rAAV-compatible human mini promoters enhance transgene expression in rat retinal ganglion cells.

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

BACKGROUND/AIMS: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. METHODS: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. RESULTS: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. CONCLUSION: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

The Role of Axonal Transport in Glaucoma.

Glaucoma is a neurodegenerative disease that affects the retinal ganglion cells (RGCs) and leads to progressive vision loss. The first pathological signs can be seen at the optic nerve head (ONH), the structure where RGC axons leave the retina to compose the optic nerve. Besides damage of the axonal cytoskeleton, axonal transport deficits at the ONH have been described as an important feature of glaucoma. Axonal transport is essential for proper neuronal function, including transport of organelles, synaptic components, vesicles, and neurotrophic factors. Impairment of axonal transport has been related to several neurodegenerative conditions. Studies on axonal transport in glaucoma include analysis in different animal models and in humans, and indicate that its failure happens mainly in the ONH and early in disease progression, preceding axonal and somal degeneration. Thus, a better understanding of the role of axonal transport in glaucoma is not only pivotal to decipher disease mechanisms but could also enable early therapies that might prevent irreversible neuronal damage at an early time point. In this review we present the current evidence of axonal transport impairment in glaucomatous neurodegeneration and summarize the methods employed to evaluate transport in this disease.

Effects of a combinatorial treatment with gene and cell therapy on retinal ganglion cell survival and axonal outgrowth after optic nerve injury.

After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.

Retina transduction by rAAV2 after intravitreal injection: comparison between mouse and rat.

Adeno-associated virus vectors (rAAV) are currently the most common vehicle used in clinical trials of retinal gene therapy, usually delivered through subretinal injections to target cells of the outer retina. However, targeting the inner retina requires intravitreal injections, a simple and safe procedure, which is effective for transducing the rodent retina, but still of low efficiency in the eyes of primates. We investigated whether adjuvant pharmacological agents may enhance rAAV transduction of the retinas of mouse and rat after intravitreal delivery. Tyrosine kinase inhibitors were highly efficient in mice, especially imatinib and genistein, and promoted transduction even of the outer retina. In rats, however, we report that they were not effective. Even with direct proteasomal inhibition in rats, the effects upon transduction were only minimal and restricted to the inner retina. Even tyrosine capsid mutant rAAVs in rats had a transduction profile similar to wtAAV. Thus, the differences between mouse and rat, in both eye size and the inner limiting membrane, compromise the efficiency of AAV vectors penetration from the vitreous into the retina, and impact the efficacy of strategies developed to enhance intravitreal retinal rAAV transduction. Further improvement of strategies, then are required.

Self-complementary and tyrosine-mutant rAAV vectors enhance transduction in cystic fibrosis bronchial epithelial cells.

