Publications by authors named "Roger L Kaspar"

45 Publications

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes.

J Clin Invest 2016 06 16;126(6):2356-66. Epub 2016 May 16.

Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16-/- mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid-derived 2 related factor 2-dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16-/- mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16-/- footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16-/- mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI84870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887188PMC
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11307-015-0875-zDOI Listing
February 2016

Differential fates of biomolecules delivered to target cells via extracellular vesicles.

Proc Natl Acad Sci U S A 2015 Mar 23;112(12):E1433-42. Epub 2015 Feb 23.

Departments of Pediatrics, Microbiology and Immunology, and Radiology, Stanford University School of Medicine, Stanford, CA 94305; and

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1418401112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378439PMC
March 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jdermsci.2015.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374015PMC
March 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mtna.2013.56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027428PMC
October 2013

Gene silencing following siRNA delivery to skin via coated steel microneedles: In vitro and in vivo proof-of-concept.

J Control Release 2013 Mar 8;166(3):211-9. Epub 2013 Jan 8.

School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB, UK.

The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified "self-delivery" siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2012.12.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594125PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

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

Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder.

J Invest Dermatol 2012 Jun 8;132(6):1627-35. Epub 2012 Mar 8.

Dermatology and Genetic Medicine, Division of Molecular Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, UK.

Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin 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 therapeutic package 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 an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2012.28DOI Listing
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nn203745nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246574PMC
December 2011

An appraisal of oral retinoids in the treatment of pachyonychia congenita.

J Am Acad Dermatol 2012 Jun 24;66(6):e193-9. Epub 2011 May 24.

Department of Dermatology, Innsbruck Medical University, Innsbruck, Austria.

Background: Pachyonychia congenita (PC), a rare autosomal-dominant keratin disorder caused by mutations in keratin genes KRT6A/B, KRT16, or KRT17, is characterized by painful plantar keratoderma and hypertrophic nail dystrophy. Available studies assessing oral retinoid treatment for PC are limited to a few case reports.

Objective: We sought to assess overall effectiveness, adverse effects, and patient perspective in patients with PC receiving oral retinoids.

Methods: In a questionnaire-based retrospective cross-sectional survey of 30 patient with PC assessing oral retinoids (10-50 mg/d for 1-240 months), we determined the clinical score, satisfaction score, visual analog pain scale, and adverse effects.

Results: In 50% of patients there was thinning of hyperkeratoses (average improvement 1.6 on a scale from -3 to +3) (95% confidence interval 1.2-1.9, P < .001). In all, 14% observed amelioration of their pachyonychia; 79% did not experience any nail change. The self-reported overall satisfaction score with oral retinoid treatment was 2 or greater in 50% of the patients (mean 4.5 on a scale of 1-10). Although 33% reported decreased and 27% increased plantar pain with treatment, 40% did not notice any pain change. All patients experienced adverse effects, and 83% reported to have discontinued medication. Risk/benefit analysis favored lower retinoid doses (≤25 mg/d) over a longer time period (>5 months), compared with higher doses (>25 mg/d) for a shorter time (≤5 months).

Limitations: The retrospective, cross-sectional study design is prone to a recall bias.

Conclusion: Oral retinoids are effective in some patients with PC. However, many patients discontinued medication because adverse effects outweighed the benefits. Careful dose titration is warranted in patients informed about potential adverse effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaad.2011.02.003DOI Listing
June 2012

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2010.426DOI Listing
May 2011

In vivo imaging of human and mouse skin with a handheld dual-axis confocal fluorescence microscope.

J Invest Dermatol 2011 May 30;131(5):1061-6. Epub 2010 Dec 30.

James H. Clark Center for Biomedical Engineering & Sciences, Department of Pediatrics and the Molecular Imaging Program, Stanford University, Stanford, California, USA.

Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2010.401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839658PMC
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2010.353DOI Listing
May 2011

Biodegradable nanoparticles with sustained release of functional siRNA in skin.

J Pharm Sci 2010 Oct;99(10):4261-6

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

A key challenge in developing RNAi-based therapeutics is efficient delivery of functional short interfering RNA (siRNA) to target cells. To address this need, we have used a supercritical CO(2) process to incorporate siRNA in biodegradable polymer nanoparticles (NPs) for in vivo sustained release. By this means we have obtained complete encapsulation of the siRNA with minimal initial burst effect from the surface of the NPs. The slow release of a fluorescently labeled siRNA mimic (siGLO Red) was observed for up to 80 days in vivo after intradermal injection into mouse footpads. In vivo gene silencing experiments were also performed, showing reduction of GFP signal in the epidermis of a reporter transgenic mouse model, which demonstrates that the siRNA retained activity following release from the polymer NPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jps.22147DOI Listing
October 2010

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope.

J Biomed Opt 2010 May-Jun;15(3):036027

Stanford University, Department of Electrical Engineering, Ginzton Laboratory, Stanford, California 94305, USA.

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.3432627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904026PMC
October 2010

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jdermsci.2009.07.008DOI Listing
November 2009

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

Nucleic Acids Res 2008 Dec 25;36(21):6752-66. Epub 2008 Oct 25.

SomaGenics, Inc, Santa Cruz, CA 95060, USA.

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkn637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588507PMC
December 2008