Cursiefen, Claus | Hadrian, Karina | Hos, Deniz - B 04

The cornea is the transparent avascular outer barrier and major refractive element of the eye. Loss of corneal transparency, e.g. due to dysfunction of corneal endothelial cells resulting in corneal swelling (edema), leads to corneal blindness and is the second most common cause for blindness worldwide. The only possible treatment so far is corneal transplantation, resulting in more than a million people suffering from corneal blindness due to shortage of donor corneas worldwide. Due to shortage of tissue donors, only 1 in 70 patients can be cured. Thus, non-surgical approaches to reduce corneal edema would be of great therapeutic value to treat corneal blindness. In this context, we recently discovered a novel suppressive role for the osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5; or tonicity-responsive enhancer binding protein; TonEBP) in corneal edema resorption, thereby identifying a novel potential therapeutic target to non-surgically combat edema-induced corneal blindness. Aim of this work is to translate these findings into a clinically applicable direction by therapeutically modulating NFAT5 in the cornea in mouse models of acute and chronic corneal edema. Furthermore, this study will investigate the impact of modulating NFAT5 on graft survival after corneal transplantation.

Recently, using an established murine model of PCI (“acute injury-induced corneal edema”) with a tamoxifen-inducible systemic NFAT5 knockout (UbcCre/NFAT5^fl/fl) and mice with a myeloid-specific NFAT5 knockout (LysMCre/NFAT5^fl/fl), we could for the first time demonstrate, that the loss of NFAT5 in uninjured corneas did not result in alterations in mean corneal thickness (MCT) as a measure of corneal edema in NFAT5 KO mice compared to wildtype mice. However, after PCI, corneas of wildtype mice showed severe edema compared to corneas of NFAT5 KO mice with an MTC comparable to uninjured corneas (Figure 2).

It was previously shown that NFAT5 regulates lymphangiogenesis in the skin via macrophages. Furthermore, we have previously demonstrated that macrophage-mediated corneal lymphangiogenesis after PCI seems to influence the resorption of corneal edema (Hos et al., 2017). Interestingly, we could not detect obvious changes in the lymphatic vessel architecture in corneas of mice lacking NFAT5, indicating that lymphangiogenesis might not be the main mechanism for the increased resorption of corneal edema in this setting. Another mechanism which might be involved in the accelerated resorption of corneal edema in NFAT5 deficient mice is increased pinocytosis of macrophages. Indeed, we were able to show a significantly higher pinocytosis capacity of BMDMs generated from LysMCre⁺/NFAT5^fl/fl mice compared to LysMCre^-/NFAT5^fl/fl mice. Based on these findings, we propose that loss of NFAT5 results in higher corneal macrophage numbers with increased pinocytotic capacity, leading to a faster resorption of corneal edema after injury (Hadrian et al., 2022).

As already mentioned, we also were the first group investigating the influence of NFAT5 on corneal hemangiogenesis and tissue vascularization using an established mouse model of suture-induced inflammatory corneal neovascularization. This model leads to an ingrowth of blood as well as lymphatic vessels into the inflamed cornea. To our knowledge, the effect of NFAT5 on blood vessels also outside the eye has not been investigated yet. Our preliminary results show decreased corneal neovascularization 1 week after suture placement in mice with a myeloid-specific NFAT5 knockout (Figure 3).

This is in line with a decreased expression of VEGF-A in corneas from LysMCre⁺/NFAT5^fl/fl mice compared to LysMCre^-/NFAT5^fl/fl mice. The observation that NFAT5 is a regulator of corneal hemangiogenesis might be of particular importance in the setting of corneal transplantation, as it is established that inhibition of corneal hemangiogenesis promotes corneal transplant survival (Hou et al., 2018; Le et al., 2018; Hou et al., 2017).

