Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy.

Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide who depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential reinducible vector DNAs in the trabecular meshwork (0.4 v.g. per cell) and negligible copies in six major internal organs (0.00002-0.005 v.g. per cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid-induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted self-complementary adeno-associated vectors (scAAV) vectors in a large animal model.

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

BACKGROUND: 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. METHODS: Four cynomolgus monkeys were injected with AdenoGFP into the anterior chamber: two monkeys received 10(9) pfu and the other two 10(7) pfu. One monkey received four consecutive injections into the same eye (10(7) pfu in each injection) over a 7-month period. In vivo gene transfer (fluorescence) and IOP were evaluated by standard clinical ophthalmic instruments (slit lamp biomicroscopy, gonioscopy and tonometry). Histopathology and cellular distribution were assessed postmortem. RESULTS: The first injection of the lower viral dose resulted in marked TM-preferred gene transfer visible non-invasively by in vivo gonioscopy. The expression of the transgene lasted for 3-4 weeks with little or no signs of clinical inflammation. Gene transfer was achieved on three sequential occasions (3-4 weeks each) but failed and induced substantial, albeit reversible, corneal abnormalities on the fourth occasion. CONCLUSIONS: Gene transfer to the TM and cornea can be monitored non-invasively in non-human primates, allowing correlation of gene transfer with physiological parameters. Because of ocular immune privilege, repeated anterior chamber administrations of adenoviral vectors expressing appropriate genes may have therapeutic potential for glaucoma.

Inefficient processing of an olfactomedin-deficient myocilin mutant: potential physiological relevance to glaucoma.

Mutations in TIGR/MYOC (myocilin), a secretory protein of unknown function, have been recently linked to glaucoma. Most known mutations map to the C-terminus, an olfactomedin-like domain. We have previously shown that, in contrast to the wild-type, a truncated form of myocilin lacking the olfactomedin domain is not secreted. In this study, we present evidence that the mutant protein is not correctly processed in the endoplasmic reticulum (ER) and accumulates into insoluble aggregates. In addition, we show that the presence of increasing amounts of mutant protein induces a fraction of the soluble, native myocilin to move to the insoluble fraction. Given the importance of such protein aggregates in the etiology of several aging-related diseases, we propose that olfactomedin-defective mutants might contribute to the pathology of glaucoma through a mechanism involving intracellular accumulation of misfolded proteins.

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.

Characterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones.

PURPOSE: To study the gene expression profile of the human trabecular meshwork (HTM). METHODS: A polymerase chain reaction (PCR)-amplified cDNA library was constructed using RNA from the TM of a 67-year-old normal, perfused human eye. A total of 1060 clones were randomly selected for sequencing of one end. These sequences were searched against nonredundant GenBank and dbEST databases for similarity comparison by using a FASTA file and the BLASTcl3 program. Relative expression patterns of those clones that matched other expressed sequence tags (ESTs) were determined using the National Center for Biotechnology Information (NCBI) Unique Human Gene Sequence Collection (UniGene) database. RESULTS: Of the 1060 clones analyzed, 519 (48.9%) had sequences identical with known genes, 125 (11.8%) matched ESTs, and 189 (17.8%) did not match any database sequences. Of the remaining clones, 31 (3%) corresponded to mitochondrial transcripts and 196 (18.5%) to repetitive and noninformative sequences. It is notable that some of the genes highly represented in this library are not ubiquitously expressed in other tissues, which suggests a potentially important role in the HTM. As evidence for the presence of true novel genes in the library, one of the clones was fully sequenced. This clone comprised a complete open reading frame of 966 nucleotides, and its deduced amino acid sequence corresponded to a protein 33% similar to the MAS-related G-protein-coupled receptor. CONCLUSIONS: The identification of the more highly expressed genes in HTM and the discovery of novel genes expressed in this tissue provides basic information for further research on the physiology of the TM and for the identification of glaucoma candidate genes.

