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

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.     

The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA,PRINS

Please cite this paper as: The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA, PRINS. Experimental Dermatology 2010; 19: 269–278. Abstract: Psoriasis Susceptibility‐Related RNA Gene Induced by Stress (PRINS) is a non‐coding RNA overexpressed in lesional and non‐lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti‐apoptotic effects in keratinocytes. siRNA‐mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon‐inducible gene with anti‐apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400‐fold upregulated in hyperproliferative lesional and ninefold upregulated in non‐lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA‐mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.     

Small-interfering RNA targeted at antiapoptotic mRNA increases keratinocyte sensitivity to apoptosis

Background Gene silencing RNA interference technology concentrates on downregulation of gene expression using specific double‐stranded RNA molecules (small‐interfering RNA, siRNA), which induce the degradation of complementary mRNA. SiRNA therapeutics is currently being tested in several clinical trials. Skin disorders are an attractive target for siRNA‐based technology because effective agents can be delivered by topical application. In several inflammatory skin disorders, the barrier function of the skin is markedly impaired and this may increase the delivery efficiency. Psoriasis is a very common skin disorder. The characteristics of this disorder are abnormal keratinocyte proliferation and inflammation. Keratinocytes from psoriatic epidermis express much higher levels of the antiapoptotic protein, Bcl‐xL, compared with normal keratinocytes. Insulin‐like growth factor 1 receptor (IGF‐1R) plays a major role in cell growth, differentiation and apoptosis in many cell types, including keratinocytes and IGF‐1R activation plays an important role in the pathogenesis of psoriasis. Keratinocytes from patients with psoriasis are more susceptible to IGF‐1‐stimulated proliferation compared with normal keratinocytes. IGF‐1R is expressed by proliferating basal and suprabasal keratinocytes and is more abundant in psoriatic lesions. Objectives To prove the validity of IGF‐1R and Bcl‐xL as useful targets for siRNA‐based therapeutics and to deliver siRNA selectively and efficiently to primary human keratinocytes; this is a primary essential step in the development of siRNA. Methods Primary normal human keratinocytes were transfected with various siRNA molecules, and transfection efficiency was monitored by fluorescent labelling. Cell growth and apoptosis induction were evaluated in the transfected cells. Results We were able to deliver efficiently siRNA targeting Bcl‐xL or IGF‐1R to primary human keratinocyte cultures. We also showed that siRNAs targeting Bcl‐xL and IGF‐1R induce growth inhibition, apoptosis and increased sensitivity to ultraviolet B in keratinocytes. Conclusions The present findings demonstrate that Bcl‐xL and IGF‐1R are valid, important targets for siRNA‐based technology directed at the suppression of keratinocyte hyperproliferation.     

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

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.     

Novel nanosome delivery system combined with siRNA targeting the antimicrobial gene DFB4: a new approach for psoriasis management?

In a recently published issue of the journal, Bracke et al. demonstrate an impressive improvement in psoriasiform features in allogeneic human skin grafts transplanted onto immune‐deficient mice. This improvement was achieved using a novel nanosome (SECosome) as a vehicle for delivering topically applied siRNA to human epidermis. Targeting the gene DFB4 with this delivery system, they prevented translation of the antimicrobial peptide, human β defensin‐2(hBD2), thus normalizing the psoriasiform epidermal phenotype of siRNA/SECosome‐treated human skin grafts. This study encourages the exploration of topical gene silencing strategies in dermatology and refocuses our attention on both, the role of hBD2 in psoriasis pathogenesis and the thorny question which animal model reflects human psoriasis most faithfully.     

SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7

Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal‐type 5 (SPINK5) gene, which encodes the protease inhibitor lympho‐epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast‐populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin‐like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin‐like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.     

Delivery and inhibition of reporter genes by small interfering RNAs in a mouse skin model

RNA interference offers the potential of a novel therapeutic approach for treating skin disorders. To this end, we investigated delivery of nucleic acids, including a plasmid expressing the reporter gene luciferase, to mouse skin by intradermal injection into footpads using in vivo bioluminescence imaging over multiple time points. In order to evaluate the ability of RNA interference to inhibit skin gene expression, reporter gene constructs were co-injected with specific or non-specific siRNAs and the in vivo effects measured. Our results revealed that specific unmodified and modified siRNAs (but not nonspecific matched controls) strongly inhibit reporter gene expression in mice. These results indicate that small interfering RNA, delivered locally as RNA directly or expressed from viral or non-viral vectors, may be effective agents for treating skin disorders.     

Development of quantitative molecular clinical end points for siRNA clinical trials

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.     

An immunohistochemical and immunoelectron microscopic study of the ontogeny of rat Langerhans cell lineage with anti-macrophage and anti-Ia monoclonal antibodies

An immunohistochemical study with anti-macrophage and anti-Ia monoclonal antibodies was performed to clarify the relationship between Langerhans cells (LC) and indeterminate cells (IC) in rat epidermis both in adulthood and in the fetal stage. On immunoelectron microscopy, a mouse anti-rat macrophage monoclonal antibody, TRPM-1, recently produced by us, reacted with IC and some LC in adult rat skin. Ontogenic study revealed that TRPM-1-positive cells first appeared in the epidermis of fetal rat heads on Day 17 of gestation and then spread caudally along the anterior-posterior axis. On Day 20 of gestation, when the distribution of the TRPM-1-positive cells over body surface became even, Ia-positive cells appeared in the epidermis and began to increase in number. Ia-positive cells with Birbeck granules were found on Day 21 of gestation. These results indicate that. TRPM-1-positive IC matured into Ia-expressing LC after being exposed to microenvironmental change during the perinatal period. The number of Ia-positive cells exceeded that of TRPM-1-positive cells at around 5 d after birth. Afterwards, there were more dendritic Ia-positive cells found in the interfollicular areas than TRPM-1-positive ones. However, local concentrations of the TRPM-1-positive IC in the follicular infundibula were frequently found in the fetal stage and occasionally in adulthood. These TRPM-1-positive cells in the follicular infundibula were thought to be a precursor pool in the epidermis for LC.     

