Long-term genetic modification of the ocular outflow tract and the retinal pigment epithelium with feline immunodeficiency viral vectors

The central goal to engineer feline immunodeficiency viral (FIV) vectors into a more modular platform for long-term ocular gene therapy was accomplished collaboratively by separating envelope, packaging and transfer function, the latter of which I optimized. FIV mediated gene transfer to the aqueous humor outflow tract and to the retina was established and long-term vector function and biocompatibility were confirmed. Transduction of the outflow tract was highly efficient and resulted in genetic modification of nearly the entire trabecular meshwork. For this purpose, I developed methods for administration and sensitive monitoring of FIV vectors in the anterior chamber and a novel subretinal injection technique. Bicistronic FIV vectors generated high levels of two different transgenes eGFP and β-galactosidase, which allowed live in vivo tracking and sensitive yet specific cell labeling in tissue specimen, respectively. An integrase mutant control vector that differed in only one amino acid resulted in no significant transduction while preserving all other biochemical properties. I developed a novel protocol for scaled-up production FIV vectors using cell factories and large-volume concentration that proved useful in the animal studies of this thesis. Especially the results achieved in the larger animal model, the domestic cat, validated the protocol for large-scale production. Transient transfection of 10 times lessDNA into 293T cells within high surface area cell factories and high volume, fixed-angle ultracentrifugation resulted in high titer vectors

Live outflow imaging in porcine eyes

Purpose: Although outflow of aqueous humor can be estimated using fluorophotometry and tonography, no method exists to assess segmental outflow directly. This has hindered investigations into outflow resistance elements that are downstream of the trabecular meshwork and into causes of failure of canal-based minimally invasive glaucoma surgeries (MIGS). Here, we describe a method to observe and quantify conventional outflow directly in ex vivo whole eyes and an organotypic culture model using standard fluorescence visualization equipment. Results: Aqueous outflow paths could be distinguished from venous and arterial vessels. Intricate vascular tree filling patterns were observed down to estimated 50 micrometer diameter. Whole eyes demonstrated significantly earlier filling of the nasal (SN, IN) than of the temporal quadrants (ST, IT). The fastest, superonasal quadrant filled 1.6 times faster than the slowest, superotemporal quadrant (p≤0.05). The inferotemporal quadrant also filled faster than the superotemporal quadrant. In contrast, perfused anterior segment cultures had lost these characteristic filling time differences (p>0.05). One eye showed no outflow after 20 minutes of pressurization. After three days of culture, the same quadrant that was the fastest in whole eyes (SN), was 2.2 times as fast as the slowest (ST) but this did not reach statistical significance with the number of eyes tested. The eye without flow at 20 minutes did not recuperate during 3 days of culture. Reverse filling was occasionally seen in d3 anterior segments (bottom row, red arrowhead). Whole eyes could not be reliably cultured for 3 days. Discussion: Canalograms using a green fluorescent chromophore can be obtained with standard visualization equipment to estimate the local outflow function. Regionally different outflow patterns and filling times can be observed that match channel size equivalents in human eyes. Outflow is impaired in fresh anterior segment cultures but then normalizes to the pattern seen in whole eyes. The nasally increased flow may have implications for placement and study of MIGS

Trabectome surgery techniques, outcomes and expanded indications

Trabectome surgery technique, outcomes and expanded indications are discusse

Angle stability and outflow in dual blade ab interno trabeculectomy with active versus passive chamber management.

PurposeTo compare intraoperative angle stability and postoperative outflow of two ab interno trabeculectomy devices that excise the trabecular meshwork with or without active aspiration and irrigation. We hypothesized that anterior segment optical coherence tomography (AS-OCT) allows for a quantitative comparison of intraoperative angle stability in a microincisional glaucoma surgery (MIGS) pig eye training model.MethodsTwelve freshly enucleated porcine eyes were measured with AS-OCT at baseline, at the beginning of the procedure and at its conclusion to determine the anterior chamber depth (ACD) and the nasal angle α in degrees. The right and left eye of pairs were randomly assigned to an active dual blade goniectome (aDBG) and a passive dual blade goniectome (pDBG) group, respectively. The aDBG had irrigation and aspiration ports while the pDBG required surgery under viscoelastic. We performed the procedures using our MIGS training system with a standard, motorized ophthalmic operating microscope. We estimated outflow by obtaining canalograms with fluorescent spheres.ResultsIn aDBG, the nasal angle remained wide open during the procedure at above 90° and did not change towards the end (100±10%, p = 0.9). In contrast, in pDBG, ACD decreased by 51±19% to 21% below baseline (p<0.01) while the angle progressively narrowed by 40±12% (p<0.001). Canalograms showed a similar extent of access to the outflow tract with the aDBG and the pDBG (p = 0.513). The average increase for the aDBG in the superonasal and inferonasal quadrants was between 27 to 31% and for the pDBG between 15 to 18%.ConclusionAS-OCT demonstrated that active irrigation and aspiration improved anterior chamber maintenance and ease of handling with the aDBG in this MIGS training model. The immediate postoperative outflow was equally good with both devices

