SBIR-STTR Award

The corneal endothelium plays a pivotal role in maintaining corneal transparency. Unlike other species, the human corneal endothelium is notorious for its limited proliferative capacity in vivo after injury, aging, and surgery. Persistet corneal endothelial dysfunction leads to sight- threatening bullous keratopathy. Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy relies upon transplantation of a cadaver donor cornea containing a healthy corneal endothelium. Because of a severe global shortage of donor corneas in conjunction with an increasing trend toward transplanting only the corneal endothelium in procedures collectively termed "endothelial keratoplasty", it is timely and paramount to develop a tissue engineering strategy to produce surgical grafts containing human corneal endothelial cells (HCEC). Using our reported in vitro model system, in which the mitotic block is mediated by contact inhibition when cell junctions mature, our preliminary studies showed that such mitotic block unlocked by the conventional engineering method based on EDTA/bFGF activates ¿-catenin/Wnt signaling and runs the risk of losing the normal phenotype to fibrous metaplasia because of endothelial-mesenchymal transition (EMT). We have further discovered such mitotic block can also uniquely be unlocked by knockdown of p120-catenin to selectively activate p120- catenin/Kaiso but not ¿-catenin/Wnt signaling. Consequently, our novel tissue engineering technology has successfully produced HCEC monolayers with a hexagonal shape and high cell density and an average size of 3.7 ¿ 0.7 mm2 (2.1 ¿ 0.4 mm in diameter) from stripped Descemet membrane of 1/8 of the corneoscleral rim normally discarded after conventional corneal transplantation. Thus, in this Phase I application, we would like to prove the concept that the size of HCEC monolayers can further be enlarged by optimizing the regimen of p120- catenin siRNA knockdown followed by additional Kaiso siRNA knockdown (Aim 1), and by addition of nocodazole to enhance p120-catenin nuclear translocation (Aim 2). Completion of these two Aims will allow us further fabricate expanded HCEC monolayers on epithelially- denuded amniotic membrane to ultimately produce 8 HCEC grafts from one donor rim and to conduct pre-clinical animal experiments in Phase II. We envision that this novel tissue engineering technology based on siRNA can also be applied to switch on and off proliferation both in vivo and ex vivo without risking the loss of normal function to EMT in other contact- inhibited tissues. Further exploration of how contact inhibition is controlled by p120- catenin/Kaiso signaling may unravel other therapeutic potentials in burgeoning regenerative medicine for treating a number of diseases characterized by the lack of regeneration due to aging, surgery, or degeneration.

Public Health Relevance:
This Phase I application proposes to develop a novel strategy of engineering human corneal endothelium based on selective activation of p120ctn/Kaiso signaling using interference RNA technology targeted at p120-catenin. Using our reported in vitro model system, we have provided strong preliminary data supporting the plausibility of further expanding human corneal endothelial monolayers by additional Kaiso siRNA knockdown with or without nocodazole. By switching on and off p120/Kaiso signaling, our novel engineering strategy may control cellular proliferation without disrupting their intercellular junctions, hence avoiding both the use of enzymatically dissociated single cells and the risk of losing the normal cell phenotype to fibrous metaplasia. Such engineered grafts may one day be used to improve the surgical procedure of endothelial keratoplasty for restoring sight in patients suffering from bullous keratopathy due to dysfunctional human corneal endothelium. Furthermore, the said technology may also be applied to engineer other similar tissues, such as the retinal pigment epithelium, in the future.

Public Health Relevance Statement:
: This Phase I application proposes to develop a novel strategy of engineering human corneal endothelium based on selective activation of p120ctn/Kaiso signaling using interference RNA technology targeted at p120-catenin. Using our reported in vitro model system, we have provided strong preliminary data supporting the plausibility of further expanding human corneal endothelial monolayers by additional Kaiso siRNA knockdown with or without nocodazole. By switching on and off p120/Kaiso signaling, our novel engineering strategy may control cellular proliferation without disrupting their intercellular junctions, hence avoiding both the use of enzymatically dissociated single cells and the risk of losing the normal cell phenotype to fibrous metaplasia. Such engineered grafts may one day be used to improve the surgical procedure of endothelial keratoplasty for restoring sight in patients suffering from bullous keratopathy due to dysfunctional human corneal endothelium. Furthermore, the said technology may also be applied to engineer other similar tissues, such as the retinal pigment epithelium, in the future.

Project Terms:
adherent junction; Affect; Aging; Animal Experiments; Aqueous Humor; base; Basement membrane; Binding (Molecular Function); Biological Models; Bromodeoxyuridine; Bullous Keratopathy; Cadaver; Caliber; catenin p120ctn protein; Cell Culture Techniques; Cell Density; Cell Line; Cell Nucleus; Cell Proliferation; Cells; Contact Inhibition; Cornea; Corneal Endothelium; Data; density; Descemet's membrane; Disease; Dose; E-Cadherin; Edetic Acid; Endothelial Cells; Engineering; Excision; Eye; Fibroblast Growth Factor 2; Functional disorder; Future; gene repression; Gene Targeting; Human; Human Engineering; improved; in vitro Model; in vivo; Injury; Intercellular Junctions; Keratoplasty; Label; Mediating; Membrane; Mesenchymal; Metaplasia; Methods; Microtubules; Mitotic; monolayer; N-Cadherin; Na(+)-K(+)-Exchanging ATPase; Natural regeneration; new technology; Nocodazole; Normal Cell; novel; novel strategies; Nuclear; Nuclear Translocation; Operative Surgical Procedures; Patients; Pattern; Phase; Phenotype; Play; pre-clinical; Procedures; Proteins; Receptor Protein-Tyrosine Kinases; Regenerative Medicine; Regimen; Reporting; response; restoration; Risk; RNA Interference; Role; Running; Shapes; Signal Pathway; Signal Transduction; Small Interfering RNA; Solutions; src-Family Kinases; Structure of retinal pigment epithelium; Technology; Therapeutic; Tissue Engineering; Tissues; Transcript; Transplantation; trend; Trypsin; Vision; Withdra