Genes upregulated in the human trabecular meshwork in response to elevated intraocular pressure.

PURPOSE: To identify genes upregulated in perfused, intact human trabecular meshwork (TM) in response to elevated intraocular pressure (IOP). METHOD: Two pairs of anterior segments of normal human eyes from postmortem donors were placed in culture and perfused 24 hours at constant flow (3 microl/min). After reaching baseline, the flow of one eye from each pair was raised to obtain an incremental pressure (deltaP) of 50 mm Hg for 6 hours. The anterior segments were then quickly frozen in liquid nitrogen, and their TMs were dissected for RNA extraction. SMART cDNA libraries were generated from control and high-pressure human TM RNAs and hybridized to sets of identical high-density cDNA gene arrays. These arrays contained 18,376 human expressed sequence tags (ESTs), corresponding to both characterized and unknown genes. Differentially expressed genes were identified by different-intensity hybridization signals and confirmed by semi-quantitative polymerase chain reaction. RESULTS: Eleven genes were found to be consistently upregulated in the human TM by elevated IOP: interleukin-6, preprotachykinin-1, secretogranin-II, cathepsin-L, stromelysin-1, thymosin-beta4, alpha-tubulin, alphaB-crystallin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein and Cu/Zn superoxide dismutase. The products of these genes are involved in vascular permeability, secretion, extracellular matrix remodeling, cytoskeleton reorganization, and reactive oxygen species scavenging. CONCLUSIONS: Elevated IOP induced specific upregulation of 11 physiologically relevant genes. On the basis of their known activities, the products of each of these genes might predict homeostatic mechanisms similar to those involved in the regulation of blood vessel permeability. We hypothesize that similar mechanisms might be involved in regulating flow through Schlemm's Canal endothelium.

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 postmortem 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 10(8) adenovirus plaque forming units (pfu) causes a reduction in C while single doses of 10(7), 10(6) and 10(5) 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.

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Gene transfer to the human trabecular meshwork by anterior segment perfusion.

PURPOSE: To determine whether adenovirus vectors are capable of transferring a foreign active protein to the perfused anterior segment of the human eye. METHODS: Primary cultures from the human trabecular meshwork tissue were exposed to replication-deficient adenovirus Av1LacZ4 carrying the reporter beta-galactosidase gene driven by the Rous Sarcoma Virus promoter. Anterior segments of six pairs of human eyes from normal donors were placed in organ culture and were perfused with culture medium at 2.5 microl/min constant flow. After 24 hours, one eye was injected once with 8 X 10(8) plaque-forming units (20 microl) of the viral vector, while the paired eye was injected with vehicle. Forty-eight hours (four pairs) and 7 days (two pairs) after injection, tissues were fixed, were assayed histochemically for transferred enzyme activity, and were analyzed morphologically. RESULTS: In monolayers, gene transfer occurs very efficiently in all distinct types of human outflow pathway cells. All human anterior segments injected with the adenovirus vector showed active gene transfer in cells of the outflow pathway: trabecular, juxtacanalicular, and inner wall of Schlemm's canal. Expression of the reporter enzyme was still present at 7 days after treatment. No activity was observed in any of the paired, vehicle-injected controls. Cell morphology and tissue architecture appeared normal in treated and control tissues, although some trabecular cell loss was observed in the corneoscleral and uveal regions of the perfused treated eyes. CONCLUSIONS: Adenoviral vectors were able to transfer active foreign genes into perfused, intact human trabecular meshwork.

Ocular adenovirus gene transfer varies in efficiency and inflammatory response.