However, for a better clinical translation, a feasible method for the local inhibition of NFAT5 at the cornea has to be established, preferably in form of eye drops. Using eye drops leads to very high local concentrations usually with few systemic side effects, has high patient compliance, and usually causes no pain. Typically, siRNAs are a common way to knock down target genes, which is also feasible in in vivo models. Therefore, we will establish an siRNA delivery system using a hybrid silicon-lipid nanoparticle system for an optimal knock down of NFAT5 at the cornea. The system contains a unique silicon-based delivery platform, which is a biocompatible hybrid of porous silicon nanoparticles and lipids (Baran-Rachwalska et al., 2020). It has been shown that this system has the ability to bind nucleic acid and deliver functional siRNA to corneal cells both in vitro and in vivo (Baran-Rachwalska et al., 2020). Therefore, we will use this system to optimize the efficacy of the siRNA knockdown of NFAT5 in the cornea.

The main indication for corneal transplantation is the loss of corneal transparency due to severe edema. This project aims to better define the novel role of NFAT5 in corneal edema regulation and transplant immunology. Translationally, our findings may provide not only the rational but also a novel therapeutic tool to non-surgically treat corneal edema and re-establish corneal transparency and vision. Further, modulation of NFAT5 may also allow enhanced graft survival after transplantation.

Based on our own previous work and the published literature,we hypothesize that
(i) local inhibition of NFAT5 by siRNA eye drops is able to reduce corneal edema, re-establish corneal transparency and reduce the need of corneal transplantation. Furthermore, we hypothesize that
(ii) modulation of NFAT5 increases corneal graft survival by reducing corneal hemangiogenesis and local alloimmune responses.

• In the first part, of the project, we will establish the successful in vivo siRNA knockdown of NFAT5 to reduce corneal edema. We will knock down NFAT5 in the cornea using siRNA-based eye drops. For an optimal delivery of the siRNA, hybrid silicon-lipid nanoparticles will be used.

Using this established injury model, we will now knock down NFAT5 using siRNA eye drops

• In the second part of this project, the impact of the knockdown of NFAT5 on chronic corneal edema will be studied. A mouse model with a Col8a2 Q455K knock-in mutation (Col8a2^Q455K/Q455K) will be used, which is already established and available in the laboratory (Jun et al., 2012; Leonard et al., 2019). This mouse line shows features strikingly similar to human FECD, including progressive alterations in endothelial cell morphology, endothelial cell loss and basement membrane guttae leading to chronic corneal edema and transparency loss. To knockdown NFAT5 in this mouse model, NFAT5 siRNA eye drops will be applied three times daily for 2 weeks. Analysis will be performed 0, 7 and 14 days after eye drops.

• In the third part of the project, the impact of NFAT5 on corneal graft survival after penetrating keratoplasty (PK) will be investigated. Two established mouse models will be used for this purpose: mice with a myeloid-specific NFAT5 knockout (LysMCre/NFAT5^fl/fl) and mice with a tamoxifen-inducible systemic NFAT5 knockout (UbcCre/NFAT5^fl/fl). In a well-established high-risk PK model recipient mice will undergo suture placement 2 weeks prior to PK to induce vascularized high-risk recipient beds. The donor tissue will be sourced from LysMCre/NFAT5^fl/fl and UbcCre/NFAT5^fl/fl mice and will be transplanted into Balb/c mice. Furthermore, corneas from wildtype mice (B10.D2) will be used as donors and transplanted into LysMCre/NFAT5^fl/fl and UbcCre/NFAT5^fl/fl as recipients. Transplant survival will be monitored and graded weekly for 8 weeks

• In the fourth part of this project, the impact of local NFAT5 knockdown using siRNA on high-risk corneal graft survival after transplantation in wildtype mice – mimicking the human high-risk setting - will be assessed. For this purpose, high-risk PK using C57Bl/6 wildtype mice as donors and Balb/c mice as recipients will be used. Three approaches using NFAT5 siRNA will be investigated to increase the graft survival after PK

3.1. Post-transplant approach: NFAT5 siRNA will be administered as eye drops in the recipient eye 3 times daily for two weeks after PK.

3.2. Pre-transplant approach: NFAT5 siRNA eye drops will be applied 3 times daily after suture placement in the recipient until PK will be performed (2 weeks)

3.3. Pre-incubation approach: the corneal graft will be pre-incubated ex vivo for 48h with NFAT5 siRNA. Afterwards, the preincubated cornea will be transplanted into high-risk recipients.