Matrix metalloproteinase-3 (MMP-3)-mediated gene therapy for glaucoma.

Approximately 80 million people globally are affected by glaucoma, with a projected increase to over 110 million by 2040. Substantial issues surrounding patient compliance remain with topical eye drops, and up to 10% of patients become treatment resistant, putting them at risk of permanent vision loss. The major risk factor for glaucoma is elevated intraocular pressure, which is regulated by the balance between the secretion of aqueous humor and the resistance to its flow across the conventional outflow pathway. Here, we show that adeno-associated virus 9 (AAV9)-mediated expression of matrix metalloproteinase-3 (MMP-3) can increase outflow in two murine models of glaucoma and in nonhuman primates. We show that long-term AAV9 transduction of the corneal endothelium in the nonhuman primate is safe and well tolerated. Last, MMP-3 increases outflow in donor human eyes. Collectively, our data suggest that glaucoma can be readily treated with gene therapy-based methods, paving the way for deployment in clinical trials.

Optimisation of AAV-NDI1 Significantly Enhances Its Therapeutic Value for Correcting Retinal Mitochondrial Dysfunction.

AAV gene therapy for ocular disease has become a reality with the market authorisation of LuxturnaTM for RPE65-linked inherited retinal degenerations and many AAV gene therapies currently undergoing phase III clinical trials. Many ocular disorders have a mitochondrial involvement from primary mitochondrial disorders such as Leber hereditary optic neuropathy (LHON), predominantly due to mutations in genes encoding subunits of complex I, to Mendelian and multifactorial ocular conditions such as dominant optic atrophy, glaucoma and age-related macular degeneration. In this study, we have optimised the nuclear yeast gene, NADH-quinone oxidoreductase (NDI1), which encodes a single subunit complex I equivalent, creating a candidate gene therapy to improve mitochondrial function, independent of the genetic mutation driving disease. Optimisation of NDI1 (ophNdi1) substantially increased expression in vivo, protected RGCs and increased visual function, as assessed by optokinetic and photonegative response, in a rotenone-induced murine model. In addition, ophNdi1 increased cellular oxidative phosphorylation and ATP production and protected cells from rotenone insult to a significantly greater extent than wild type NDI1. Significantly, ophNdi1 treatment of complex I deficient patient-derived fibroblasts increased oxygen consumption and ATP production rates, demonstrating the potential of ophNdi1 as a candidate therapy for ocular disorders where mitochondrial deficits comprise an important feature.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Optimized OPA1 Isoforms 1 and 7 Provide Therapeutic Benefit in Models of Mitochondrial Dysfunction.

Optic Atrophy 1 (OPA1) is a mitochondrially targeted GTPase that plays a pivotal role in mitochondrial health, with mutations causing severe mitochondrial dysfunction and typically associated with Dominant Optic Atrophy (DOA), a progressive blinding disease involving retinal ganglion cell loss and optic nerve damage. In the current study, we investigate the use of codon-optimized versions of OPA1 isoform 1 and 7 as potential therapeutic interventions in a range of in vitro and in vivo models of mitochondrial dysfunction. We demonstrate that both isoforms perform equally well in ameliorating mitochondrial dysfunction in OPA1 knockout mouse embryonic fibroblast cells but that OPA1 expression levels require tight regulation for optimal benefit. Of note, we demonstrate for the first time that both OPA1 isoform 1 and 7 can be used independently to protect spatial visual function in a murine model of retinal ganglion cell degeneration caused by mitochondrial dysfunction, as well as providing benefit to mitochondrial bioenergetics in DOA patient derived fibroblast cells. These results highlight the potential value of OPA1-based gene therapy interventions.

Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells.

Retinal ganglion cells (RGCs) are known to be involved in several ocular disorders, including glaucoma and Leber hereditary optic neuropathy (LHON), and hence represent target cells for gene therapies directed towards these diseases. Restricting gene therapeutics to the target cell type in many situations may be preferable compared to ubiquitous transgene expression, stimulating researchers to identify RGC-specific promoters, particularly promoter sequences that may also be appropriate in size to fit readily into recombinant adeno associated viral (AAV) vectors, the vector of choice for many ocular gene therapies. In the current study we analysed EGFP expression driven by various sequences of the putative human NEFH promoter in order to define sequences required for preferential expression in RGCs. EGFP expression profiles from four different potential NEFH promoter constructs were compared in vivo in mice using retinal histology and mRNA expression analysis. Notably, two efficient promoter sequences, one comprising just 199 bp, are presented in the study.

