Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin.

Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.

The Efficacy and Biopharmaceutical Properties of a Fixed-Dose Combination of Disulfiram and Benzyl Benzoate.

Scabies and hair lice are parasitic diseases that affect human skin and hair, respectively. The incidence and resistances of these infections are increasing. Tenutex® (disulfiram and benzyl benzoate emulsion) is an alternative to standard insecticides to avoid resistances. The aim of the work is to evaluate the transdermal absorption and the in vitro efficacy against scabies and hair lice after different exposition times. Dermatomed human skin was used to assess the dermal absorption using a validated High Performance Liquid Chromatography (HPLC) method. HEK001 keratinocytes were used to evaluate the cytotoxicity of benzyl benzoate. Only benzyl benzoate was able to cross the skin, but it did not show cytotoxicity at any of the tested concentrations. The product efficacy was tested on Psoroptes ovis after direct contact and after administration on sheep skin explants at different contact times. Permethrin/malathion-resistant strains of Pediculus humanis capitis adults and eggs were directly exposed to Tenutex, and the vitality and hatchability, respectively, were evaluated. The anti-scabies study demonstrated that exposure for 6 or 24 h completely eradicated the parasite. The pediculicidal activity of Tenutex exhibited superior efficacy than standard treatment on resistant lice. The positive results obtained suggest that Tenutex® is a good treatment option, especially in drug resistance situations.

RNAi-based modulation of IFN-γ signaling in skin.

Aberrant activation of interferon (IFN)-γ signaling plays a key role in several autoimmune skin diseases, including lupus erythematosus, alopecia areata, vitiligo, and lichen planus. Here, we identify fully chemically modified small interfering RNAs (siRNAs) that silence the ligand binding chain of the IFN-γ receptor (IFNGR1), for the modulation of IFN-γ signaling. Conjugating these siRNAs to docosanoic acid (DCA) enables productive delivery to all major skin cell types local to the injection site, with a single dose of injection supporting effective IFNGR1 protein reduction for at least 1 month in mice. In an ex vivo model of IFN-γ signaling, DCA-siRNA efficiently inhibits the induction of IFN-γ-inducible chemokines, CXCL9 and CXCL10, in skin biopsies from the injection site. Our data demonstrate that DCA-siRNAs can be engineered for functional gene silencing in skin and establish a path toward siRNA treatment of autoimmune skin diseases.

Future Perspectives of Oral Delivery of Next Generation Therapies for Treatment of Skin Diseases.

Gene therapies have conspicuously bloomed in recent years as evidenced by the increasing number of cell-, gene-, and oligo-based approved therapies. These therapies hold great promise for dermatological disorders with high unmet need, for example, epidermolysis bullosa or pachyonychia congenita. Furthermore, the recent clinical success of clustered regularly interspaced short palindromic repeats (CRISPR) for genome editing in humans will undoubtedly contribute to defining a new wave of therapies. Like biologics, naked nucleic acids are denatured inside the gastrointestinal tract and need to be administered via injections. For a treatment to be effective, a sufficient amount of a given regimen needs to reach systemic circulation. Multiple companies are racing to develop novel oral drug delivery approaches to circumvent the proteolytic and acidic milieu of the gastrointestinal tract. In this review, we provide an overview of the evolution of the gene therapy landscape, with a deep focus on gene and oligonucleotide therapies in clinical trials aimed at treating skin diseases. We then examine the progress made in drug delivery, with particular attention on the peptide field and drug-device combinations that deliver macromolecules into the gastrointestinal tract. Such novel devices could potentially be applied to administer other therapeutics including genes and CRISPR-based systems.

Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products.

BACKGROUND: We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function. RESULTS: We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas. CONCLUSIONS: The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions.

Genes encoding WFDC- and Kunitz-type protease inhibitor domains: are they related?

We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).

Overexpression of elafin in ovarian carcinoma is driven by genomic gains and activation of the nuclear factor kappaB pathway and is associated with poor overall survival.

