Publications by authors named "Robyn P Hickerson"

30 Publications

  • Page 1 of 1

Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies.

Transl Vis Sci Technol 2020 12 29;9(13):44. Epub 2020 Dec 29.

Centre for Dermatology and Genetic Medicine, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.

Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.

Methods: Corneal expression of the firefly luciferase transgene () was achieved via insertion into the locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.

Results: The mouse exclusively expresses in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the transgene ().

Conclusions: This novel mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.
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http://dx.doi.org/10.1167/tvst.9.13.44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774114PMC
December 2020

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.

Hum Mol Genet 2016 Mar 11;25(6):1176-91. Epub 2016 Jan 11.

School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK,

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.
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http://dx.doi.org/10.1093/hmg/ddw001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764196PMC
March 2016

Imaging Functional Nucleic Acid Delivery to Skin.

Methods Mol Biol 2016 ;1372:1-24

Molecular Imaging Program at Stanford (MIPS), E150 Clark Center, Stanford University School of Medicine, 318 Campus Drive, Stanford, CA, 94305, USA.

Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells and even subcellular structures. Here we describe the animal models, reporter genes, imaging approaches and general strategies for delivery of nucleic acids to cells in the skin for local expression (e.g., plasmid DNA) or gene silencing (e.g., siRNA) with the intent of developing nucleic acid-based therapies to treat diseases of the skin.
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http://dx.doi.org/10.1007/978-1-4939-3148-4_1DOI Listing
August 2016

Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin.

Mol Imaging Biol 2016 Feb;18(1):34-42

TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA.

Purpose: Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results.

Procedures: To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers.

Results: Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression.

Conclusions: Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.
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http://dx.doi.org/10.1007/s11307-015-0875-zDOI Listing
February 2016

Gene expression profiling in pachyonychia congenita skin.

J Dermatol Sci 2015 Mar 14;77(3):156-65. Epub 2015 Jan 14.

TransDerm Inc., Santa Cruz, CA 95060, USA. Electronic address:

Background: Pachyonychia congenita (PC) is a skin disorder resulting from mutations in keratin (K) proteins including K6a, K6b, K16, and K17. One of the major symptoms is painful plantar keratoderma. The pathogenic sequelae resulting from the keratin mutations remain unclear.

Objective: To better understand PC pathogenesis.

Methods: RNA profiling was performed on biopsies taken from PC-involved and uninvolved plantar skin of seven genotyped PC patients (two K6a, one K6b, three K16, and one K17) as well as from control volunteers. Protein profiling was generated from tape-stripping samples.

Results: A comparison of PC-involved skin biopsies to adjacent uninvolved plantar skin identified 112 differentially-expressed mRNAs common to patient groups harboring K6 (i.e., both K6a and K6b) and K16 mutations. Among these mRNAs, 25 encode structural proteins including keratins, small proline-rich and late cornified envelope proteins, 20 are related to metabolism and 16 encode proteases, peptidases, and their inhibitors including kallikrein-related peptidases (KLKs), and serine protease inhibitors (SERPINs). mRNAs were also identified to be differentially expressed only in K6 (81) or K16 (141) patient samples. Furthermore, 13 mRNAs were identified that may be involved in pain including nociception and neuropathy. Protein profiling, comparing three K6a plantar tape-stripping samples to non-PC controls, showed changes in the PC corneocytes similar, but not identical, to the mRNA analysis.

Conclusion: Many differentially-expressed genes identified in PC-involved skin encode components critical for skin barrier homeostasis including keratinocyte proliferation, differentiation, cornification, and desquamation. The profiling data provide a foundation for unraveling the pathogenesis of PC and identifying targets for developing effective PC therapeutics.
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http://dx.doi.org/10.1016/j.jdermsci.2015.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374015PMC
March 2015

In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation.

J Control Release 2014 Dec 30;196:355-62. Epub 2014 Oct 30.

Centre for Dermatology and Genetic Medicine, Division of Molecular Medicine, University of Dundee, Dundee DD1 5EH, Scotland, UK. Electronic address:

Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.
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http://dx.doi.org/10.1016/j.jconrel.2014.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275573PMC
December 2014

Keratin 16 regulates innate immunity in response to epidermal barrier breach.

