June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Development of SERP1 and SERP2 anti-inflammatory serpins from Myxoma virus for use in gene therapy for ocular inflammation
Author Affiliations & Notes
  • Virginia White
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Sriram Ambadapadi
    Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
  • Matthew Amontree
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Brianna M Bowman
    Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, United States
  • Cristhian J Ildefonso
    Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, United States
  • Alexandra Lucas
    Medicine, University of Florida College of Medicine, Gainesville, Florida, United States
  • D Grant McFadden
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Alfred S Lewin
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Virginia White, None; Sriram Ambadapadi, None; Matthew Amontree, None; Brianna Bowman, None; Cristhian Ildefonso, None; Alexandra Lucas, None; D Grant McFadden, None; Alfred Lewin, None
  • Footnotes
    Support  National Eye Institute (R01EY026268), NEI core grant to the University of Florida (P30 EY02172), and the Shaler Richardson Professorship endowment
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1929. doi:
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      Virginia White, Sriram Ambadapadi, Matthew Amontree, Brianna M Bowman, Cristhian J Ildefonso, Alexandra Lucas, D Grant McFadden, Alfred S Lewin; Development of SERP1 and SERP2 anti-inflammatory serpins from Myxoma virus for use in gene therapy for ocular inflammation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1929.

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

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Abstract

Purpose : Inflammation of the retina is a contributing factor in several of the leading causes of vision loss, such as diabetic retinopathy, age-related macular degeneration (AMD), and recurrent uveitis. SERP1 and SERP2 are serine protease inhibitors produced by myxoma virus, a poxvirus infecting rabbits. SERP1 is unique among poxviral serpins in that it is secreted as a glycoprotein late during infection. SERP1 has the ability to inhibit multiple targets in the inflammatory cascade, giving it potent anti-inflammatory action. SERP2 is an inhibitor of granzyme B, caspase-1, and caspase-8. Both purified serpin proteins display potent anti-inflammatory activity in vascular injury and transplant models. We hypothesize that these serpins can be used in anti-inflammatory gene therapies for ocular inflammatory diseases.

Methods : The SERP1 and SERP2 genes were fused to green fluorescent protein (GFP) with a self-cleaving porcine teschovirus-1 P2A peptide using the Gibson assembly method. These fused genes were cloned in plasmids containing AAV2 terminal repeats. HEK293T cells were transfected with either SERP1-P2A-GFP or SERP2-P2A-GFP using linear polyethyleneimine (PEI).

Results : SERP1-P2A-GFP and SERP2-P2A-GFP gene expression was determined by visualization of GFP via fluorescence microscopy after 48 hours. Western blot confirmed the expression and cleavage of the secreted SERP1-P2A-GFP in the conditioned media and of SERP2-P2A-GFP in the cell lysate. The inhibitory properties of these genes were measured using in vitro assays for IL-1β secretion and urokinase plasminogen activator (uPA) activity assay.

Conclusions : We have successfully generated two novel AAV vectors that can deliver viral serpin genes from Myxoma virus. These genes have potent anti-inflammatory activities in vitro.
Future studies include in vivo efficacy studies of intravitreal injection of AAV-vectored SERP1 and SERP2 in the endotoxin-induced uveitis mouse model. Gene expression and safety will be determined by electroretinography (ERG), fluorescence fundoscopy, and optical coherence tomography (OCT). If successful, this could lead to novel gene therapies for ocular inflammatory diseases, relieving a significant public health burden.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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