Acyclovir bioavailability in human skin.

Clinical experience demonstrates that oral acyclovir (ACV) is superior to topical ACV in treating recurrent cutaneous herpes simplex virus type 1 (HSV-1) infections. Cutaneous HSV-1 infections are complex in their pathology, affecting the basal epidermis in skin as well as establishing a latency phase in sensory ganglia. In vitro and in vivo human skin model systems were used in the present study to quantitate ACV disposition and absorption in skin and blood following two routes of administration and to investigate whether bioavailability differences were the result of insufficient drug delivery. Physiochemical and physiologic parameters determined from these experiments were used to develop a mathematical model to predict ACV disposition and absorption in human subjects. Model predictions and in vivo data agree; topical administration of commercial 5% ACV ointment and cream result in a 48 times greater total epidermal ACV concentration than after oral administration. Mathematical modeling of the ACV concentration gradient through the epidermis revealed, however, that the drug concentration in the target site of HSV-1 infections, the basal epidermis, is 2-3 times less after topical administration than after oral administration. Thus, the observed lack of clinical efficacy with topical ACV therapy in the recurring HSV-1 infection likely reflects the insufficient delivery of the drug to the target site of the HSV-1 infection, the basal epidermis.     

The role of polymorphonuclear leukocyte infiltration in herpes simplex virus infection of murine skin

Abstract We undertook the present study to investigate the role of polymorphonuclear leukocytes (PMN) in defending skin against herpes simplex virus (HSV) infection. For this purpose, we established a mouse model of cutaneous HSV infection. The hind limb footpad skin of 4-week-old ICR mice was abraded linearly once with a feather edge file and infected with various strains of HSV with different virulence. In uninfected control mice, PMN appeared at the abraded skin lesion within 24 h, and were eliminated from the epidermis after 3 days. Mice inducted with a highly virulent strain of HSV demonstrated wide and severe erythematous lesions of the footpad skin and histologically, virus antigen-positive ballooning degenerated keratinocytes were observed. However, in infections with attenuated strains of HSV, the epidermis was regenerated and a viral antigen was discharged within 5 days, together with any infiltrated PMN. Macrophages and NK cells numbered less than PMN. In mice treated with anti-PMN antiserum before HSV infection, PMN infiltration was significantly suppressed 1 day after infection, and these animals developed a severe cutaneous disease even if infected with an attenuated virus. These results indicate the importance of PMN in the control of HSV cutaneous infections, especially in the primary infectious phase. Received: 17 April 1998 / Received after revision: 3 September 1998 / Accepted: 11 September 1998     

Heterozygous HPS1 mutations in a case of Hermansky-Pudlak syndrome with giant melanosomes

We report a Japanese man with Hermansky-Pudlak syndrome, having oculocutaneous albinism with a bleeding diathesis. Gene analysis of the patient's peripheral blood cells revealed that he was a compound heterozygote for HPS1 gene mutations. One of the mutations was a novel frameshift mutation at codon 321 (a G insertion) in exon 11 ( approximately 962-963insG), and the other was a 5' splice-junction mutation of IVS5 (IVS5 + 5G-->A). The content of eumelanin in the patient's hairs was significantly reduced. Histological analysis using light and electron microscopy revealed that melanocytes in the patient's epidermis contained an appreciable number of giant melanosomes. Cultured melanocytes from the patient's skin also contained giant melanosomes. Our finding of mutations in the HPS1 gene in relation to abnormalities in melanosome morphology and melanin production shed light on the role and function of the HPS1 gene product in the synthesis of melanosomes and melanin pigment.     

Novel mutations of TGM1 in a child with congenital ichthyosiform erythroderma

We report novel mutations in the transglutaminase (TGase) 1 gene (TGM1) in a Japanese boy with non-bullous congenital ichthyosiform erythroderma (NBCIE). The patient showed fine, grey or light-brown scales on an erythematous skin. An in situ TGase activity assay detected markedly reduced TGase activity in the patient's epidermis. Electron microscopy revealed incomplete thickening of the cornified cell envelope during keratinization in the epidermis. Sequencing of the entire exons and exon-intron borders of TGM1 revealed that the proband was a compound heterozygote for two novel mutations, 9008delA and R388H. In lamellar ichthyosis, most previously reported TGM1 mutations have been located in the central core domain or upstream of the TGase 1 molecule. In the present NBCIE patient, the frameshift mutation 9008delA resulting in a premature termination codon at the tail of the TGase 1 peptide was in the beta-barrel 2 domain (C-terminal end domain) of the peptide, far from the active sites of the TGase 1 molecule, and the mis-sense mutation R388H was in the core domain.     

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

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.