Genetic Medicine for Hearing Loss: OTOF as Exemplar.

Millions of people worldwide have disabling hearing loss because one of their genes generates an incorrect version of some specific protein the ear requires for hearing. In many of these cases, delivering the correct version of the gene to a specific target cell within the inner ear has the potential to restore cochlear function to enable high-acuity physiologic hearing. Purpose: In this review, we outline our strategy for the development of genetic medicines with the potential to treat hearing loss. We will use the example of otoferlin gene (OTOF)-mediated hearing loss, a sensorineural hearing loss due to autosomal recessive mutations of the OTOF gene.

Characterization of gene-activated human acid-beta-glucosidase: crystal structure, glycan composition, and internalization into macrophages.

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-beta-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K(m) and V(max)) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Simultaneous but not prior inhibition of VEGF165 enhances the efficacy of photodynamic therapy in multiple models of ocular neovascularization.

PURPOSE: To investigate the effect of the combined treatment of photodynamic therapy and specific VEGF165 inhibition with pegaptanib sodium (Macugen; Eyetech Pharmaceuticals, Lexington, MA) on ocular neovascularization. METHODS: Photodynamic therapy's (PDT's) effects on the integrity of pegaptanib sodium were analyzed by HPLC, a VEGF165-binding assay, and a VEGF165-induced tissue factor gene expression assay. The effects of mono- or combined treatment on vessel growth and regression were determined in a murine corneal neovascularization model. The effects of combined treatment on vessel growth were also determined in a murine choroidal neovascularization model. RESULTS: PDT did not affect the chemical composition of pegaptanib sodium nor the efficacy of pegaptanib sodium in the inhibition of VEGF165 binding to Flt-1 and VEGF165-induced gene expression. In an animal model of effects on existing ocular neovascular lesions (corneal neovascularization), PDT monotherapy yielded an initial regression of these vessels, but there followed a rapid regrowth. In contrast, pegaptanib sodium monotherapy yielded little regression but potently abrogated further vessel growth. The combination of pegaptanib sodium and PDT resulted in the regression of the neovascular lesions, as observed with PDT alone, but also prevented significant vessel regrowth, leading to a significantly greater reduction in lesion size than did each monotherapy. In addition, there was a significantly greater effect of the combination of pegaptanib sodium and PDT on lesion size in choroidal neovascularization than with each monotherapy. Pretreatment with pegaptanib sodium appeared to decrease the efficacy of PDT-induced vessel regression in corneal neovascularization, and as such the enhanced efficacy over monotherapy when the agents were delivered simultaneously was not observed. CONCLUSIONS: Although the combined simultaneous treatment of ocular neovascularization with PDT and pegaptanib sodium may provide a more effective approach for the regression and overall treatment of CNV associated with AMD, the order of addition of these treatments may play a role in achieving optimal efficacy.

Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice.

PURPOSE: Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo. METHODS: The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes. RESULTS: In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice. CONCLUSIONS: EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.

Inhibitory effect of an antibody to cryptic collagen type IV epitopes on choroidal neovascularization.

PURPOSE: The wet form of age-related macular degeneration (AMD) occurs as a consequence of abnormal blood vessel growth from the choroid into the retina. Pathological angiogenesis during tumor growth and ocular disease has been associated with specific exposure of cryptic extracellular matrix epitopes. We investigated the presence of cryptic collagen IV epitopes in a murine model of choroidal neovascularization (CNV), and tested the effect on blood vessel growth of H8, a humanized antibody directed against a cryptic collagen type IV epitope. METHODS: To induce experimental CNV in adult C57BL/6 mice, Bruch's membrane was ruptured using a diode laser. Subsequently, mice were treated with daily intraperitoneal (i.p.) injections of either H8 (10 mg/kg or 30 mg/kg) or an isotype-matched antibody control. Two weeks postinjection, choroidal flat mounts were immunostained with the blood vessel marker platelet/endothelial cell adhesion molecule-1 (PECAM-1) and H8. CNV was visualized using fluorescence microscopy and the CNV lesion area measured using Open Lab software. RESULTS: Collagen type IV and the cryptic epitope were observed at the site of laser-induced lesions. Staining with H8 was first observed three days post injury, two days after MMP2 expression in CNV lesions, becoming most intense five days following laser injury and extending beyond the area of neovascularization. At 14 days post injury, H8 staining was reduced in intensity, colocalized with the area of CNV, and was nearly absent from the underlying choroidal vessels. In addition, mice treated with H8 had a significant dose-dependent decrease in the area of CNV as compared to isotype-matched antibody controls. CONCLUSIONS: Results suggest that exposure of cryptic collagen type IV epitopes is associated with the incidence of CNV and that the humanized antibody H8 may provide a new treatment for CNV.

An antisense oligodeoxynucleotide against vascular endothelial growth factor in a nonhuman primate model of iris neovascularization.