PURPOSE: To study the effects of adenoviral gene transfer to the tissues of the anterior segment in vitro by rat and monkey lens organ cultures and in vivo by single injection into the anterior chamber of rabbits. METHODS: In vitro, intact lens cultures were exposed to 1 to 4 x 10(8) pfu Av1LacZ4 and Av1Luc1 in TC199 medium containing no serum or growth factors. Av1LacZ4 and Av1Luc1 are replication-deficient adenovirus vectors, carrying the reporter genes Escherichia coli LacZ and firefly luciferase, respectively. In vivo, the anterior chambers of eight rabbits were injected once with 20 mumol Av1LacZ4 (8 x 10(8) pfu) and evaluated 48 hours after injection. Enzyme activity of the reporter genes was measured biochemically and histochemically. RESULTS: In organ cultures, adenovirus delivers reporter genes efficiently to the ciliary processes but penetrates poorly into the capsulated lenses. Viral receptors, however, are present in rat lens epithelium, as in primary trabecular meshwork and other lens cell lines. In vivo, gene transfer was evident in corneal endothelium, iris anterior surface, and trabecular meshwork. Presence of the virus did not affect lens transparency or provoke external discomfort signs. Infected corneal endothelial cells were swollen and partly detached; 3 of 8 infected eyes showed a severe inflammatory response in chamber angle, anterior uvea, and limbal conjunctiva. CONCLUSIONS: These findings reveal the distinct gene transfer potential of each of the tissues of the anterior segment and emphasize the need to address the inflammatory response to these first-generation adenoviral vectors.

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse.

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens-specific promoter.

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

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-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. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-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 zeta-crystallin gene disclosed a dinucleotide deletion 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 zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.

DNA binding factors which interact with the Sp1 site of the chicken delta 1-crystallin promoter are developmentally regulated.

Transcription of the delta 1-crystallin gene is developmentally regulated in the embryonic chicken lens. Previous work defined a positive transcription regulatory element between positions -120 and -43 of the delta 1-crystallin promoter. This region contains a putative Sp1 binding site (-78 to -71), adjacent to a CAAT box (-70 to -67). Gel retardation assays using lens nuclear extracts revealed two protein-DNA complexes which involved the Sp1 site. The formation of the complexes increased from day 6 to day 11 of embryogenesis (period of lens organogenesis) peaked between days 11 and 15, then decreased in a non-parallel manner until hatching (day 21). A point mutation in the Sp1 binding site of the delta 1-crystallin promoter abolished formation of one of the complexes (complex 1, slower in mobility), while point mutations in the CAAT box had no effect on the formation of either complex. Studies using purified Sp1 protein and increasing amounts of embryonic chicken lens nuclear extracts showed cooperativity in the formation of both complexes, more remarkable with complex 1.

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.

Transcriptional stimulation of the delta 1-crystallin gene by insulin-like growth factor I and insulin requires DNA cis elements in chicken.

Insulin-like growth factor I (IGF-I) and insulin regulate expression of the endogenous delta 1-crystallin gene in embryonic lens cells that express receptors for both peptides. To further analyze the transcriptional component of this hormonal effect, transient transfections of lens cells were prepared with DNA constructs containing deletions of the delta 1-crystallin promoter and the chloramphenicol acetyltransferase reporter gene. A 77-nucleotide DNA segment of the delta 1-crystallin promoter from nucleotide positions-120 to -43 confers sensitivity to insulin and IGF-I. The hormonal effect is dose-dependent, and maximal stimulation of promoter activity (2- to 2.5-fold induction) is obtained with 10(-8) M IGF-I and 10(-7) M insulin. Mobility-shift DNA-binding analysis shows specific binding of nuclear protein(s) to the delta 1-crystallin promoter DNA between positions -120 and +23, which appears to be regulated by IGF-I. An SP1-binding motif is involved in this DNA-protein interaction. The bivalent IgG fraction of an anti-insulin receptor antiserum (B-10), known to mimic insulin action in other systems, stimulates promoter activity to the same extent as insulin.