Ocular hypotension, actin stress fiber disruption and phagocytosis increase by RKI-1447, a Rho-kinase inhibitor

Objective: The Rho GTPase/Rho kinase pathway is an important target in glaucoma treatment. This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor developed for cancer treatment, in a porcine ex vivo pigmentary glaucoma model. Materials and Methods: Twenty-eight fresh porcine anterior chambers were perfused with pigment medium (1.67×107 pigment particles/ml) for 48 hours before being subjected to the RKI-1447 (n=16) or the vehicle control (n=12). Another twelve eyes with normal medium perfusion served as the control. The intraocular pressure (IOP) was recorded at two-minute intervals and the outflow facility was calculated. To investigate the intracellular mechanism of the IOP reduction, primary trabecular meshwork cells were exposed to RKI-1447 or the vehicle control and then analyzed for changes in cytoskeleton, motility, and phagocytosis. Results: Compared to the baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P=0.003) at 48 hours. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14+/-2.59 mmHg to 13.38+/-0.91 mmHg (P=0.02). Pigment perfusion reduced the outflow facility from 0.27+/-0.03 at baseline to 0.18+/-0.02 at 48 hours (P<0.001). This was partially reversed with RKI-1447. RKI-1447 exhibited no apparent changes in the micro- or macroscopic appearance, including histology. Primary TM cells exposed to RKI-1447 showed a significant disruption of the actin cytoskeleton both in the presence and absence of pigment exposure (P<0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI reversed. Conclusions: RKI-1447 is a novel ROCK inhibitor that significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it especially useful for the treatment of secondary glaucomas with an increased phagocytosis load but also for other open angle glaucomas

Impact of Pigment Dispersion on Trabecular Meshwork Cells

Purpose: To investigate the effect of pigment dispersion on trabecular meshwork (TM) cells. Methods: Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose, migrate, and contract were quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. Results: TM cells readily phagocytosed pigment granules. Pigment induced stress fiber formation (pigment (P): 60.1 ± 0.3%, n=10, control (C): 38.4 ± 2.5%, n=11, P<0.001) and contraction at 24 hours onward (P<0.01). Phagocytosis declined (P: 68.7 ± 1.3%, C: 37.0 ± 1.1%, n=3, P<0.001) and migration was reduced after 6 hours (P: 28.0.1 ± 2.3, n=12, C: 40.6 ± 3.3, n=13, P<0.01). Microarray analysis revealed that Rho, IGF-1, and TGFβ signaling cascades were central to these responses. Conclusions: TM cell exposure to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigment glaucoma

Stratification of phaco-trabectome surgery results using a glaucoma severity index

The outcomes of phacoemulsification combined with trabectome surgery was analyzed using a glaucoma severity index based on preoperative intraocular pressure (IOP), number of preoperative medications, and visual field damage. Despite a less absolute indication to lower IOP, a substantial pressure reduction was seen in patients with more advanced glaucoma

A porcine ex vivo model of pigmentary glaucoma

AbstractPigment dispersion syndrome can lead to pigmentary glaucoma (PG), a poorly understood condition of younger, myopic eyes with fluctuating, high intraocular pressure (IOP). The absence of a model similar in size and behavior to human eyes has made it difficult to investigate its pathogenesis. Here, we present a porcine ex vivo model that recreates the features of PG including intraocular hypertension, pigment accumulation in the trabecular meshwork and relative failure of phagocytosis. Inin vitromonolayer cultures as well as inex vivoeye perfusion cultures, we found that the trabecular meshwork (TM) cells that regulate outflow, form actin stress fibers and have a decreased phagocytosis. Gene expression microarray and pathway analysis indicated key roles of RhoA in regulating the TM cytoskeleton, motility, and phagocytosis thereby providing new targets for PG therapy.</jats:p