Proc Natl Acad Sci U S A 2013 Nov 11;110(48):19537-42. Epub 2013 Nov 11.

Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205.

Mutations in the type I keratin 16 (Krt16) and its partner type II keratin 6 (Krt6a, Krt6b) cause pachyonychia congenita (PC), a disorder typified by dystrophic nails, painful hyperkeratotic calluses in glabrous skin, and lesions involving other epithelial appendages. The pathophysiology of these symptoms and its relationship to settings in which Krt16 and Krt6 are induced in response to epidermal barrier stress are poorly understood. We report that hyperkeratotic calluses arising in the glabrous skin of individuals with PC and Krt16 null mice share a gene expression signature enriched in genes involved in inflammation and innate immunity, in particular damage-associated molecular patterns. Transcriptional hyper-activation of damage-associated molecular pattern genes occurs following de novo chemical or mechanical irritation to ear skin and in spontaneously arising skin lesions in Krt16 null mice. Genome-wide expression analysis of normal mouse tail skin and benign proliferative lesions reveals a tight, context-dependent coregulation of Krt16 and Krt6 with genes involved in skin barrier maintenance and innate immunity. Our results uncover a role for Krt16 in regulating epithelial inflammation that is relevant to genodermatoses, psoriasis, and cancer and suggest a avenue for the therapeutic management of PC and related disorders.
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http://dx.doi.org/10.1073/pnas.1309576110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845144PMC
November 2013

Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device.

Mol Ther Nucleic Acids 2013 Oct 22;2:e129. Epub 2013 Oct 22.

1] TransDerm, Santa Cruz, California, USA [2] Current address: University of Dundee, Dundee, UK.

Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.Molecular Therapy-Nucleic Acids (2013) 2, e129; doi:10.1038/mtna.2013.56; published online 22 October 2013.
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http://dx.doi.org/10.1038/mtna.2013.56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027428PMC
October 2013

Intravital fluorescence imaging of small interfering RNA-mediated gene repression in a dual reporter melanoma xenograft model.

Nucleic Acid Ther 2012 Dec 25;22(6):438-43. Epub 2012 Oct 25.

TransDerm Inc., Santa Cruz, California, USA.

Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription-quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo.
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http://dx.doi.org/10.1089/nat.2012.0364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507521PMC
December 2012

Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells.

Proc Natl Acad Sci U S A 2012 Aug 30;109(33):13171-6. Epub 2012 Jul 30.

Department of Chemistry, Stanford University, Stanford, CA 94305, USA.

The polyanionic nature of oligonucleotides and their enzymatic degradation present challenges for the use of siRNA in research and therapy; among the most notable of these is clinically relevant delivery into cells. To address this problem, we designed and synthesized the first members of a new class of guanidinium-rich amphipathic oligocarbonates that noncovalently complex, deliver, and release siRNA in cells, resulting in robust knockdown of target protein synthesis in vitro as determined using a dual-reporter system. The organocatalytic oligomerization used to synthesize these co-oligomers is step-economical and broadly tunable, affording an exceptionally quick strategy to explore chemical space for optimal siRNA delivery in varied applications. The speed and versatility of this approach and the biodegradability of the designed agents make this an attractive strategy for biological tool development, imaging, diagnostics, and therapeutic applications.
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http://dx.doi.org/10.1073/pnas.1211361109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421195PMC
August 2012

Inhibition of CD44 gene expression in human skin models, using self-delivery short interfering RNA administered by dissolvable microneedle arrays.

Hum Gene Ther 2012 Aug 5;23(8):816-23. Epub 2012 Jun 5.

TransDerm, Santa Cruz, CA 95060, USA.

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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http://dx.doi.org/10.1089/hum.2011.211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413896PMC
August 2012

Visualization of plasmid delivery to keratinocytes in mouse and human epidermis.

Sci Rep 2011 15;1:158. Epub 2011 Nov 15.

Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.
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http://dx.doi.org/10.1038/srep00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240989PMC
September 2013

In vivo sustained release of siRNA from solid lipid nanoparticles.

ACS Nano 2011 Dec 18;5(12):9977-83. Epub 2011 Nov 18.

Department of Chemistry, Stanford University, 333 Campus Drive, Stanford, California 94305-5080, United States.

Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with a challenge of being delivered in a sustained manner. Nanoparticle drug delivery systems allow for incorporating and controlled release of therapeutic payloads. We demonstrate that solid lipid nanoparticles can incorporate and provide sustained release of siRNA. Tristearin solid lipid nanoparticles, made by nanoprecipitation, were loaded with siRNA (4.4-5.5 wt % loading ratio) using a hydrophobic ion pairing approach that employs the cationic lipid DOTAP. Intradermal injection of these nanocarriers in mouse footpads resulted in prolonged siRNA release over a period of 10-13 days. In vitro cell studies showed that the released siRNA retained its activity. Nanoparticles developed in this study offer an alternative approach to polymeric nanoparticles for encapsulation and sustained delivery of siRNA with the advantage of being prepared from physiologically well-tolerated materials.
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http://dx.doi.org/10.1021/nn203745nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246574PMC
December 2011

Use of self-delivery siRNAs to inhibit gene expression in an organotypic pachyonychia congenita model.

J Invest Dermatol 2011 May 20;131(5):1037-44. Epub 2011 Jan 20.

TransDerm Inc, Santa Cruz, California, USA.

Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.
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http://dx.doi.org/10.1038/jid.2010.426DOI Listing
May 2011

Development of quantitative molecular clinical end points for siRNA clinical trials.

J Invest Dermatol 2011 May 30;131(5):1029-36. Epub 2010 Dec 30.

TransDerm, Santa Cruz, California 95060, USA.

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.
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http://dx.doi.org/10.1038/jid.2010.372DOI Listing
May 2011

Development of skin-humanized mouse models of pachyonychia congenita.

J Invest Dermatol 2011 May 9;131(5):1053-60. Epub 2010 Dec 9.

Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT, Madrid, Spain.

Molecular characterization and assessment of therapeutic outcomes for inherited cutaneous disorders requires faithful preclinical models. In this study we report the establishment of two different skin-humanized pachyonychia congenita (PC) model systems, based on permanent engraftment of bioengineered skin equivalents generated from patient skin cells onto immunodeficient mice. Using keratinocytes and fibroblasts isolated from unaffected skin biopsies of two PC patients carrying the p.Asn171Lys mutation of the keratin 6a gene (KRT6A), we were able to regenerate PC-derived human skin that appeared phenotypically normal, but developed sustained PC features after the use of an acute hyperproliferative stimulus (i.e., tape stripping). In contrast, the use of keratinocytes from an affected area (i.e., plantar callus) from a different patient carrying the KRT6A mutation p.Asn171Asp led to a full recapitulation of the phenotype that included marked acanthosis and epidermal blistering after minor trauma. The ability to generate large numbers of PC skin-engrafted mice will enable the testing of novel pharmacological or gene-based therapies for this as yet untreatable disease.
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http://dx.doi.org/10.1038/jid.2010.353DOI Listing
May 2011

Silencing of reporter gene expression in skin using siRNAs and expression of plasmid DNA delivered by a soluble protrusion array device (PAD).

Mol Ther 2010 Sep 22;18(9):1667-74. Epub 2010 Jun 22.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.
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http://dx.doi.org/10.1038/mt.2010.126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956931PMC
September 2010

Nanoparticle formation of organic compounds with retained biological activity.

J Pharm Sci 2010 Jun;99(6):2750-5

Department of Chemistry, Stanford University, 333 Campus Drive, Stanford, California 94305-5080, USA.

Many pharmaceuticals are formulated as powders to aid drug delivery. A major problem is how to produce powders having high purity, controlled morphology, and retained bioactivity. We demonstrate the use of supercritical carbon dioxide as an antisolvent for meeting this need for two model drug systems, quercetin, a sparingly soluble antioxidant, and short interfering RNA (siRNA), which can silence genes. In both cases we achieve retention of bioactivity as well as a narrow particle size distribution in which the particles are free of impurities.
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http://dx.doi.org/10.1002/jps.22035DOI Listing
June 2010

First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.

Mol Ther 2010 Feb 24;18(2):442-6. Epub 2009 Nov 24.

Department of Dermatology, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112-5550, USA.