Recombinant adeno-associated virus (rAAV) vector platforms have shown considerable therapeutic success in gene therapy for inherited disorders. In cystic fibrosis (CF), administration of first-generation rAAV2 was safe, but clinical benefits were not clearly demonstrated. Therefore, next-generation vectors that overcome rate-limiting steps in rAAV transduction are needed to obtain successful gene therapy for this devastating disease. In this study, we evaluated the effects of single-strand or self-complementary (sc) rAAV vectors containing single or multiple tyrosine-to-phenylalanine (Y-F) mutations in capsid surface-exposed residues on serotypes 2, 8 or 9. For this purpose, CF bronchial epithelial (CFBE) cells were transduced with rAAV vectors, and the transgene expression of enhanced green fluorescence protein (eGFP) was analyzed at different time points. The effects of vectors on the cell viability, host cell cycle and in association with co-adjuvant drugs that modulate intracellular vector trafficking were also investigated. Six rAAV vectors demonstrated greater percentage of eGFP+ cells compared to their counterparts at days 4, 7 and 10 post-transduction: rAAV2 Y(272,444,500,730)F, with 1.95-, 3.5- and 3.06-fold increases; rAAV2 Y(252,272,444,500,704,730)F, with 1.65-, 2.12-, and 2-fold increases; scrAAV2 WT, with 1.69-, 2.68-, and 2.32-fold increases; scrAAV8 Y773F, with 57-, 6.06-, and 7-fold increases; scrAAV9 WT, with 7.47-, 4.64-, and 3.66-fold increases; and scrAAV9 Y446F, with 8.39-, 4.62-, and 4.4-fold increases. At days 15, 20, and 30 post-transduction, these vectors still demonstrated higher transgene expression than transfected cells. Although the percentage of eGFP+ cells reduced during the time-course analysis, the delta mean fluorescence intensity increased. These vectors also led to increased percentage of cells in G1-phase without eliciting any cytotoxicity. Prior administration of bortezomib or genistein did not increase eGFP expression in cells transduced with either rAAV2 Y(272,444,500,730)F or rAAV2 Y(252,272,444,500,704,730)F. In conclusion, self-complementary and tyrosine capsid mutations on rAAV serotypes 2, 8, and 9 led to more efficient transduction than their counterparts in CFBE cells by overcoming the intracellular trafficking and second-strand DNA synthesis limitations.

rAAV8-733-Mediated Gene Transfer of CHIP/Stub-1 Prevents Hippocampal Neuronal Death in Experimental Brain Ischemia.

Brain ischemia is a major cause of adult disability and death, and it represents a worldwide health problem with significant economic burden for modern society. The identification of the molecular pathways activated after brain ischemia, together with efficient technologies of gene delivery to the CNS, may lead to novel treatments based on gene therapy. Recombinant adeno-associated virus (rAAV) is an effective platform for gene transfer to the CNS. Here, we used a serotype 8 rAAV bearing the Y733F mutation (rAAV8-733) to overexpress co-chaperone E3 ligase CHIP (also known as Stub-1) in rat hippocampal neurons, both in an oxygen and glucose deprivation model in vitro and in a four-vessel occlusion model of ischemia in vivo. We show that CHIP overexpression prevented neuronal degeneration in both cases and led to a decrease of both eIF2α (serine 51) and AKT (serine 473) phosphorylation, as well as reduced amounts of ubiquitinated proteins following hypoxia or ischemia. These data add to current knowledge of ischemia-related signaling in the brain and suggest that gene therapy based on the role of CHIP in proteostasis may provide a new venue for brain ischemia treatment.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

BACKGROUND/AIMS: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. METHODS: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. RESULTS: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. CONCLUSION: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

Single tyrosine mutation in AAV8 vector capsid enhances gene lung delivery and does not alter lung morphofunction in mice.

BACKGROUND/AIMS: Vectors derived from adeno-associated viruses (AAVs) are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. METHODS: Male C57BL/6 mice (20-25 g, n=24) were randomly assigned into three groups: control group animals received intratracheal (i.t.) instillation of saline (50 µl), wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t.) AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP), and inflammatory cytokines and growth factor expression were analyzed. RESULTS: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. CONCLUSION: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

Shared Molecular Pathways in Glaucoma and Other Neurodegenerative Diseases: Insights from RNA-Seq Analysis and miRNA Regulation for Promising Therapeutic Avenues.

Advances in RNA-sequencing technologies have led to the identification of molecular biomarkers for several diseases, including neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's diseases and Amyotrophic Lateral Sclerosis. Despite the nature of glaucoma as a neurodegenerative disorder with several similarities with the other above-mentioned diseases, transcriptional data about this disease are still scarce. microRNAs are small molecules (~17-25 nucleotides) that have been found to be specifically expressed in the CNS as major components of the system regulating the development signatures of neurodegenerative diseases and the homeostasis of the brain. In this review, we sought to identify similarities between the functional mechanisms and the activated pathways of the most common neurodegenerative diseases, as well as to discuss how those mechanisms are regulated by miRNAs, using RNA-Seq as an approach to compare them. We also discuss therapeutically suitable applications for these disease hallmarks in clinical future studies.

Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.

Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration.

Purpose: Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods: Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results: In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions: Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

Gene Therapy Strategies for Glaucomatous Neurodegeneration.

Glaucoma leads to irreversible vision loss and current therapeutic strategies are often insufficient to prevent the progression of the disease and consequent blindness. Elevated intraocular pressure is an important risk factor, but not required for the progression of glaucomatous neurodegeneration. The demise of retinal ganglion cells represents the final common pathway of glaucomatous vision loss. Still, lifelong control of intraocular pressure is the only current treatment to prevent severe vision loss, although it frequently fails despite best practices. This scenario calls for the development of neuroprotective and pro-regenerative therapies targeting the retinal ganglion cells as well as the optic nerve. Several experimental studies have shown the potential of gene modulation as a tool for neuroprotection and regeneration. In this context, gene therapy represents an attractive approach as a persistent treatment for glaucoma. Viral vectors engineered to promote overexpression of a broad range of cellular factors have been shown to protect retinal ganglion cells and/or promote axonal regeneration in experimental models. Here, we review the mechanisms involved in glaucomatous neurodegeneration and regeneration in the central nervous system. Then, we point out the current limitations of gene therapy platforms and review a myriad of studies that use viral vectors to manipulate genes in retinal ganglion cells, as a strategy to promote neuroprotection and regeneration. Finally, we address the potential of combining neuroprotective and regenerative gene therapies as an approach to glaucomatous neurodegeneration.

Mitotherapy: Unraveling a Promising Treatment for Disorders of the Central Nervous System and Other Systemic Conditions.

Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer's disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach.

Retinal Genomic Fabric Remodeling after Optic Nerve Injury.

Glaucoma is a multifactorial neurodegenerative disease, characterized by degeneration of the retinal ganglion cells (RGCs). There has been little progress in developing efficient strategies for neuroprotection in glaucoma. We profiled the retina transcriptome of Lister Hooded rats at 2 weeks after optic nerve crush (ONC) and analyzed the data from the genomic fabric paradigm (GFP) to bring additional insights into the molecular mechanisms of the retinal remodeling after induction of RGC degeneration. GFP considers three independent characteristics for the expression of each gene: level, variability, and correlation with each other gene. Thus, the 17,657 quantified genes in our study generated a total of 155,911,310 values to analyze. This represents 8830x more data per condition than a traditional transcriptomic analysis. ONC led to a 57% reduction in RGC numbers as detected by retrograde labeling with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI). We observed a higher relative expression variability after ONC. Gene expression stability was used as a measure of transcription control and disclosed a robust reduction in the number of very stably expressed genes. Predicted protein-protein interaction (PPI) analysis with STRING revealed axon and neuron projection as mostly decreased processes, consistent with RGC degeneration. Conversely, immune response PPIs were found among upregulated genes. Enrichment analysis showed that complement cascade and Notch signaling pathway, as well as oxidative stress and kit receptor pathway were affected after ONC. To expand our studies of altered molecular pathways, we examined the pairwise coordination of gene expressions within each pathway and within the entire transcriptome using Pearson correlations. ONC increased the number of synergistically coordinated pairs of genes and the number of similar profiles mainly in complement cascade and Notch signaling pathway. This deep bioinformatic study provided novel insights beyond the regulation of individual gene expression and disclosed changes in the control of expression of complement cascade and Notch signaling functional pathways that may be relevant for both RGC degeneration and remodeling of the retinal tissue after ONC.

High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors.

