Iacovos P. Michael

In silico Analysis of the Human Kallikrein Gene 6

Kallikreins are a family of 15 serine proteases clustered together on the long arm of chromosome 19. Recent reports have linked kallikreins to malignancy. The human kallikrein gene 6 (KLK6) is a newly characterized member of the human kallikrein gene family. Recent work has focused on the possible role of this gene and its protein product as a tumor marker and its involvement in diseases of the central nervous system. In this study, we performed extensive in silico analyses of KLK6 expression from different databases using various bioinformatic tools. These data enabled us to construct and verify the longest transcript for this kallikrein, to identify several polymorphisms among published sequences and to summarize the 21 single-nucleotide polymorphisms of the gene. Our expressed sequence tag (EST) analyses suggest the existence of seven new splice variants of the gene, in addition to the already reported ones. Most of these variants were identified in libraries from cancerous tissues. KLK6 orthologues were identified from three other species with approximately 86% overall homology with rat and mouse orthologues. We also utilized several databases to compare KLK6 gene expression in normal and cancerous tissues. The serial analysis of gene expression and EST expression profiles showed upregulation of the gene in female genital (ovarian and uterine) and gastrointestinal (gastric, colon, esophageal and pancreatic) cancers. Significant downregulation was observed in breast cancers and brain tumors, in relation to their normal counterparts.

Identification of New Splice Variants and Differential Expression of the Human Kallikrein 10 Gene, a Candidate Cancer Biomarker

The human kallikrein gene 10 (KLK10) is a member of the kallikrein gene family on chromosome 19q13.4. This gene was identified by its downregulation in breast cancer, and preliminary evidence suggests that it may act as a tumor suppressor. A computer-based analysis was performed on EST and SAGE clones from the Cancer Genome Anatomy Project and other databases. Experimental verification of differential expression of KLK10 in cancer was performed by PCR using gene-specific primers. The mRNA and EST analysis allowed the construction of the longest transcript of the gene and characterization of a 5′ extension of the reported mRNA. In addition, seven new splice variants of KLK10 were identified. One of these variants, named KLK10 splice variant 3 (KLK10-SV3) which starts with a novel first exon, was experimentally verified. This variant is predicted to encode for the same protein as the ‘classical’ KLK10 mRNA, since the first exon is untranslated. One variant mRNA partially matches with the sequence of KLK10, while the rest of the mRNA matches with a portion of the polycystic kidney disease gene, found on chromosome 15. This variant could not be experimentally verified in either normal or cancerous tissues. There are 39 reported single nucleotide polymorphisms (SNPs) for the gene, in which three result in amino acid substitutions. SAGE analysis shows a clear upregulation of KLK10 in ovarian, pancreatic, colon, and gastric cancers. The gene is, however, downregulated in breast and prostate cancers. A three-fold decrease in expression levels was noted in actinic keratosis, compared to normal skin from the same patient. The differential regulation of KLK10 in ovarian and prostate cancers was experimentally verified by RT-PCR analysis. In addition, a significant number of clones were isolated from carcinomas of the head and neck. Fewer clones were found in carcinomas of the skin, brain and prostate. Orthologues were identified in three other species, with the highest degree of homology observed with the mouse and rat orthologues (42% in each). In conclusion new splice variants of the KLK10 gene were identified. These in silico analyses show a differential expression of the gene in various malignancies and provide the basis for directing experimental efforts to investigate the possible role of the gene as a cancer biomarker.

KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome.

Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in SPINK5 encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. Spink5 -/- mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which Klk5 and Spink5 were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in Spink5 -/- mice. By repeated intercrossing between Klk5 -/- mice with Spink5 -/- mice, we generated Spink5 -/- Klk5 -/- animals. We showed that Klk5 knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using in situ zymography and specific protease substrates, we showed that Klk5 knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in Spink5 -/- Klk5 -/-. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in Spink5 -/- Klk5 -/- skin. Notably, Il-1β, Il17A and Tslp levels were normalized. Our results provide in vivo evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in Spink5 -/- skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.

The Kallikrein Gene 5 Splice Variant 2 Is a New Biomarker for Breast and Ovarian Cancer