The rare skin disorder pachyonychia congenita (PC) is an autosomal dominant syndrome that includes a disabling plantar keratoderma for which no satisfactory treatment is currently available. We have completed a phase Ib clinical trial for treatment of PC utilizing the first short-interfering RNA (siRNA)-based therapeutic for skin. This siRNA, called TD101, specifically and potently targets the keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA. The safety and efficacy of TD101 was tested in a single-patient 17-week, prospective, double-blind, split-body, vehicle-controlled, dose-escalation trial. Randomly assigned solutions of TD101 or vehicle control were injected in symmetric plantar calluses on opposite feet. No adverse events occurred during the trial or in the 3-month washout period. Subjective patient assessment and physician clinical efficacy measures revealed regression of callus on the siRNA-treated, but not on the vehicle-treated foot. This trial represents the first time that siRNA has been used in a clinical setting to target a mutant gene or a genetic disorder, and the first use of siRNA in human skin. The callus regression seen on the patient's siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.
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http://dx.doi.org/10.1038/mt.2009.273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839285PMC
February 2010

Rapamycin selectively inhibits expression of an inducible keratin (K6a) in human keratinocytes and improves symptoms in pachyonychia congenita patients.

J Dermatol Sci 2009 Nov 21;56(2):82-8. Epub 2009 Aug 21.

TransDerm, Inc., Santa Cruz, CA, USA.

Background: The macrolide sirolimus (rapamycin) selectively blocks translation of mRNAs containing a terminal 5' oligopyrimidine (TOP) tract by altering the activity of mammalian target of rapamycin (mTOR) and inhibiting downstream mTOR pathway components involved in TOP mRNA translation. The skin disorder pachyonychia congenita (PC) is caused by mutations in the inducible keratins (K) including K6a, K6b, K16 and K17. Published sequence data suggest the 5' untranslated regions of K6a and K6b mRNAs contain 5' TOP motifs and therefore may be sensitive to rapamycin treatment.

Objective: Determine if mTOR inhibitors (rapamycin, temsirolimus or everolimus) are viable drug candidates for treatment of PC and other disorders caused by inappropriate expression of K6a and K6b.

Methods: 5' RACE analysis was used to map the transcriptional start sites for K5, K6a, K6b, K14, K16 and K17. The sensitivity of these keratins to mTOR inhibitors was determined by Western and qPCR analysis following treatment of a human HaCaT keratinocyte cell line with rapamycin, temsirolimus or everolimus. A small off-label study was undertaken using orally administered rapamycin in three PC patients and the effects were monitored by clinical examination, photography, a validated Dermatology Life Quality Index (DLQI) and a pain and activity diary.

Results: Sequence comparison and 5' RACE analysis of the 5' untranslated regions of K6a and K6b revealed putative TOP regulatory elements. Treatment of a human HaCaT keratinocyte cell line with mTOR inhibitors (rapamycin, temsirolimus or everolimus) resulted in selective K6a repression. Furthermore, treatment of this HaCaT cell line with siRNAs targeting components of the mTOR pathway altered the levels of K6a expression. To test the ability of rapamycin to ameliorate PC symptoms, an off-label study was conducted. PC patient clinical responses to oral rapamycin showed a therapeutic response in callus character as well as subjective improvement. Of particular note, rapamycin greatly reduced the presence of painful cutaneous thromboses after reaching therapeutic serum levels. The well-known rapamycin side effects led to the early withdrawal of all of the patients from the study.

Conclusion: Rapamycin selectively blocks K6a expression in human keratinocytes. The improvement of symptoms in PC patients following rapamycin treatment suggests rapamycin (or rapamycin analogs) may be a therapeutic option, particularly if topical formulations can be developed that avoid the side effects associated with systemic administration.
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http://dx.doi.org/10.1016/j.jdermsci.2009.07.008DOI Listing
November 2009

Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding.

RNA 2009 Aug 24;15(8):1597-604. Epub 2009 Jun 24.

Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois 60607, USA.

Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays.
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http://dx.doi.org/10.1261/rna.1681609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714759PMC
August 2009

Following movement of the L1 stalk between three functional states in single ribosomes.

Proc Natl Acad Sci U S A 2009 Feb 3;106(8):2571-6. Epub 2009 Feb 3.

Department of Physics, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA.