The corneal endothelium plays a pivotal role in maintaining corneal transparency. Unlike in other species, the human corneal endothelium is notorious for its limited proliferative capacity in vivo after diseases, injury, aging, and surgery Persistent corneal endothelial dysfunction leads to sight-threatening bullous keratopathy. Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy relies upon transplantation of a cadaver donor cornea containing a healthy corneal endothelium. Due to a severe global shortage of donor corneas, in conjunction with an increasing trend toward transplanting only the corneal endothelium in procedures collectively termed 'endothelial keratoplasties,' it is timely and paramount to develop a tissue engineering strategy to produce surgical grafts containing human corneal endothelial cells (HCECs). Using our reported in vitro model system, in which the mitotic block is mediated by contact inhibition when cell junctions mature, we have shown that the conventional engineering methods using EDTA/bFGF to generate single HCECs activates -catenin/Wnt signaling and the loss of the normal HCEC phenotype to endothelial-mesenchymal transition (EMT). In contrast, our novel engineering method based on transient knockdown by p120 catenin (p120) and Kaiso siRNAs unlocks the mitotic block by activating p120/Kaiso signaling but not -catenin/Wnt signaling. We have further optimized this p120-Kaiso knockdown regimen by switching to a serum-free medium containing bFGF and LIF and discovered that our method further activates RohA-ROCK-canonical BMP signaling to reprogram HCECs to neural-crest like progenitors which proliferates to maintain the normal HCEC phenotype without EMT. Consequently, our novel tissue engineering technology can successfully produce from Descemet membrane stripped from 1/8 of the corneoscleral rim (normally discarded after conventional corneal transplantation) one HCEC monolayer with a hexagonal shape, comparable in vivo cell density, and an average size of 11.0 ¿ 0.6 mm in diameter. That is, the technology will add at least an additional 8 transplantable grafts per one donor cornea. In this Phase II application, we propose to establish reproducible GMP engineering of HCEC grafts on an implantable collagen membrane, with a new packing system to transport these grafts (Aim 1), and to examine the safety and efficacy of these engineered HCEC grafts through the surgical procedure of DMEK in an in vivo NIH mini pig model of endothelial dysfunction (Aim 2). Completion of these two Aims will allow the Company to gather sufficient pre-clinical data needed for an IND submission to the FDA. Ultimately, the Company can capture a unique market opportunity by fulfilling an unmet global need. One day, this new tissue engineering technology can also be deployed to engineer other similar monolayer tissues such as retinal pigment epithelium (RPE) for submacular transplantation in treating retinal blinding diseases characterized by dysfunctional RPE. Furthermore, successful commercialization of this technology will stimulate the scientific community to re-think how 'contact inhibition' can safely be perturbed to our benefit, i.e., by maintaining the normal phenotype, and whether this new regenerative approach can circumvent the need to reprogramming directly from embryonic stem cells or induced pluripotent stem cells.

Thesaurus Terms:
Abbreviations;Acronyms;Aging;Anterior;Antibodies;Antigens;Atelocollagen;Base;Biological Models;Blindness;Bullous Keratopathy;Businesses;Cadaver;Caliber;Cattle;Cell Cycle;Cell Density;Cell Line;Cells;Cephalic;Clinical;Clinical Data;Collagen;Collagen Type Iv;Commercialization;Communities;Confocal Microscopy;Contact Inhibition;Cornea;Corneal Endothelium;Data;Debridement;Density;Descemet's Membrane;Disease;Doctor Of Philosophy;Dose;Edetic Acid;Embryonic Stem Cell;Endothelial Cells;Endothelial Dysfunction;Engineering;Excision;Eye;F-Actin;Fibroblast Growth Factor 2;Frequencies (Time Pattern);G1 Phase;Glossary;Growth;Growth Factor;Human;Human Engineering;Hydration Status;Immunofluorescence Immunologic;In Vitro;In Vitro Model;In Vivo;Induced Pluripotent Stem Cell;Injury;Innovation;Intercellular Junctions;Investigation;Keratoplasty;Laminin;Lif Gene;Marketing;Matrigel;Mechanics;Mediating;Membrane;Mesenchymal;Methods;Miniature Swine;Mitotic;Modeling;Monitor;Monolayer;Morphology;N-Cadherin;Na(+)-K(+)-Exchanging Atpase;Neural Crest;Neural Crest Cell;Ngfr Protein;Novel;Novel Strategies;Operative Surgical Procedures;Outcome;Patients;Pattern;Phase;Phenotype;Physiologic Intraocular Pressure;Play;Pre-Clinical;Procedures;Progenitor;Proliferating;Public Health Relevance;Pump;Regenerative;Regimen;Reporting;Resort;Retina;Retinal;Risk;Rna Interference;Role;Running;Safety;Serum-Free Culture Media;Shapes;Signal Transduction;Small Interfering Rna;Solutions;Source;Structure Of Retinal Pigment Epithelium;Success;System;Technology;Thick;Time;Tissue Engineering;Tissues;Transplantation;Transportation;Trend;Trypsin;Ultrasound Microscopy;United States National Institutes Of Health;Universities;Vision;Withdrawal;Yang;