Findings from a Genotyping Study of Over 1000 People with Inherited Retinal Disorders in Ireland.

The Irish national registry for inherited retinal degenerations (Target 5000) is a clinical and scientific program to identify individuals in Ireland with inherited retinal disorders and to attempt to ascertain the genetic cause underlying the disease pathology. Potential participants first undergo a clinical assessment, which includes clinical history and analysis with multimodal retinal imaging, electrophysiology, and visual field testing. If suitable for recruitment, a sample is taken and used for genetic analysis. Genetic analysis is conducted by use of a retinal gene panel target capture sequencing approach. With over 1000 participants from 710 pedigrees now screened, there is a positive candidate variant detection rate of approximately 70% (495/710). Where an autosomal recessive inheritance pattern is observed, an additional 9% (64/710) of probands have tested positive for a single candidate variant. Many novel variants have also been detected as part of this endeavor. The target capture approach is an economic and effective means of screening patients with inherited retinal disorders. Despite the advances in sequencing technology and the ever-decreasing associated processing costs, target capture remains an attractive option as the data produced is easily processed, analyzed, and stored compared to more comprehensive methods. However, with decreasing costs of whole genome and whole exome sequencing, the focus will likely move towards these methods for more comprehensive data generation.

Reduced humidity experienced by mice in vivo coincides with reduced outflow facility measured ex vivo.

Mice are routinely used to study aqueous humour dynamics. However, physical factors such as temperature and hydration affect outflow facility in enucleated eyes. This retrospective study examined whether differences in temperature and relative humidity experienced by living mice within their housing environment in vivo coincide with differences in outflow facility measured ex vivo. Facility data and environmental records were collected for one enucleated eye from 116 mice (C57BL/6J males, 9-15 weeks old) at two institutions. Outflow facility was reduced when relative humidity was below the lower limit of 45% recommended by the UK Code of Practice, but there was no detectable effect of temperature on outflow facility. Even when accounting for effects of humidity, there were differences in outflow facility measured between institutions and between individual researchers at the same institution. These data indicate that humidity, as well as additional environmental factors experienced by living mice within their housing environment, may significantly affect outflow facility measured ex vivo.

Intracameral Delivery of AAV to Corneal Endothelium for Expression of Secretory Proteins.

AAV9 drives gene expression in a highly selective manner within the corneal endothelium of mice following intracameral inoculation into the anterior chamber of the eye. In principle, this allows genes encoding protein constituents of the secretome (representing up to 20% of the human proteome) to be delivered directly into the aqueous humor. From here the secreted protein moves with the natural flow of the aqueous humor via a pressure gradient and is directed toward the outflow tissues. Such a delivery can be employed to modulate outflow facility and intraocular pressure through interactions at the trabecular meshwork and Schlemm's canal. We provide a protocol for the delivery of AAV to the corneal endothelium, using a CMV-driven eGFP reporter gene as a marker.

Age-related changes in eye morphology and aqueous humor dynamics in DBA/2J mice using contrast-enhanced ocular MRI.

PURPOSE: Here, we are testing the hypothesis that dynamic contrast enhanced MRI (DCE-MRI) is a useful approach for non-invasively evaluating age-related changes in aqueous humor outflow and its contribution to elevated intraocular pressure in the DBA/2J model of pigmentary glaucoma. METHODS: A rodent-specific 7 T MRI was used to assess eye anatomy (anterior chamber (AC) and vitreous body (VB) morphology, eye size, lens size) and aqueous humor dynamics (via intravenous administration of Gd-DTPA and Gd-BOPTA contrast agents) in C57BL/6 and DBA/2J mice at 3 and 9 months of age. RESULTS: Gd-MRI was used to demonstrate an anterior solute pathway into the mouse AC. Topical latanoprost treatment in C57BL/6J mice reduced Gd-BOPTA accumulation in the AC. Age-related increases in AC area, AC depth and eye size were observed in DBA/2J mice compared to C57BL/6J mice. The rate of Gd-DTPA accumulation and peak Gd-DTPA intensity was lowest in 9-month old DBA/2J mice compared to 3-month old DBA/2J mice and C57BL/6J mice at both ages. Leakage of Gd-DTPA posteriorly into the VB was also observed in 9-month old DBA/2J mice. CONCLUSIONS: These studies support the idea that age-related changes in aqueous humor outflow contribute to elevated intraocular pressure (IOP) in the DBA/2J model of pigmentary glaucoma. Gd-MRI is a valuable tool for better understanding of mechanisms and dynamics of aqueous humor circulation in normal and glaucomatous mouse eyes or following topical administration of medicines to reduce IOP.