The L1 stalk is a mobile domain of the large ribosomal subunit E site that interacts with the elbow of deacylated tRNA during protein synthesis. Here, by using single-molecule FRET, we follow the real-time dynamics of the L1 stalk and observe its movement relative to the body of the large subunit between at least 3 distinct conformational states: open, half-closed, and fully closed. Pretranslocation ribosomes undergo spontaneous fluctuations between the open and fully closed states. In contrast, posttranslocation ribosomes containing peptidyl-tRNA and deacylated tRNA in the classical P/P and E/E states, respectively, are fixed in the half-closed conformation. In ribosomes with a vacant E site, the L1 stalk is observed either in the fully closed or fully open conformation. Several lines of evidence show that the L1 stalk can move independently of intersubunit rotation. Our findings support a model in which the mobility of the L1 stalk facilitates binding, movement, and release of deacylated tRNA by remodeling the structure of the 50S subunit E site between 3 distinct conformations, corresponding to the E/E vacant, P/E hybrid, and classical states.
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http://dx.doi.org/10.1073/pnas.0813180106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650305PMC
February 2009

Stability study of unmodified siRNA and relevance to clinical use.

Oligonucleotides 2008 Dec;18(4):345-54

TransDerm Inc., Santa Cruz, California, USA.

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.
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http://dx.doi.org/10.1089/oli.2008.0149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829675PMC
December 2008

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita.

J Dermatol Sci 2008 Sep 20;51(3):151-7. Epub 2008 May 20.

Department of Dermatology and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States.

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.
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http://dx.doi.org/10.1016/j.jdermsci.2008.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587483PMC
September 2008

Single-nucleotide-specific siRNA targeting in a dominant-negative skin model.

J Invest Dermatol 2008 Mar 11;128(3):594-605. Epub 2007 Oct 11.

TransDerm Inc., Santa Cruz, California, USA.

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.
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http://dx.doi.org/10.1038/sj.jid.5701060DOI Listing
March 2008

Development of therapeutic siRNAs for pachyonychia congenita.

J Invest Dermatol 2008 Jan 30;128(1):50-8. Epub 2007 Aug 30.

Epithelial Genetics Group, Human Genetics Unit, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.
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http://dx.doi.org/10.1038/sj.jid.5701040DOI Listing
January 2008

The antibiotic viomycin traps the ribosome in an intermediate state of translocation.

Nat Struct Mol Biol 2007 Jun 21;14(6):493-7. Epub 2007 May 21.

Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.

During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
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http://dx.doi.org/10.1038/nsmb1243DOI Listing
June 2007

Observation of intersubunit movement of the ribosome in solution using FRET.

J Mol Biol 2007 Jul 20;370(3):530-40. Epub 2007 Apr 20.

Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used Förster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.
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http://dx.doi.org/10.1016/j.jmb.2007.04.042DOI Listing
July 2007

SiRNA-mediated selective inhibition of mutant keratin mRNAs responsible for the skin disorder pachyonychia congenita.

Ann N Y Acad Sci 2006 Oct;1082:56-61

TransDerm, Santa Cruz, California 95060, USA.

RNA interference offers a novel approach for treating genetic disorders including the rare monogenic skin disorder pachyonychia congenita (PC). PC is caused by mutations in keratin 6a (K6a), K6b, K16, and K17 genes, including small deletions and single nucleotide changes. Transfection experiments of a fusion gene consisting of K6a and a yellow fluorescent reporter (YFP) resulted in normal keratin filament formation in transfected cells as assayed by fluorescence microscopy. Similar constructs containing a single nucleotide change (N171K) or a three-nucleotide deletion (N171del) showed keratin aggregate formation. Mutant-specific small inhibitory RNAs (siRNAs) effectively targeted these sites. These studies suggest that siRNAs can discriminate single nucleotide mutations and further suggest that "designer siRNAs" may allow effective treatment of a host of genetic disorders including PC.
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http://dx.doi.org/10.1196/annals.1348.059DOI Listing
October 2006

mRNA helicase activity of the ribosome.

Cell 2005 Jan;120(1):49-58

Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, we show that ribosomes are able to disrupt downstream helices, including a perfect 27 base pair helix of predicted T(m) = 70 degrees . Using helices of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. We conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.
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http://dx.doi.org/10.1016/j.cell.2004.11.042DOI Listing
January 2005