briefly, at 24 hours after plating, AD293 cells
were transfected with KRT9 firefly luciferase reporter constructs
(pfLUC-flKRT9/WT, pfLUC-ex1KRT9/WT, pfLUC-ex1KRT9/R163W,
pfLUC-ex1KRT9/R163Q, pfLUC-ex1KRT9/M157V, or pfLUC-ex1KRT9/
M157T), a Renilla luciferase expression plasmid, and 0–6.25nM of the
indicated siRNA using Lipofectamine 2000 (Invitrogen). The Dual-
Luciferase Reporter Assay (Promega, Southampton, UK) was performed
24hours after transfection following the manufacturer’s instructions. See
Supplementary Materials and Methods online for detailed protocols.
ImmunoblottingAD293 cells were transfected with 100 ng of pKRT9-WT/FlagHA,
100 ng of pKRT9-R163W/StrepHA, pKRT9-R163Q/StrepHA, pKRT9-
M157V/StrepHA, or pKRT9-M157T/StrepHA, and the indicated
siRNAs at final concentrations of 0.25 or 0.5 nM using Lipofectamine
2000 (Invitrogen). Lysates were generated in 1� denaturingNuPAGE LDS sample buffer (Invitrogen) 48 hours after transfection,
resolved by SDS-PAGE, and transferred to nitrocellulose membranes.
Membranes were cut, blocked with western blot blocking buffer
(3% BSA, Tris-buffered saline/0.5% Tween 20, 1:100 Biotin Blocking
Buffer (IBA GmbH, Göttingen, Germany)) for 3 hours at room
temperature, and incubated with primary antibodies diluted in
western blot blocking buffer overnight at 4 1C. The top halves ofall the membranes (450 kDa) were simultaneously probed with
1:1,000 mouse a-Flag M2 (F1804, Sigma-Aldrich, Gillingham, UK)and 1:1,000 rabbit a-Strep-tag II (ab76949, Abcam, Cambridge, UK).The bottom portions of the membranes (p50 kDa) were simulta-neously probed with 1:1,000 rabbit a-Strep-tag II and 1:10,000mouse a-b-actin (A1978, Sigma-Aldrich). Membranes were washedextensively and probed with Alexa Fluor 680 goat a-rabbit IgG(A-21076; Invitrogen, Life Technologies, Paisley, UK) and IRDye
800–conjugated goat a-mouse IgG (610-132-121; RocklandImmunochemicals, Gilbertsville, PA) secondary antibodies, diluted
1:5,000 in 3% BSA/Tris-buffered saline/0.5% Tween 20 for 1 hour at
room temperature. Membranes were washed and scanned in the
700- and 800-nm channels using the Odyssey Infrared Imaging
System (LI-COR Biotechnology UK, Cambridge, UK). See Supple-
mentary Materials and Methods online for detailed protocols.
Colored images of each immunoblot were generated using Photo-
shop (Adobe Systems, San Jose, CA). Quantitative immunoblot
analyses were performed using the GelEval software (www.frogdance.
dundee.ac.uk) as described in the Supplementary Materials and
Methods online.
Mouse footpad injections and in vivo imaging
CD1 female mice (Charles River, Wilmington, MA or Harlan
Laboratories, Hillcrest, UK) were used for these experiments follow-
ing the guidelines for Animal Care of the National Institutes of Health
(NIH), Stanford University, TransDerm, and UK animal welfare act.
Mouse footpad injections were administered as described previously
(Hickerson et al., 2008; Smith et al., 2008; Gonzalez-Gonzalez
et al., 2010). Briefly, a total volume of 80 ml phosphate-bufferedsaline containing 15 mg siRNA (NSC4, siLUC, or siR163Q-13) and15 mg of firefly luciferase expression plasmid (pfLUC-ex1KRT9/WT orpfLUC-ex1KRT9/R163Q) was intradermally injected with a 29-gauge
needle into the footpads of anesthetized mice. At 24 hours after
injection, luciferin (150 mg/kg body weight) was administered by
intraperitoneal injection and the mice were imaged 10 minutes after
injection using the IVIS200 in vivo imaging system (Xenogen product
from Caliper Life Sciences, Alameda, CA). The resulting light
emissions were quantied using LivingImage software 3.1 (Caliper
Life Sciences, Runcorn, UK). For the pfLUC-ex1KRT9/R163Q;
NSC4:siLUC and pfLUC-ex1KRT9/WT;NSC4:siLUC, the injections
were administered four times, with each treatment group containing
4 or 6 animals (22 animals/treatment). pfLUC-ex1KRT9/R163Q;
NSC4:siR163Q-13 and pfLUC-ex1KRT9/WT;NSC4:siR163Q-13 in-
jections were also administered four times, and each treatment group
contained 5 or 6 animals (23 animals/treatment). Statistical analyses
of these data were performed using R as described in the text.
CONFLICT OF INTERESTThe authors state no conflict of interest.
ACKNOWLEDGMENTSThis work was supported by grants from the Medical Research Council(Programme grant G0802780 to WHIM and FJDS), MRC Milstein AwardG0801742 (to Paul A. Campbell, WHIM, and FJDS), and TranslationalMedicine Research Collaboration funding (to DMLP). We thank PatrickPedrioli for his valuable help with our statistical analyses and use of R.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
1634 Journal of Investigative Dermatology (2012), Volume 132
DM Leslie Pedrioli et al.siRNA Therapeutics for EPPK
http://www.dharmacon.com/designcenter/designcenterpage.aspxhttp://www.dharmacon.com/designcenter/designcenterpage.aspxwww.frogdance.dundee.ac.ukwww.frogdance.dundee.ac.ukhttp://www.nature.com/jidhttp://www.nature.com/jid
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http://www.jidonline.org
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