Target 5000: Target Capture Sequencing for Inherited Retinal Degenerations.

There are an estimated 5000 people in Ireland who currently have an inherited retinal degeneration (IRD). It is the goal of this study, through genetic diagnosis, to better enable these 5000 individuals to obtain a clearer understanding of their condition and improved access to potentially applicable therapies. Here we show the current findings of a target capture next-generation sequencing study of over 750 patients from over 520 pedigrees currently situated in Ireland. We also demonstrate how processes can be implemented to retrospectively analyse patient datasets for the detection of structural variants in previously obtained sequencing reads. Pathogenic or likely pathogenic mutations were detected in 68% of pedigrees tested. We report nearly 30 novel mutations including three large structural variants. The population statistics related to our findings are presented by condition and credited to their respective candidate gene mutations. Rediagnosis rates of clinical phenotypes after genotyping are discussed. Possible causes of failure to detect a candidate mutation are evaluated. Future elements of this project, with a specific emphasis on structural variants and non-coding pathogenic variants, are expected to increase detection rates further and thereby produce an even more comprehensive representation of the genetic landscape of IRDs in Ireland.

A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors.

Significant advances in gene therapy have enabled exploration of therapies for inherited retinal disorders, many of which are in preclinical development or clinical evaluation. Gene therapy for retinal conditions has led the way in this growing field. The loss of retinal ganglion cells (RGCs) is a hallmark of a number of retinal disorders. As the field matures innovations that aid in refining therapies and optimizing efficacy are in demand. Gene therapies under development for RGC-related disorders, when delivered with recombinant adeno associated vectors (AAV), have typically been expressed from ubiquitous promoter sequences. Here we describe how a novel promoter from the murine Nefh gene was selected to drive transgene expression in RGCs. The Nefh promoter, in an AAV2/2 vector, was shown to drive preferential EGFP expression in murine RGCs in vivo following intravitreal injection. In contrast, EGFP expression from a CMV promoter was observed not only in RGCs, but throughout the inner nuclear layer and in amacrine cells located within the ganglion cell layer (GCL). Of note, the Nefh promoter sequence is sufficiently compact to be readily accommodated in AAV vectors, where transgene size represents a significant constraint. Moreover, this promoter should in principle provide a more targeted and potentially safer alternative for RGC-directed gene therapies.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma.

Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm's canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3.Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

Panel-Based Population Next-Generation Sequencing for Inherited Retinal Degenerations.

Inherited retinopathies affect approximately two and a half million people globally, yet the majority of affected patients lack clear genetic diagnoses given the diverse range of genes and mutations implicated in these conditions. We present results from a next-generation sequencing study of a large inherited retinal disease patient population, with the goal of providing clear and actionable genetic diagnoses. Targeted sequencing was performed on 539 individuals from 309 inherited retinal disease pedigrees. Causative mutations were identified in the majority (57%, 176/309) of pedigrees. We report the association of many previously unreported variants with retinal disease, as well as new disease phenotypes associated with known genes, including the first association of the SLC24A1 gene with retinitis pigmentosa. Population statistics reporting the genes most commonly implicated in retinal disease in the cohort are presented, as are some diagnostic conundrums that can arise during such studies. Inherited retinal diseases represent an exemplar group of disorders for the application of panel-based next-generation sequencing as an effective tool for detection of causative mutations.

Corrigendum to "Efficient gene delivery to photoreceptors using AAV2/rh10 and rescue of the Rho-/- mouse".

[This corrects the article DOI: 10.1038/mtm.2015.16.].

Autoregulated paracellular clearance of amyloid-ß across the blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting neural tissue from damaging blood-borne agents. The barrier is characterized by endothelial tight junctions that limit passive paracellular diffusion of polar solutes and macromolecules from blood to brain. Decreased brain clearance of the neurotoxic amyloid-ß (Aß) peptide is a central event in the pathogenesis of Alzheimer's disease (AD). Whereas transport of Aß across the BBB can occur via transcellular endothelial receptors, the paracellular movement of Aß has not been described. We show that soluble human Aß(1-40) monomers can diffuse across the paracellular pathway of the BBB in tandem with a decrease in the tight junction proteins claudin-5 and occludin in the cerebral vascular endothelium. In a murine model of AD (Tg2576), plasma Aß(1-40) levels were significantly increased, brain Aß(1-40) levels were decreased, and cognitive function was enhanced when both claudin-5 and occludin were suppressed. Furthermore, Aß can cause a transient down-regulation of claudin-5 and occludin, allowing for its own paracellular clearance across the BBB. Our results show, for the first time, the involvement of the paracellular pathway in autoregulated Aß movement across the BBB and identify both claudin-5 and occludin as potential therapeutic targets for AD. These findings also indicate that controlled modulation of tight junction components at the BBB can enhance the clearance of Aß from the brain.