Vectors derived from adeno-associated viruses (AAVs) have become important gene delivery tools for the treatment of many inherited ocular diseases in well-characterized animal models. Previous studies have determined that the viral capsid plays an essential role in the cellular tropism and efficiency of transgene expression. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome- mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo. Because the tyrosine residues are highly conserved in other AAV serotypes, in this study we evaluated the intraocular transduction characteristics of vectors containing point mutations in surface- exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9. Several of these novel AAV mutants were found to display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts. For the first time, we show efficient transduction of the ganglion cell layer by AAV serotype 8 or 9 mutant vectors, thus providing additional tools besides AAV2 for targeting these cells. These enhanced AAV vectors have a great potential for future therapeutic applications for retinal degenerations and ocular neovascular diseases.

Neuroprotection from optic nerve injury and modulation of oxidative metabolism by transplantation of active mitochondria to the retina.

Mitochondrial dysfunctions are linked to a series of neurodegenerative human conditions, including Parkinson's disease, schizophrenia, optic neuropathies, and glaucoma. Recently, a series of studies have pointed mitotherapy - exogenous mitochondria transplant - as a promising way to attenuate the progression of neurologic disorders; however, the neuroprotective and pro-regenerative potentials of isolated mitochondria in vivo have not yet been elucidated. In this present work, we tested the effects of transplants of active (as well-coupled organelles were named), liver-isolated mitochondria on the survival of retinal ganglion cells and axonal outgrowth after optic nerve crush. Our data show that intravitreally transplanted, full active mitochondria incorporate into the retina, improve its oxidative metabolism and electrophysiological activity at 1 day after transplantation. Moreover, mitotherapy increases cell survival in the ganglion cell layer at 14 days, and leads to a higher number of axons extending beyond the injury site at 28 days; effects that are dependent on the organelles' structural integrity. Together, our findings support mitotherapy as a promising approach for future therapeutic interventions upon central nervous system damage.

A subacute model of glaucoma based on limbal plexus cautery in pigmented rats.

Glaucoma is a neurodegenerative disorder characterized by the progressive functional impairment and degeneration of the retinal ganglion cells (RGCs) and their axons, and is the leading cause of irreversible blindness worldwide. Current management of glaucoma is based on reduction of high intraocular pressure (IOP), one of its most consistent risk factors, but the disease proceeds in almost half of the patients despite such treatments. Several experimental models of glaucoma have been developed in rodents, most of which present shortcomings such as high surgical invasiveness, slow learning curves, damage to the transparency of the optic media which prevents adequate functional assessment, and variable results. Here we describe a novel and simple method to induce ocular hypertension in pigmented rats, based on low-temperature cauterization of the whole circumference of the limbal vascular plexus, a major component of aqueous humor drainage and easily accessible for surgical procedures. This simple, low-cost and efficient method produced a reproducible subacute ocular hypertension with full clinical recovery, followed by a steady loss of retinal ganglion cells and optic axons, accompanied by functional changes detected both by electrophysiological and behavioral methods.

Does the use of recombinant AAV2 in pulmonary gene therapy damage lung function?

Forty-eight BALB/c mice were divided into two groups of 24 animals each. In the control group (CTRL) saline was intratracheally instilled, while the virus group (VR) received rAAV2-GFP (4 x 10(9) particles). These groups were subdivided into four sub-groups (n=6). Pulmonary mechanical parameters were analyzed after 3 weeks (VR1d3w) and at 1 (VR2d1w), 2 (VR2d2w) and 3 weeks (VR2d3w) after a second AAV2 dose. Fractions of the area of alveolar collapse and the amount of polymorpho- and mononuclear cells were determined by point-counting technique. Viral transduction was evaluated by immunohistochemistry. Lung mechanical data were similar in all groups. However, there was an increase in airway and lung parenchyma cellularity and in the fraction of area of alveolar collapse in the VR2d2w group, which nonetheless decreased with time. There was no evidence of apoptosis in any group. In conclusion, the gene transfer vector AAV2 induces, in the lung, a discrete inflammatory reaction that does not affect either baseline lung mechanics or airway hyperresponsiveness.