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Abstract

Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.

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References

Aruffo

, Stamenkovic

, Melnick

et al. 1990. CD44 is the principal cell surface receptor for hyaluronate. Cell, 61:1303–1313.

Burnett

J.C.

, Rossi

J.J.

, Tiemann

2011. Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol. J., 6:1130–1146.

Cao

, Longley

M.A.

, Wang

X.J.

, Roop

D.R.

2001. An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy. J. Cell Biol., 152:651–656.

Chen

, Roop

D.R.

2005. Mouse models in preclinical studies for pachyonychia congenita. J. Investig. Dermatol. Symp. Proc., 10:37–46.

Chen

S.H.

, Zhaori

2011. Potential clinical applications of siRNA technique: benefits and limitations. Eur. J. Clin. Invest., 41:221–232.

Coulman

, Allender

, Birchall

2006. Microneedles and other physical methods for overcoming the stratum corneum barrier for cutaneous gene therapy. Crit. Rev. Ther. Drug Carrier Syst., 23:205–258.

Frank-Kamenetsky

, Grefhorst

, Anderson

N.N.

et al. 2008. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. U.S.A., 105:11915–11920.

Geusens

, Sanders

, Prow

et al. 2009. Cutaneous short-interfering RNA therapy. Expert Opin. Drug Deliv., 6:1333–1349.

Gill

H.S.

, Denson

D.D.

, Burris

B.A.

, Prausnitz

M.R.

2008. Effect of microneedle design on pain in human volunteers. Clin. J. Pain, 24:585–594.

10.

Gonzalez

, Gilaberte-Calzada

2008. In vivo reflectance-mode confocal microscopy in clinical dermatology and cosmetology. Int. J. Cosmet. Sci., 30:1–17.

11.

Gonzalez-Gonzalez

, Ra

, Hickerson

R.P.

et al. 2009. siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model. Gene Ther., 16:963–972.

12.

Gonzalez-Gonzalez

, Speaker

T.J.

, Hickerson

R.P.

et al. 2010b. Silencing of reporter gene expression in skin using siRNAs and expression of plasmid DNA delivered by a soluble protrusion array device (PAD) Mol. Ther., 18:1667–1674.

13.

Gonzalez-Gonzalez

, Kim

Y.C.

, Speaker

T.J.

et al. 2011. Visualization of plasmid delivery to keratinocytes in mouse and human epidermis. Sci. Rep., 1:158.

14.

Guido

M.C.

, Clemente

C.F.

, Moretti

A.I.

et al. 2011. Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia. Shock, 37:77–84.

15.

Haggerty

J.G.

, Bretton

R.H.

, Milstone

L.M.

1992. Identification and characterization of a cell surface proteoglycan on keratinocytes. J. Invest. Dermatol., 99:374–380.

16.

Hickerson

R.P.

, Smith

F.J.

, Reeves

R.E.

et al. 2008. Single-nucleotide specific siRNA targeting in a dominant-negative skin model. J. Invest. Dermatol., 128:594–605.

17.

Hickerson

R.P.

, Flores

M.A.

, Leake

et al. 2011a. Use of self-delivery siRNAs to inhibit gene expression in an organotypic pachyonychia congenita model. J. Invest. Dermatol., 131:1037–1044.

18.

Hickerson

R.P.

, Leachman

S.A.

, Pho

L.N.

et al. 2011b. Development of quantitative molecular clinical end points for siRNA clinical trials. J. Invest. Dermatol., 131:1029–1036.

19.

Hsu

, Mitragotri

2011. Delivery of siRNA and other macromolecules into skin and cells using a peptide enhancer. Proc. Natl. Acad. Sci. U.S.A., 108:15816–15821.

20.

Huang

W.R.

, Fan

X.X.

, Tang

2011. SiRNA targeting EGFR effectively prevents posterior capsular opacification after cataract surgery. Mol. Vis., 17:2349–2355.

21.

Kaspar

R.L.

, McLean

W.H.

, Schwartz

M.E.

2009. Achieving successful delivery of nucleic acids to skin: 6th Annual Meeting of the International Pachyonychia Congenita Consortium. J. Invest. Dermatol., 129:2085–2087.

22.

Kaspar

R.L.

, Leachman

S.A.

, McLean

W.H.

, Schwartz

M.E.

2011. Toward a treatment for pachyonychia congenita: Report on the 7th Annual International Pachyonychia Congenita Consortium meeting. J. Invest. Dermatol., 131:1011–1014.

23.

Khavari

P.A.

, Rollman

, Vahlquist

2002. Cutaneous gene transfer for skin and systemic diseases. J. Intern. Med., 252:1–10.

24.

Kugelman

L.C.

, Ganguly

, Haggerty

J.G.

et al. 1992. The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44. J. Invest. Dermatol., 99:886–891.

25.

Kuwahara

, Nishina

, Yoshida

et al. 2011. Efficient in vivo delivery of siRNA into brain capillary endothelial cells along with endogenous lipoprotein. Mol. Ther., 19:2213–2221.

26.

Leachman

S.A.

, Hickerson

R.P.

, Hull

P.R.

et al. 2008. Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita. J. Dermatol. Sci., 51:151–157.

27.

Leachman

S.A.

, Hickerson

R.P.

, Schwartz

M.E.

et al. 2010. First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder. Mol. Ther., 18:442–446.

28.

Leslie Pedrioli

D.M.

, Fu

D.J.

, Gonzalez-Gonzalez

et al. 2012. Generic and personalized RNAi therapeutics for a dominant-negative epidermal fragility disorder. J. Invest. Dermatol.(in press).

29.

McLean

W.H.

, Moore

C.B.

2011. Keratin disorders: From gene to therapy. Hum. Mol. Genet., 20:R189–R197.

30.

Naor

, Sionov

R.V.

, Ish-Shalom

1997. CD44: Structure, function, and association with the malignant process. Adv. Cancer Res., 71:241–319.

31.

Nehal

K.S.

, Gareau

, Rajadhyaksha

2008. Skin imaging with reflectance confocal microscopy. Semin. Cutan. Med. Surg., 27:37–43.

32.

Pfutzner

, Vogel

J.C.

2000. Advances in skin gene therapy. Expert Opin. Investig. Drugs, 9:2069–2083.

33.

Prausnit

M.R.

, Mitragotri

, Langer

2004. Current status and future potential of transdermal drug delivery. Nat. Rev. Drug Discov., 3:115–124.

34.

, Piyawattanametha

, Gonzalez-Gonzalez

et al. 2011. In vivo imaging of human and mouse skin with a handheld dual-axis confocal fluorescence microscope. J. Invest. Dermatol., 131:1061–1066.

35.

Schurr

M.J.

, Foster

K.N.

, Centanni

J.M.

et al. 2009. Phase I/II clinical evaluation of StrataGraft: A consistent, pathogen-free human skin substitute. J. Trauma, 66:866–873discussion 873–874.

36.

Screaton

G.R.

, Bell

M.V.

, Jackson

D.G.

et al. 1992. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. U.S.A., 89:12160–12164.

37.

Shim

M.S.

, Kwon

Y.J.

2010. Efficient and targeted delivery of siRNA in vivo. FEBS J., 277:4814–4827.

38.

Tadin-Strapps

, Peterson

L.B.

, Cumiskey

A.M.

et al. 2011. siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids. J. Lipid Res., 52:1084–1097.

39.

Vaishnaw

A.K.

, Gollob

, Gamba-Vitalo

et al. 2010. A status report on RNAi therapeutics. Silence, 1:14.

40.

Wang

, Tammi

1992. Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages. Histochemistry, 98:105–112.

41.

Yasaka

, Furue

, Tamaki

1995. CD44 expression in normal human skin and skin tumors. J. Dermatol., 22:88–94.

42.

Zhou

, Haggerty

J.G.

, Milstone

L.M.

1999. Growth and differentiation regulate CD44 expression on human keratinocytes. In Vitro Cell Dev. Biol. Anim., 35:228–235.

43.

Zimmermann

T.S.

, Lee

A.C.

, Akinc

et al. 2006. RNAi-mediated gene silencing in non-human primates. Nature, 441:111–114.

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Inhibition of CD44 Gene Expression in Human Skin Models,Using Self-Delivery Short Interfering RNA Administered by Dissolvable Microneedle Arrays

Abstract

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Supplementary Material