Ovarian cancer is a leading cause of cancer mortality in women. The aim of this study was to elucidate whether whey acidic protein (WAP) genes on chromosome 20q13.12, a region frequently amplified in this cancer, are expressed in serous carcinoma, the most common form of the disease. Herein, we report that a trio of WAP genes (HE4, SLPI, and Elafin) is overexpressed and secreted by serous ovarian carcinomas. To our knowledge, this is the first report linking Elafin to ovarian cancer. Fluorescence in situ hybridization analysis of primary tumors demonstrates genomic gains of the Elafin locus in a majority of cases. In addition, a combination of peptidomimetics, RNA interference, and chromatin immunoprecipitation experiments shows that Elafin expression can be transcriptionally upregulated by inflammatory cytokines through activation of the nuclear factor kappaB pathway. Importantly, using a clinically annotated tissue microarray composed of late-stage, high-grade serous ovarian carcinomas, we show that Elafin expression correlates with poor overall survival. These results, combined with our observation that Elafin is secreted by ovarian tumors and is minimally expressed in normal tissues, suggest that Elafin may serve as a determinant of poor survival in this disease.

Urokinase-type plasminogen activator receptor: a beacon of malignancy?

Discriminating between benign and malignant disease is a pivotal diagnostic issue in the care of women with pelvic masses. Identification of serum biomakers that can rellably make the distinction would aid in the proper referral for patient care and may provide leads in the development of early detection strategies.

Evolution of kallikrein-related peptidases in mammals and identification of a genetic locus encoding potential regulatory inhibitors.

The human kallikrein locus on chromosome 19 consists of 15 genes encoding serine proteases. Here we review studies on their evolution, which demonstrate that there are several taxon-unique KLK1 paralogs in mouse, rat and horse, but not in primates and many other mammals. Furthermore, the duplication yielding KLK2 and prostate-specific antigen (KLK3) appears to be specific to primates, but a functional progenitor to KLK2 is expressed in the dog prostate. The linkage to a locus of possible regulatory protease inhibitors on chromosome 20 is discussed, as is the potential role of the kallikrein locus in innate immunity.

The evolution of a genetic locus encoding small serine proteinase inhibitors.

We previously identified a locus on human chromosome 20 that encompasses 14 genes of postulated WFDC-type proteinase inhibitors with a potential role in innate immunity. In an extended study, homologous loci are here described on mouse chromosome 2, rat chromosome 3, and dog chromosome 24. As in humans, the murine and canine loci are divided into two sub-loci separated by 0.2Mb. The majority of genes are conserved in all species, but there are also species-specific gains and losses of genes, e.g., several duplications have yielded four SLPI genes in the rat and, most surprisingly, there is no murine elafin gene. Two human pseudogenes were identified due to the discovery of functional rodent genes. The conservation of different WFDC domains varies considerably, and it is hypothesized that this reflects a dual role of WFDC inhibitors in natural immunity, which is directed both against microbes and proinflammatory cells.

Molecular cloning of complementary DNA encoding mouse seminal vesicle-secreted protein SVS I and demonstration of homology with copper amine oxidases.

The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans-presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.

A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein.

A locus containing 14 genes, encoding protein domains that have homology with whey acidic protein (WAP), has been identified in a region of 678 kb on human chromosome 20q12-13.1. Among them are genes of the known or postulated protease inhibitors elafin, secretory leucocyte protease inhibitor, human epididymis gene product 4, eppin, and huWAP2. Nucleotide sequences of full-length transcripts were obtained from cDNA fragments generated by rapid amplification of cDNA ends. Characteristic features of the genes are that the upstream promoter regions are devoid of TATA-boxes and that the coding nucleotides are divided into distinct exons for the signal peptide and for each WAP domain. In most cases, there is also a separate exon encompassing a few terminal codons and the 3' untranslated nucleotides. There are also examples of mixed type inhibitors, that encode inhibitor domains of both WAP and Kunitz types. Several of the genes appear to be expressed ubiquitously, but, in most cases, the highest transcript levels are found in epididymis followed by testis and trachea. Some of the genes also display high transcript levels in neural tissues. Potential biological roles of protein products could be in host defence against invading micro-organisms or in the regulation of endogenous proteolytic enzymes, of which those originating from the kallikrein gene locus on chromosome 19 are of particular interest.

Identification of a novel protease inhibitor gene that is highly expressed in the prostate.

A novel gene was identified 52 kb upstream of the gene encoding the protease inhibitor elafin (PI3) on human chromosome 20q12-13.1. The transcript of the new gene, denoted huWAP2, was characterized by rapid amplification of cDNA ends and DNA sequencing. The size is 774 bp and it gives rise to a polypeptide of 111 amino acid residues that is homologous to elafin and similar WAP-type protease inhibitors. By RT-PCR it was shown that the gene is highly expressed in prostate, skin, lung, and esophagus.