IL-18 Immunotherapy for Neovascular AMD: Tolerability and Efficacy in Nonhuman Primates.

PURPOSE: Age-related macular degeneration is the most common form of central retinal blindness in the elderly. Of the two end stages of disease, neovascular AMD-although the minority form-is the most severe. Current therapies are highly successful at controlling progression of neovascular lesions; however, a significant number of patients remain refractory to treatment and the development of alternative and additive therapies to anti-VEGFs is essential. METHODS: In order to address the translational potential of interleukin (IL)-18 for use in neovascular AMD, we initiated a nonhuman primate tolerability and efficacy study for the use of intravitreally (IVT) administered clinical grade human IL-18 (SB-485232). Cynomolgus monkeys were injected IVT with increasing doses of human IL-18 (two each at 1000, 3000, and 10,000 ng per eye). In tandem, 21 monkeys were administered nine laser burns in each eye prior to receiving IL-18 as an IVT injection at a range of doses. Fundus fluorescein angiography (FFA) was performed on days 8, 15, and 22 post injection and the development of neovascular lesions was assessed. RESULTS: We show intravitreal, mature, recombinant human IL-18 is safe and can reduce choroidal neovascular lesion development in cynomolgus monkeys. CONCLUSIONS: Based on our data comparing human IL-18 to current anti-VEGF-based therapy, clinical deployment of IL-18 for neovascular AMD has the potential to lead to a new adjuvant immunotherapy-based treatment for this severe form of central blindness.

Efficient gene delivery to photoreceptors using AAV2/rh10 and rescue of the Rho(-/-) mouse.

As gene therapies for various forms of retinal degeneration progress toward human clinical trial, it will be essential to have a repertoire of safe and efficient vectors for gene delivery to the target cells. Recombinant adeno-associated virus (AAV) serotype 2/2 has been shown to be well tolerated in the human retina and has provided efficacy in human patients for some inherited retinal degenerations. In this study, the AAV2/8 and AAV2/rh10 serotypes have been compared as a means of gene delivery to mammalian photoreceptor cells using a photoreceptor specific promoter for transgene expression. Both AAV2/8 and AAV2/rh10 provided rescue of the retinal degeneration present in the rhodopsin knockout mouse, with similar levels of benefit as evaluated by molecular, histological, and functional readouts. Transgene expression levels were significantly higher (fivefold) 1 week postsubretinal injection when employing AAV2/8 for rhodopsin gene delivery compared to AAV2/rh10, and were indistinguishable by 6 weeks postadministration of vector. This study reports the use of the AAV2/rh10 serotype to provide rescue in a degenerating retina and provides a comparative evaluation of AAV2/rh10 with respect to AAV2/8, a serotype regarded as providing efficient delivery to photoreceptors.

Electroretinographic assessment of rod- and cone-mediated bipolar cell pathways using flicker stimuli in mice.

Mouse full-field electroretinograms (ERGs) are dominated by responses of photoreceptors and depolarizing (ON-) bipolar cells, but not much of hyperpolarizing (OFF-) bipolar cells under conventional recording conditions. Here we investigate a novel ERG protocol in mice for functional assessment of the major ON- and OFF-bipolar cell pathways using flicker stimuli for a high luminance with varying frequency up to 30 Hz. Wild-type (WT) and functionally specific transgenic mice (Cnga3(-/-), no cone photoreceptor function; rho(-/-), no rod photoreceptor function; mGluR6(-/-), no ON-bipolar cell function) were examined. The Cnga3(-/-) flicker ERG was similar to the WT flicker ERG at very low stimulus frequencies, whereas ERGs were comparable between WT and rho(-/-) mice at 5 Hz and above. Between 5 and 15 Hz, ERGs in mGluR6(-/-) mice differed in configuration and amplitude from those in WT and rho(-/-) mice; in contrast, response amplitudes above 15 Hz were comparable among WT, rho(-/-) and mGluR6(-/-) mice. In summary, we found three frequency ranges with these conditions that are dominated by activity in the rod pathways (below 5 Hz), cone ON-pathway (between 5 and 15 Hz), and cone OFF-pathway (above 15 Hz) that enables a quick overview of the functionality of the major bipolar cell pathways.