The presence of more than one mRNA form for the same gene is common among kallikreins, and many of the kallikrein splice variants may hold significant clinical value. The human kallikrein gene 5 (KLK5) is a member of the human kallikrein gene family of serine proteases on chromosome 19q13.4. KLK5 has been shown to be differentially expressed in a variety of endocrine tumors including ovarian, breast and prostate cancer. Utilizing Expressed Sequence Tag database analysis and reverse transcriptase polymerase chain reaction, we identified a new alternatively spliced form of KLK5(KLK5-splice variant 2, KLK5-SV2). This variant mRNA is 1,438 bp in length; formed of 195 bp of 5′ untranslated region, 882 bp of protein coding sequence and a 3′ untranslated region of 326 nucleotides. KLK5-SV2 has 7 exons, the first 2 of which are untranslated, and 6 intervening introns. KLK5-SV2 is different from the classic form of the KLK5 mRNA in its 5′ untranslated region, where the first 5′ untranslated exon of the classic form is split into 2 exons with an intervening intron of 135 nucleotides. KLK5-SV2 is expressed in a variety of tissues, with higher expression levels in the mammary gland, cervix, salivary gland and trachea. The steroid hormone receptor-positive breast cancer cell line BT-474 was used to examine the effect of different steroids on the expression levels of KLK5-SV2. Expression levels were significantly higher after stimulation with androgens, but not estrogens, progestins, aldosterone or corticosteroids. While relatively high levels of expression were found in all 10 normal breast tissues examined, no expression was detected in 16 breast cancer tissues, and expression was significantly lower than normal in the remaining 4 cancers. Expression levels comparable to normal were found in only 1 breast cancer cell line. Weak to no expression was detected in 3 other breast cancer cell lines. KLK5-SV2 was not detectable in any of the 10 normal ovarian tissues examined. It was, however, expressed at relatively high levels in 10 out of 20 ovarian cancer tissues, and lower levels were found in 4 other cancers. No expression was detected in the remaining 6 cancers. High expression levels were also detected in the CAOV-3 ovarian cancer cell line. KLK5-SV2 is a potential biomarker for breast and ovarian cancers.

Molecular Cloning of a New Gene Which Is Differentially Expressed in Breast and Prostate Cancers

Objective: The chromosomal region 19q13 is non-randomly rearranged in many solid tumors. Methods: Using the positional candidate gene approach, we cloned a new gene, tentatively named cancer-associated gene (CAG), which is differentially expressed in breast and prostate cancers. Results: The gene is formed of 3 exons and 2 intervening introns. Its coding region is 1,047 bp in length and is predicted to encode a 348-amino-acid polypeptide. The new gene maps to chromosome 19q13.4 and is located 14 kb telomeric to the kallikrein gene locus (KLK14 gene) and 17 kb centromeric from the Siglec family of genes (Siglec-9). The gene is expressed in a wide variety of tissues including the brain, colon, kidney and pancreas. The CAG protein shows a high degree of conservation among species and phylogenetically is most closely related to its mouse ortholog. In silico analysis indicates that this gene is differentially expressed in a variety of tumors including brain, colon, ovarian and prostate cancers. Conclusions: Our preliminary experimental data show that CAG is upregulated in prostate cancer tissues compared to normal prostatic tissues. CAG also appears to be downregulated in breast cancer tissues. The physiological function of the CAG protein is currently unknown.

Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor‐A (VEGF) biologics, termed VEGF “Sticky‐traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky‐traps could locally inhibit angiogenesis to at least the same extent as the original VEGF‐trap that also gains whole‐body access. Sticky‐traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky‐trap remained localized to various regions of the eye, such as the inner‐limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky‐trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre‐maturity.

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno‐associated virus/piggyBac transposase gene delivery system

Liver‐targeted gene therapy based on recombinant adeno‐associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult‐focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver‐targeting properties of rAAV with stable piggyBac‐mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild‐type mice, with a 20‐fold increase in stably gene‐modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase–deficient Spfash mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver‐tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase‐mediated integration. (Hepatology 2015;62:417–428

700. Stable Correction of Severe Metabolic Liver Disease Phenotypes in the Growing Murine Liver Using a Hybrid rAAV/piggyBac Transposon Gene Delivery System

Liver-targeted gene therapy using adeno-associated virus-based vectors (rAAV) shows therapeutic promise in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver where rapid clearance of rAAV episomal genomes occurs in concert with hepatocellular proliferation. This phenomenon has been shown to underpin progressive loss of therapeutic efficacy, initially achieved in the neonatal period, in mouse models of early-onset urea cycle defects (UCDs). We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an EGFP expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks post-treatment compared to traditional rAAV gene delivery. We then modeled the therapeutic potential of the system in the context of severe early onset UCDs. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase (OTC) deficient Spfash mouse and neonatal lethal argininosuccinate synthetase (ASS) knock-out mouse. The system was next utilized in a murine model of chronic liver disease, progressive familial intrahepatic cholestasis type 3 (PFIC3), in which intervention prior to disease onset is necessary due to the development of chronic liver pathology which impedes vector delivery and hepatocyte transduction. Stable persistent expression extending into adulthood was achieved with increased mean biliary phosphatidylcholine concentration and dramatically reduced liver fibrosis. Finally, transposon integration patterns were analysed, revealing 127,286 unique integration sites which conformed to previously published piggyBac data. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system we achieved stable therapeutic protection in two UCD mouse models and a murine model with chronic liver pathology. This strategy has the potential for treating a wide array of inherited liver diseases with early onset. A clinically conceivable early application of this technology in the management of severe UCDs could be as a bridging therapy while awaiting liver transplantation. Further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.