Eye lens zeta-crystallin relationships to the family of "long-chain" alcohol/polyol dehydrogenases. Protein trimming and conservation of stable parts.

zeta-Crystallin of guinea pig lens is distantly related to the family of zinc-containing alcohol/polyol dehydrogenases. The amino acid residues binding the catalytic zinc atom in the alcohol dehydrogenase are exchanged in zeta-crystallin, explaining lack of known enzyme activity, and those residues binding the noncatalytic zinc in the dehydrogenase are located in a segment absent from the crystallin. Mammalian alcohol dehydrogenase, polyol dehydrogenase, and zeta-crystallin therefore constitute a series of proteins exhibiting successive changes in subunit metal content, from two to one and probably zero zinc atoms, respectively. In common with tetrameric dehydrogenases, the crystallin lacks a loop structure present in the dimeric dehydrogenase. Significantly, the crystallin is tetrameric, and a correlation between extra subunit interactions and lack of the loop segment is indicated. The lacking segment in crystallin is extended, encompassing a second loop in the dehydrogenase. The greatest conservation corresponds to the coenzyme-binding domain of the dehydrogenases, the central parts of which are remarkably similar to those in the crystallin. Glycine is by far the most conserved residue and corresponds to positions at bends in the conformation of the alcohol dehydrogenase. The conservation of the stable parts of the fold, the absence of the loop structure, the lack of the metal atoms, and the presence of only a small proportion of oxidation-sensitive cysteine residues in crystallin (5 versus 15 in the beta 1 dehydrogenase subunit) suggest an increased stability of the lens protein and a derivation from the alcohol dehydrogenase family. This is compatible with the recruitment of stable enzyme structures for lens crystallin functions, with trimming of protein structures through these dehydrogenases or a yet unknown enzyme, and with multiple changes in the dehydrogenase family.

Developmental regulation of hypomethylation of delta-crystallin genes in chicken embryo lens cells.

Sequences in the two delta-crystallin genes become hypomethylated when they are expressed in the chick lens. This system is particularly advantageous for studying temporal changes in hypomethylation, since lens tissue can be isolated at all developmental stages. In previous work we have shown that most HpaII sites become hypomethylated within the delta 1-crystallin gene long after delta-crystallin gene activation. One site is hypomethylated when crystallin mRNA begins to be synthesized at high levels at 50 h; we show here that this site maps to the 3' end (intron 15) of the delta 1-crystallin gene. In addition, we have examined the methylation status of HpaII and HhaI sites found near the 5' end of the delta 1-crystallin gene. Two HhaI sites adjacent to a viral core enhancer sequence in intron 2 are also first hypomethylated at 50 h. These findings point to regions of the delta 1 gene that should be investigated further for functional significance in regulating delta-crystallin transcription.

Zeta-crystallin, a novel protein from the guinea pig lens is related to alcohol dehydrogenases.

zeta-Crystallin is a major component of the water-soluble proteins of the guinea pig lens. We have constructed a lens cDNA library from one- to seven-day-old guinea pigs in the plasmid Bluescript KS+ and used the 16 amino acid (aa) sequence of a CNBr peptide to design an oligodeoxyribonucleotide probe. Analysis of two positive clones and direct sequence of the 5' end of the RNA resulted in the completion of a most probably full-length mRNA comprising 1842 nucleotides (nt). The ATG start codon occurs 83 nt downstream from the 5' end. The open reading frame, ending with a stop codon at nt position 1070, predicts a protein of 328 aa with a calculated Mr of 35,071. Comparison of the amino acid sequence with the National Biomedical Research Foundation protein data base reveals a significant similarity of zeta-crystallin with the enzyme of the alcohol dehydrogenase family.

Primary structure of zeta-crystallin protein from guinea pig. Its similarity to the enzyme alcohol dehydrogenase.

The primary structure of zeta-crystallin, a guinea pig lens specific protein, was obtained by cloning the copy of its mRNA from a 0-1 week old cDNA lens library. The protein is 328 amino acids long and 34% of its secondary structure appears in alpha-helical conformation. Comparison of the zeta-crystallin sequence with the sequences of the protein data base bank, revealed the similarity of this lens protein to the enzymes of the long-chain zinc-containing alcohol dehydrogenase family.