GENETIC TREATMENT FOR GLAUCOMA

 

In primary congenital glaucoma (PCG), there are developmental defects in the conventional aqueous outflow pathway, comprising of the trabecular meshwork (TM) and Schlemm’s canal (SC).

Studies have shown the central role played by ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis.

Further information about these pathways can provide new candidates for SC-targeted glaucoma therapy.

The angiopoietin (ANGPT)-TEK (tunica interna endothelial cell kinase, also known as TIE2) system is an endothelial growth factor pathway comprised of the receptor tyrosine kinase TEK, which is highly expressed by SC endothelial cells and its ligands, the angiopoietins.

Heterozygous loss of function variants in TEK or its primary ligand ANGPT1 have been linked to PCG in children, and ANGPT1 and ANGPT2 have been associated with primary open-angle glaucoma (POAG) in adults.

ANGPT1-TEK signaling is required for SC development in mice, suggesting a potential mechanism for IOP elevation. In addition, the pathway is an essential regulator of IOP homeostasis in adult mice and nonhuman primates, which rapidly develop ocular hypertension and glaucoma after inhibition of ANGPT-TEK signaling.

In parallel with ANGPT-TEK signaling, SC and the TM are regulated by numerous pathways, many of which are likely to regulate outflow and provide IOP-lowering therapeutic targets.

SVEP1 (Sushi, von Willebrand factor type A, EGF, and pentraxin domain containing protein 1), a gene encoding a large extracellular matrix protein also known as Polydom is expressed in the TM. It has been reported to bind ANGPT1 in vitro.

SVEP1 is essential for lymphatic development and was linked to PCG via a large family containing five generations of affected individuals also harboring a heterozygous loss of function variant in TEK22 and has been associated with POAG in a large multi-ethnic cohort.

Using mouse models, it was shown that deletion of Angpt1 or Svep1 from the neural crest tissues leads to major defects in the adjacent SC, ocular hypertension and phenotypes similar to PCG. These studies demonstrate the importance of TM-SC crosstalk and confirm that TM-expressed molecules are essential drivers of SC development and function.

A study from USA has shown that treatment of Angpt1ΔNC mice with intra-peritoneal Hepta-ANGPT1 from birth to P14 partially compensated for the loss of endogenous ANGPT1 and allowed the development of a functional SC.

Despite the discontinuation of treatment at P14, adult Hepta-ANGPT1 treated mice had reduced IOP compared to vehicle-treated Angpt1ΔNC controls, increased SC size and blunted loss of RGCs, indicating lasting effect of this developmental rescue.

In WT mice, developmental Hepta-ANGPT1 boosted SC development, leading to an enlarged SC with elevated PROX1 expression. Retention or increase in PROX1 expression in treated eyes was important confirmation that the enlarged canal retained its differentiated phenotype and was conducting outflow as PROX1 is an important marker of the differentiated SC phenotype and reduced aqueous humor outflow leads to decreased PROX1 expression.

Several other signaling molecules identified in the single-cell transcriptomics dataset have been implicated in glaucoma and offer exciting opportunities for future studies and drug development directly targeting the conventional outflow pathway.

 

SUPRACHOROIDAL INJECTION OF POLYZWITTERION HYDROGEL FOR TREATING GLAUCOMA

 

The aqueous outflow passages consist of the conventional and the unconventional pathways. The former route is through the trabecular meshwork and Schlemm’s canal, while in the latter approximately 15% of the aqueous flows out through the ciliary muscle, supraciliary muscle and suprachoroidal space.

The unconventional outflow pathways itself includes two routes, the uveoscleral pathway and the uveovortex pathway. The uveoscleral pathway indicates drainage of aqueous humor through the sclera, which is finally absorbed by the orbital blood vessels, while in the uveovortex pathway aqueous humor enters the choroid and drains through the vortex vein.

The suprachoroidal space (SCS) is the potential space between the choroid and the sclera, which forms part of the uveovortex pathway in the circulation of aqueous humor.

Recently, SCS injection of commercial hyaluronic acid (HA) hydrogel and modified HA hydrogel has been reported to reduce IOP for a period of time in rabbits. However, the HA hydrogel exhibited a short retention time in the SCS, and there was also minor hemorrhage and fibrosis at the injection site. In vivo injection of collagenase could also expand the SCS of rabbit eyes, which was usually well tolerated, but the duration of IOP reduction was also not long enough.

In recent years, zwitterionic hydrogels have been applied in a wide range of biomedical engineering fields. These zwitterionic hydrogels have excellent antifouling ability to resist adsorption and adhesion of proteins and cells, thus mitigating inflammation and the formation of fibrosis after implantation.

In an experiment reported by Hao and colleagues from Tianjin Key Laboratory of Retinal Functions and Diseases, polycarboxybetaine (PCB-OAA) macromonomer, which could in situ form a hydrogel in vitreous cavity by crosslinking with dithiothreitol (DTT) through the Michael addition reaction was injected into the SCS.

