Teresa Borrás

Altered secretion of a TIGR/MYOC mutant lacking the olfactomedin domain

TIGR/MYOC, a novel 504 amino acids (aa) protein of unknown function, has recently been linked to glaucoma. The protein is both intra- and extracellular and most known mutations map to its C-terminus, an olfactomedin-like domain. To investigate the properties of a TIGR/MYOC peptide lacking this important domain, we constructed a replication-deficient adenovirus with the first 344 aa and over-expressed the truncated protein in primary human trabecular meshwork cells and perfused human anterior segment cultures. The truncated mutant contains the entire N-terminus plus 98 aa of the olfactomedin-like domain. We found that the delivered truncated mutant accumulates inside the cell, reduces secretion of endogenous TIGR/MYOC and induces an increase in outflow facility at 48 h post-infection. Based on these findings, we hypothesize that TIGR/MYOC might have a dual role in trabecular meshwork function. This dual role might be that of an intracellular modulator of vesicular transport as well as that of a secreted protein involved in extracellular matrix conformation. Both functions could have a direct effect in maintaining aqueous humor outflow facility.

Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo.

Glaucoma is a group of chronic eye diseases often associated with an elevated intraocular pressure (IOP). If not controlled, the condition leads to blindness. The eye tissue responsible for maintaining aqueous humor resistance and thus normal IOP is the trabecular meshwork (TM). Adenoviral vectors are capable of transducing the TM in several rodent species. Because of the relevance of the non‐human primate model in the study of glaucoma, gene transfer to the eyes of cynomolgus monkeys was investigated.

Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma

Obstruction of the aqueous humor outflow from the anterior chamber of the eye leads to an elevation of intraocular pressure in glaucoma, the second major cause of blindness worldwide. Our goal is to be able to modulate aqueous humor outflow resistance by gene transfer to the cells of the trabecular meshwork (TM). We have previously shown that adenoviral vectors are able to transfer a reporter gene to the TM of post-mortem human donors. However, assessing gene therapy for glaucoma requires models that can monitor changes in aqueous humor outflow facility (C = flow/pressure). In this study we used four replication-deficient adenoviruses in two such perfusion models. In the first model, whole porcine eyes were infected, perfused at constant pressure and flow changes recorded for 5 h. In the second one, anterior segments from human eyes were infected, perfused at constant flow and pressure changes recorded for 3 days. A single dose of 108 adenovirus plaque forming units (pfu) causes a reduction in C while single doses of 107, 106 and 105 p.f.u. do not affect outflow facility and retain positive gene transfer. These findings indicate that adenovirus, at effective doses, could become useful vectors for gene therapy of glaucoma.

Expression of optineurin, a glaucoma-linked gene, is influenced by elevated intraocular pressure

Optineurin (optic neuropathy inducing protein; OPTN) was recently linked to 16.7% of families with primary open-angle glaucoma. The function of OPTN in the eye is not known, but is present in the trabecular meshwork, which is responsible for maintenance of intraocular pressure (IOP). To gain insight into the role of OPTN in the development of glaucoma we studied its expression in response to factors known to be associated with the disease: elevated IOP, tumor necrosis factor-alpha (TNFalpha), and dexamethasone (DEX). We performed the treatments in human organ cultures under conditions mimicking physiological pressure. We find OPTN significantly upregulated after 2, 4, and 7 days of sustained elevated IOP. OPTN expression is also induced 2.3-fold by TNFalpha and 2.6-fold by prolonged DEX treatment. These results demonstrate that OPTN is part of the transcriptome responding to glaucomatous insults and support the protective role of this protein in the trabecular meshwork.

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene☆

Abstract A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.

Evidence for positive and negative regulation in the promoter of the chicken δ1-crystallin gene

We investigated the role of sequences flanking the transcription initiation site of the δ1-crystallin gene in transient transfection assays of primary embryonic chicken lens epithelial cells or fibroblasts. Varying lengths of the 5′ flanking sequence of the δ1-crystallin gene (containing some untranslated sequence from exon 1) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene in the pSVOCAT plasmid. A plasmid carrying the bacterial β-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter was used as an internal control. Standardized results showed that the sequence located between −120 to −43 exhibited strong promoter activity; however, the promoter activity was markedly reduced (20-fold) when the upstream sequence between −603 and −120 was included in the construct. The δ1-crystallin promoter displayed little lens preference. This upstream sequence did not reduce the activity of the Simian virus 40 (SV40) early promoter (with or without its enhancer) or the Herpes thymidine kinase promoter in transfection tests, indicating some specificity in its effect. Evidence for a δ1-crystallin negative trans-acting factor was provided by competition experiments. Our data raise the possibility that expression of the δ1-crystallin gene involves a negative cis-acting transcription element, a speculation which may deserve further attention in view of the gradual decrease in δ-crystallin synthesis in the developing lens.

Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

The trabecular meshwork tissue controls the drainage of the aqueous humor of the eye. A dysfunctional trabecular meshwork leads to an altered fluid resistance, which results in increased intraocular pressure (IOP). IOP is the major risk factor of glaucoma, the second‐leading cause of blindness in the developed world. In the search for genes altered by glaucomatous insults, we identified angiopoietin‐like7 (ANGPTL7), a member of the ANGPTL family. Although structurally related to the angiopoietins, ANGPTL7′s function is poorly understood. Because ANGPTL7 is secreted and because extracellular matrix (ECM) deposition and organization is critical for aqueous humor resistance, we investigated the effect of ANGPTL7 on relevant trabecular meshwork ECM genes and proteins. We find that overexpression of ANGPTL7 in primary human trabecular meshwork cells altered the expression of fibronectin, collagens type I, IV & V, myocilin, versican, and MMP1. ANGPTL7 also interfered with the fibrillar assembly of fibronectin. Finally, we find that silencing ANGPTL7 during the glucocorticoid insult significantly affected the expression of other steroid‐responsive proteins. These results indicate that ANGPTL7 modulates the trabecular meshwork’s ECM as well as the response of this tissue to steroids. Together with previous findings, these properties strengthen ANGPTL7′s candidacy for the regulation of IOP and glaucoma.

Individual molecular response to elevated intraocular pressure in perfused postmortem human eyes

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma. In the clinic, the response to elevated pressure and thus the risk for development of glaucoma differs among individuals. We took advantage of our ability to subject postmortem human eyes from the same individual to physiological and elevated pressure in a perfused outflow model and compared individual patterns of gene expression under pressure. The architecture of the trabecular meshwork, tissue responsible for the maintenance of IOP, was conserved. We performed two sets of experiments. The first set (n = 5, 10 eyes) used Affymetrix GeneChips, identified the 20 most pressure-altered genes in each individual, and compared their pressure response in the other four. The second set (n = 5, 10 eyes) selected 21 relevant trabecular meshwork genes and examined, by real-time TaqMan-PCR, the rank of their abundance and of their pressure differential expression in each individual. The majority of the up- and downregulated top-changers of each individual showed an individual response trend. Few genes were general responders. Individual responders included STATH, FBN2, TF, OGN, IL6, IGF1, CRYAB, and ELAM1 (marker for glaucoma). General responders included MMP1, MMP10, CXCL2, and PDPN. In addition, we found that although the relative abundance of selected genes was very similar among nonstressed individuals, the response to pressure of those same genes had a marked individual component. Our results offer the first molecular insight on the variation of the individual response to IOP observed in the clinical setting.

The transcripts of zeta-crystallin, a lens protein related to the alcohol dehydrogenase family, are altered in a guinea-pig hereditary cataract

Zeta-Crystallin, a major component of the guinea-pig lens proteins, is distantly related to the enzymes of the zinc-containing alcohol dehydrogenase family (ADH). Analysis of the structural similarities between zeta-crystallin and ADH reveals that while characteristics important in maintaining the tertiary structure of the molecule appear conserved, the amino acids binding the catalytic zinc atom are absent in zeta-crystallin. Significantly, zeta-crystallin does not have ADH activity. Previous studies showed that the zeta-crystallin protein is modified in the lens of guinea-pigs affected with an autosomal dominant hereditary cataract. We have further investigated the molecular origin of the lens defect by examining the steady-state levels of zeta-crystallin transcripts in normal and mutant eyes. Our data indicate that no normal zeta-crystallin mRNA is present in the lens of the homozygous animals; instead, a cross-hybridizing lower molecular weight mRNA is detected at significantly reduced concentrations. Heterozygous lenses exhibit both mRNA species.