June 2020
Volume 61, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2020
Development of HC-HA/PTX3 as a Biologic for Proliferative Vitreoretinopathy (PVR) Treatment
Author Affiliations & Notes
  • Hua He
    TissueTech, Inc., Miami, Florida, United States
  • Ajay E. Kuriyan
    Flaum Eye Institute, University of Rochester Medical Center; Center for Visual Sciences, University of Rochester, Rochester, New York, United States
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Esdras Arrieta
    Center for Visual Sciences, University of Rochester, Rochester, New York, United States
  • Megha Mahabole
    TissueTech, Inc., Miami, Florida, United States
  • Yuan Zhang
    TissueTech, Inc., Miami, Florida, United States
  • Kristy Hamlin
    TissueTech, Inc., Miami, Florida, United States
  • Victoria Graham
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Jean-Marie A Parel
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Harry W Flynn
    Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Scheffer C.G. Tseng
    TissueTech, Inc., Miami, Florida, United States
    Ocular Surface Center and Ocular Surface Research Education Foundation, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Hua He, TissueTech, Inc. (E); Ajay Kuriyan, None; Esdras Arrieta, None; Megha Mahabole, TissueTech, Inc. (E); Yuan Zhang, TissueTech, Inc. (E); Kristy Hamlin, TissueTech, Inc. (E); Victoria Graham, None; Jean-Marie Parel, None; Harry Flynn, None; Scheffer C.G. Tseng, TissueTech, Inc. (I), TissueTech, Inc. (P)
  • Footnotes
    Support  NIH grant R43EY025447, R44EY025447
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3061. doi:
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      Hua He, Ajay E. Kuriyan, Esdras Arrieta, Megha Mahabole, Yuan Zhang, Kristy Hamlin, Victoria Graham, Jean-Marie A Parel, Harry W Flynn, Scheffer C.G. Tseng; Development of HC-HA/PTX3 as a Biologic for Proliferative Vitreoretinopathy (PVR) Treatment. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3061.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : PVR is characterized by membranes developed on the retinal surface after retinal detachments, mainly due to proliferation and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). Despite surgical intervention, the visual outcome remains poor. We would like to develop HC-HA/PTX3, a unique matrix purified from human amniotic membrane with anti-inflammatory and anti-scarring activities, as a biologic for PVR.

Methods : HC-HA/PTX3 was manufactured from human amniotic membrane by 2 – 4 runs of CsCl gradient ultracentrifugation in the presence of 4 M Guanidine HCl. The identity and purity were verified by HA and protein quantitation, Western blot, agarose gel electrophoresis, and the potency was verified with M2 macrophage polarization assay using the cloned monocyte precursor (expression of monocyte markers: CCR2, CD62L, CX3CR1, Ly6C) from RAW264.7 cells. The cell viability was determined by WST-1 assay, and the EMT assay was performed by pSmad2/3 immunostaining and fibronectin ELISA using ARPE-19 cells with TGF-β stimulation.

Results : We have purified and qualified HC-HA/PTX3 under GMP for IND-enabling pre-clinical studies and used it as the Reference Material for the potency assay. We have validated that purified and lyophilized HC-HA/PTX3 was stable after storage at -20°C for at least two years. Multiple clones of CCR2l/CX3CR1h/Ly6C1+ RAW264.7 cells were obtained and exhibited differentiation into M1 macrophage with IFN-γ/LPS stimulation suitable for the M2 polarization assay. We prepared a two-tiered bank (Master Cell and Working Cell) from the selected clone and validated that these cells (up to passage 4) were stable (no significant changes in viability and differentiation capacity) in liquid nitrogen for at least four years. In vitro, HC-HA/PTX3 (up to 200 µg/ml) was not toxic to human RPE cells and down-regulated nuclear translocation of SMAD2/3 and fibronectin production. In vivo, intravitreal injection of HC-HA/PTX3 (up to 150 µg/per eye) was safe to rabbit eyes as judged by histopathology and ERG and intravitreal injection of HC-HA/PTX3 (37.5 - 150 µg per eye) reduced RPE-induced PVR in rabbits.

Conclusions : HC-HA/PTX3 manufactured under GMP is safe and effective in both in vitro testing and pre-clinical studies. Hence, these findings will allow us to further develop it as a novel biologic for managing PVR through human trials.

This is a 2020 ARVO Annual Meeting abstract.

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