OBJECTIVE: To evaluate an antisense oligodeoxynucleotide (AS-ODN) targeted against vascular endothelial growth factor for its effects on ocular angiogenesis and its intraocular localization in a nonhuman primate model of iris neovascularization. METHODS: Bilateral laser retinal vein occlusion was performed in monkeys, followed by intravitreal injections of a vascular endothelial growth factor-specific AS-ODN or control. Serial fluorescein angiograms were graded in a masked manner to measure iris neovascularization. Localization was determined using a fluorescent-labeled AS-ODN and confocal microscopy on fixed tissue. RESULTS: Intravitreally injected vascular endothelial growth factor-specific AS-ODN localized to the retina, in the ganglion cell layer, inner nuclear layer, outer plexiform layer, photoreceptor outer segments, and retinal pigment epithelium. In 8 animals tested with 3microM ODN, AS-ODN-treated eyes had a significant reduction in iris neovascularization compared with control fellow eyes (P = .006, MIXOR analysis). Overall, in 17 animals tested across a range of ODN concentrations (0.1-50.0microM), AS-ODN-treated eyes were more likely to have lower iris neovascularization grades (P = .006, McNemar test) and the absence of iris neovascularization (P< .001, mixed-effects logistic regression model). CONCLUSION: Antisense ODNs that target vascular endothelial growth factor delivered to the retina via intravitreal injection reduced iris neovascularization in this model. Clinical Relevance Antisense ODNs against vascular endothelial growth factor may have therapeutic potential for neovascular eye diseases.

Melanoma-associated antigens in esophageal adenocarcinoma: identification of novel MAGE-A10 splice variants.

PURPOSE: The melanoma-associated antigens (MAGEs) are tumor-specific antigens recognized by cytotoxic T lymphocytes. In this study, expression of MAGE family A members was evaluated during the development of esophageal adenocarcinoma (EA) as potential targets for immunotherapy. EXPERIMENTAL DESIGN: MAGE-A mRNA expression was evaluated in 46 samples including Barrett's metaplasia (BM), dysplasia, and EA using oligonucleotide microarrays. Expression of MAGE-A proteins was confirmed by immunohistochemistry on tissue microarrays containing 59 EA, 11 dysplasia, and 9 BM samples and by Western blot. To further evaluate MAGE-A10 expression, reverse transcription-polymerase chain reaction (RT-PCR) products were sequenced, and protein expression was determined using a specific antibody. RESULTS: Overexpression of MAGE-A1, MAGE-A2b, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, and MAGE-A12 was found in EAs relative to BM on oligonucleotide microarrays. MAGE-A3 overexpression was confirmed by real-time RT-PCR in 21.4% (6 of 28) of esophageal tumors. Immunohistochemistry on tissue microarray revealed MAGE-A proteins in 20.3% (12 of 59) of EAs and MAGE-A10 staining in 16.9% (10 of 59) of EAs. MAGE-A expression was confirmed by Western blot in several esophageal tumors and in two EA cell lines, Flo-1 and Seg-1, whereas Flo-1 also expressed MAGE-A10. Tumors produced from these cell lines in nude mice retained MAGE-A expression. Interestingly, RT-PCR in primary tumors expressing MAGE-A10 protein revealed additional PCR products that were identified as novel MAGE-A10 alternative splice variants using DNA sequencing. CONCLUSIONS: This is the first report of these MAGE-A10 alternative splice sequences, and characterization of MAGE-A expression may provide potential targets for immunotherapy in patients with EA.

L-type amino acid transporter-1 overexpression and melphalan sensitivity in Barrett's adenocarcinoma.

The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P <.001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P <.05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P <.001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1.

In situ gene expression analysis of cancer using laser capture microdissection, microarrays and real time quantitative PCR.

The simultaneous development of laser capture microdissection (LCM) and high-throughput mRNA analysis platforms has provided a significant technological advance in the world of cancer biology. The combination of such technologies provides a unique and powerful opportunity to directly assess the in situ molecular genetic events that are associated with initiation and progression of human malignancies. Despite these technological advances, the integration of LCM with high-throughput gene expression analysis has been met with various challenges. The goal of this review is to highlight some of the obstacles that we have faced and continue to face as we try to optimally apply LCM to in situ gene expression analysis.

RGS5 expression is a quantitative measure of pericyte coverage of blood vessels.

Pericytes play a key role in the process of vascular maturation and stabilization however, the current methods for quantifying pericyte coverage of the neovasculature are laborious and subjective in nature. In this study, we have developed an objective, sensitive, and high-throughput method for quantifying pericyte coverage of angiogenic vessels by analyzing the expression of the pericyte-specific gene, the regulator of G-protein signaling 5 (RGS5). We determined that RGS5 expression was up-regulated during a defined developmental time period in which nascent vessel sprouts acquired a pericyte covering. Furthermore, RGS5 expression was dramatically reduced in vessels with poor pericyte coverage compared to normal angiogenic vasculature. Finally, we determined that the susceptibility of nascent vessels to regression by vascular endothelial growth factor (VEGF) inhibition was significantly reduced following RGS5 up-regulation, further implicating RGS5 in pericyte-endothelial cell interactions and the vascular maturation process. These studies establish the use of RGS5 gene expression as a quantitative and robust measure of pericyte coverage of neovasculature. This method provides a tool for vascular biologists studying pericyte-endothelial cell interactions and vascular maturation in both normal and pathological conditions, such as diabetic retinopathy and cancer.