About 100 μL of HA (which acted as control) and PCB-OAA hydrogels were injected into the SCS, using a microneedle of 1.5 mm length to avoid damage to the retina and other ocular tissues.

Before the operation, the averaged IOP value for each group was 12 ± 0.5 mmHg by continuous measurement for 1 week. At 3 days post-surgery it was observed that the HA hydrogel group and PCB-OAA hydrogel group both showed a IOP reduction of approximately 4 mmHg compared with the normal group, which was at the lowest level, proving that HA and PCB-OAA hydrogels were successfully injected into the SCS space. In the following 4 weeks, the IOP of the HA group increased steadily over time and approached the normal level. However, in the PCB-OAA hydrogel group, IOP maintained a lower value than HA group and still showed about 2 mmHg reduction at 28 days post-operation. Then at 8 weeks, IOP in the PCB-OAA hydrogel group increased to a normal level. These results showed that injecting PCB-OAA hydrogels into SCS could reduce IOP for approximate 8 weeks, superior to the performance of HA hydrogel.

Ultrasound biomicroscopy (UBM) was performed during the study period to assess the SCS. The largest expanded SCS was observed both in the HA and PCB-OAA hydrogel group 3 days post-operation, which was coincident with the highest IOP reduction (4 mmHg). Additionally, it was worth noting that the SCS space gradually became smaller during the subsequent 8 weeks.

It respectively took 4 and 8 weeks for HA and PCB-OAA hydrogel to achieve complete SCS collapse.

Histopathological examination following completion of the study showed both HA and PCB-OAA hydrogel groups had complete retinal structure, indicating that the suprachoroidal injection had no negative effect on the morphology of the retina. There was no pathological inflammation and fibrosis around SCS in the HA and PCB-OAA hydrogel groups compared with the normal group.

HA INJECTION

Technologies such as this polyzwitterion hydrogel can be used in the future to treat ocular hypertension and glaucoma without high cost or resorting to daily medication or complicated surgical intervention.

FPF MAPPING IN DIAGNOSIS OF GLAUCOMA

 

It has been shown that mitochondrial metabolic dysfunction in conditions such as glaucoma, can be used as a marker for the pre-apoptotic cellular state of the eye. Mitochondrial dysfunction and altered energy dynamics drive the production of reactive oxygen species (ROS). This biochemical mechanism is implicated in the apoptosis of retinal ganglion cells (RGCs).

Mitochondrial dysfunction with production of ROS results in conversion of mitochondrial flavoproteins to their oxidized state, which produces an increase in their blue light-stimulated fluorescence. This finding has established flavoprotein fluorescence (FPF) as a potential biomarker for mitochondrial dysfunction. This technology of FPF-mapping could be developed into a non-invasive method to image the eye for pre-perimetric glaucoma.

RGCs, which are highly concentrated within the macula, experience impairment of mitochondrial activity as a result of increased IOP. Studies have shown changes in the macula act as an early marker of glaucoma in a sub-set of patients.

In a study performed at the New York Eye and Ear Infirmary of Mount Sinai, retinal FPF was measured as a surrogate for mitochondrial dysfunction in ocular hypertension (OHT) and open-angle glaucoma (POAG) eyes to investigate its potential as a biomarker for substructural glaucomatous injury.

The study demonstrated significantly increased FPF and FPF/RGC+ thickness of the macula in OHT compared to controls. As OHT eyes demonstrate a lack of cpRNFL thinning or visual field defects, it is notable that that increased mitochondrial dysfunction at the macula can be detected prior to clinical evidence of structural or functional deterioration.

In POAG, the ratio of FPF to RGC+ thickness, representing the FPF per mitochondrion within the ganglion and inner plexiform layers, was found to be significantly increased compared to controls.

FPF maps furthermore demonstrated discrete foci of increased FPF, suggesting that mitochondrial dysfunction may occur in distinct patterns or at particular regions of the macula.

 

FPF-mapping has also been used in diabetic patients.

In the following image, a 65 y/o Hispanic male was diagnosed with PDR and CSME. (a, b) Color fundus picture shows macular edema, hard exudates, and hemorrhages. (c) OCT CMT preinjection (574 μm); (d) OCT CMT postinjection (541 μm). (e) FPF map preinjection shows an increase in green-yellow coloring suggesting greater mitochondrial dysfunction (oxidative stress) compared to the (f) blue-green postinjection FPF map. gsu = grayscale units.

 

 

 

 

 

ROLE OF OCT IN DIAGNOSIS OF GLAUCOMA

While OCT of the retinal nerve fiber layer and macula is being increasingly used in the diagnosis of glaucoma, it is interesting to note the recommendations for the diagnosis of glaucoma based on these tests. The guidelines by most authoritative organizations do not recommend OCT for diagnosis. At best, it can be used for follow-up and monitoring of glaucoma patients.

AMERICAN ACADEMY OF OPHTHALMOLOGY GUIDELINES:

ASIA PACIFIC GLAUCOMA GUIDELINES:

EUROPEAN GLAUCOMA SOCEITY GUIDELINES:

NHS UK GUIDELINES: