SBIR-STTR Award

Proliferative vitreoretinopathy (PVR) is characterized by membranes that develop on the surface of the retina after rhegmatogenous retinal detachments (RRDs). Because PVR membranes exert tractional forces on the retina, they are the leading cause of failure after RRD surgery. Despite additional surgical interventions, the visual outcome still remains poor. Prevention of PVR during the initial RRD surgery is preferable and will avoid such recurrent retinal detachment or even blindness. Unfortunately, all previous attempts to prevent PVR formation using different pharmacologic agents have been unsuccessful. The two main pathological hallmarks of PVR membranes are proliferation and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells. RPE cells play a pivotal role in maintaining photoreceptor function and are normally differentiated and mitotically quiescent due to contact inhibition. RRDs allow RPE cells to be dispersed in the vitreous cavity and become exposed to growth factors (e.g., EGF, FGF, TGF-ß, VEGF and TNF-¿) and inflammatory cytokines (e.g., IFN-¿), which result in proliferation and EMT into fibroblasts or myofibroblasts. Our recent discovery provides insight into the mystery of how growth factors control the proliferation and EMT of RPE cells. Using post-confluent ARPE-19 cells in an in vitro model, we demonstrated that proliferation is coupled with EMT when contact inhibition is perturbed by EGTA followed by exposure to EGF and/or FGF-2, which activate canonical Wnt signaling. In contrast, proliferation ceases with full-blown EMT followed by exposure to TGF-ß1, which activates Smad/ZEB signaling. This baseline characterization establishes a framework for discovering new drugs which can prevent PVR by targeting both proliferation and EMT. Transplantation of cryopreserved amniotic membrane (AM) has become a standard surgical procedure for ocular surface reconstruction to deliver anti-inflammatory, anti-scarring and anti-angiogenic actions to promote wound healing. Recently, we have purified and characterized an active AM matrix component termed heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) complex, which retains AM's aforementioned therapeutic actions. In this SBIR Phase I application, we propose to prove the concept that HC-HA/PTX3 can inhibit both canonical Wnt signaling and TGF-ß-induced Smad/ZEB signaling in RPE cells to inhibit proliferation and EMT, respectively. Successful completion of these aims will allow us to move to Phase II in order to gather critical pre-clinical safety and efficacy data for the IND submission to the FDA so that we may examine its therapeutic potential as a new class of biologics to prevent PVR in human patients.

Public Health Relevance Statement:


Public Health Relevance:
Our published and preliminary studies support the hypothesis that the HC-HA/PTX3 complex, purified from human amniotic membrane, can suppress both proliferation and EMT of human RPE cells. In this Phase I application, we propose to prove the concept so that we may proceed with pre-clinical safety and efficacy studies in Phase II to gather critical data for the IND application to the FDA. Our ultimate goal is to develop HC- HA/PTX3 as a new class of biologics to prevent PVR in human patients. Furthermore, the success of achieving this product development program will pave the way to deploy HC-HA/PTX3 as a new class of biologics to treat inflammatory/scarring diseases in the eye and other parts of the body.

Project Terms:
Anti-inflammatory; Anti-Inflammatory Agents; beta catenin; Blindness; Body part; Bromodeoxyuridine; Cells; Cicatrix; Collagen; Complex; Contact Inhibition; Coupled; cytokine; Data; Disease; Dose; EGF gene; Egtazic Acid; Epithelial; Exhibits; Exposure to; Eye; Failure (biologic function); Fibroblast Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Gel; Genes; Goals; Growth Factor; Health; Human; Hyaluronic Acid; improved; in vitro Model; Inflammatory; insight; Interferon Type II; Interferons; jun Oncogene; Label; Link; Membrane; Mesenchymal; migration; Modeling; Molecular Weight; monolayer; Myofibroblast; N-Cadherin; Na(+)-K(+)-Exchanging ATPase; novel therapeutics; Nuclear Translocation; ocular surface; Operative Surgical Procedures; Outcome; Patients; Pharmaceutical Preparations; Phase; Phenotype; Photoreceptors; Play; pre-clinical; prevent; Prevention; product development; programs; Proliferative Vitreoretinopathy; protein expression; PTX3 protein; Publishing; reconstruction; Recurrence; Reporting; Retina; Retinal Detachment; Role; RPE65 protein; Safety; Scheme; Signal Pathway; Signal Transduction; Small Business Innovation Research Grant; Structure of retinal pigment epithelium; success; Surface; Testing; Therapeutic; Therapeutic Agents; TNF gene; Transforming Growth Factor beta; Transplantation; Trypsin Inhibitors; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factors; Vimentin; Visual; Wound Hea

Prevention of Proliferative Vitreoretinopathy by HC-HA/PTX3 Summary Proliferative vitreoretinopathy (PVR) is characterized by membranes that develop on the surface of the retina after rhegmatogenous retinal detachments (RRD), during which time RPE cells are dispersed into the vitreous cavity where they lose contact inhibition and are exposed to multiple growth factors and cytokines. This pathological setting promotes proliferation and EMT of RPE cells to fibroblasts or myofibroblasts that produce intravitreal membranes. These PVR membranes exert tractional forces on the retina and become the leading cause of failure after RRD surgery. Despite additional surgical interventions, the visual outcome still remains poor. Prevention of PVR during the initial RRD surgery could improve the visual success rate. Unfortunately, all previous attempts using different agents have been unsuccessful. Using an in vitro RPE cell culture model, we have reported that following perturbation of contact inhibition of RPE cells, EGF and FGF-2 upregulate while TGF-β1 downregulates canonical Wnt signaling in the proliferative phase, but TGF-β1 promotes canonical TGF-β/Smad/ZEB signaling in the irreversible scarring phase of EMT. We have successfully purified and characterized HC-HA/PTX3 from amniotic membrane (AM) and have reported that this unique matrix is responsible for AM's anti-inflammatory, anti-scarring and anti- angiogenic therapeutic actions. HC-HA/PTX3 is formed by tight association between pentraxin 3 (PTX3) and HC-HA, which consists of high molecular weight hyaluronic acid (HA) covalently linked to heavy chain 1 (HC1) of inter-α-trypsin inhibitor (I I) through the catalytic action of tumor necrosis factor-stimulated gene-6 (TSG-6). Through Phase I support, we have proven the concept that HC-HA/PTX3 can be a novel “biologic” to prevent PVR by inhibiting proliferation and EMT in the aforementioned in vitro model that has been optimized to better mimic in vivo pathological processes of PVR regarding cell density, growth factor stimulation, and measurement methods. We have developed the potency assay that is required as an in-process control of the manufacturing of HC-HA/PTX3 from different donors, demonstrated the safety (i.e., lack of cytotoxicity) and the efficacy of HC- HA/PTX3 over a wide range of doses, demonstrated the efficacy of HC-HA/PTX3, but not HA, in inhibiting proliferation and gel contraction caused by both ARPE-19 cells and primary human RPE cells, and delineated the mode of action of HC-HA/PTX3 in inhibiting the aforementioned Wnt and TGF-β signaling. These accomplishments allow us to propose in this Phase II application to scale up the manufacturing of HC-HA/PTX3 by combining AM and umbilical cord (UC) from the same donor (Aim 1), to establish the release criteria and the stability of the HC-HA/PTX3 formulation via reproducible GMP manufacturing (Aim 2), and to determine the safety and efficacy of intravitreal injection of HC-HA/PTX3 in our recently-established rabbit PVR model (Aim 3). Collectively, we would like to gather necessary and sufficient pre-clinical data for an IND submission to the FDA so that the Company can capture a unique business opportunity by deploying this novel biologic to fulfill the unmet global need of treating this severe retinal blinding disease.

Public Health Relevance Statement:
Narrative Our published data and the Phase I progresses have proven the concept that the HC-HA/PTX3 complex purified from human amniotic membrane can suppress proliferation, migration, gel contraction, and EMT of human RPE cells. Using an improved in vitro RPE culture model to better mimic the in vivo pathological processes of PVR, we have developed the potency assay as an in-process control of the manufacturing process of HC-HA/PTX3, demonstrated the safety and the efficacy of HC-HA/PTX3 in inhibiting both abnormal proliferation and EMT in the optimized in vitro RPE model, and delineated the mode of action of HC-HA/PTX3 in inhibiting the pathological Wnt and TGF-β signaling. In addition, we have reproduced a rabbit PVR model so that in this Phase II application we may scale up the manufacturing of HC-HA/PTX3 by combining both AM and umbilical cord (UC) from the same donor (Aim 1), establish the release criteria and the shelf-life of the HC- HA/PTX3 formulation via reproducible GMP manufacturing (Aim 2), and determine the safety and efficacy of intravitreal injection of HC-HA/PTX3 in the established rabbit PVR model (Aim 3). The successful completion of these three Aims will allow us to gather sufficient pre-clinical data for an IND submission to the FDA so that the Company may deploy HC-HA/PTX3 as a biologic to treat this serious retinal blinding disease.

NIH Spending Category:
Eye Disease and Disorders of Vision; Neurosciences; Prevention

Project Terms:
Anti-inflammatory; Anti-Inflammatory Agents; Biological Assay; Businesses; Cell Culture Techniques; Cell Density; Cells; Cicatrix; Clinical Data; Complex; Contact Inhibition; cytokine; cytotoxicity; Data; Dialysis procedure; Disease; Dose; EGF gene; Epithelial; Failure; FGF2 gene; Fibroblasts; Formulation; Freeze Drying; Gel; Genes; Growth; Histologic; Human; Hyaluronic Acid; improved; In Vitro; in vitro Model; in vivo; innovation; inter-alpha-inhibitor; intravitreal injection; Life; Link; manufacturing process; manufacturing scale-up; Measurement; Membrane; Mesenchymal; Methods; migration; Modeling; Molecular Weight; Monitor; Myofibroblast; novel; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Pathologic Processes; Phase; pre-clinical; prevent; Prevention; Process; Production; Proliferative Vitreoretinopathy; PTX3 protein; Publishing; Reporting; Retina; Retinal; Retinal Detachment; Safety; scale up; Signal Transduction; Specimen; Structure of retinal pigment epithelium; success; Surface; tartrate-resistant acid phosphatase; Therapeutic; Time; TNF gene; Transforming Growth Factor beta; Ultracentrifugation; Umbilical cord structur