June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Evaluation of Influenza Associated Virus (IAV)-based replication-incompetent for Ocular Delivery of microRNAS
Author Affiliations & Notes
  • Coralia Luna
    Ophthalmology, Duke University, Durham, NC
  • Benjamin tenOever
    Microbiology, Mount Sinai School of Medicine, New York, NY
  • Guorong Li
    Ophthalmology, Duke University, Durham, NC
  • Sonja Schmid
    Microbiology, Mount Sinai School of Medicine, New York, NY
  • Pratap Challa
    Ophthalmology, Duke University, Durham, NC
  • David Epstein
    Ophthalmology, Duke University, Durham, NC
  • Jianming Qiu
    Ophthalmology, Duke University, Durham, NC
  • Pedro Gonzalez
    Ophthalmology, Duke University, Durham, NC
  • Footnotes
    Commercial Relationships Coralia Luna, None; Benjamin tenOever, None; Guorong Li, None; Sonja Schmid, None; Pratap Challa, None; David Epstein, None; Jianming Qiu, None; Pedro Gonzalez, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2727. doi:
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    • Get Citation

      Coralia Luna, Benjamin tenOever, Guorong Li, Sonja Schmid, Pratap Challa, David Epstein, Jianming Qiu, Pedro Gonzalez; Evaluation of Influenza Associated Virus (IAV)-based replication-incompetent for Ocular Delivery of microRNAS. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2727.

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

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Abstract
 
Purpose
 

Safe and efficient delivery to cells of interest in the eye remains a significant challenge to the therapeutic potential of microRNAs (miRNAs) for the treatment of glaucoma. IAV-based replication-incompetent vectors are a promising new class of vectors that avoids the toxicity associated with saturation of the endogenous miRNA biogenesis system. Our objective was to evaluate the potential of these vectors for in vivo delivery of miRNAs to ocular tissues relevant to glaucoma.

 
Methods
 

To generate IAV-based replication-incompetent vectors expressing miR-124 or GFP, fibroblasts were transfected with plasmids encoding the RNA dependent RNA polymerase components (PA, PB1, PB2 and NP), hemaggluttinin (HA) and the eight viral RNA segments. The supernatant from these cells was transferred to a Madin-Darby Canine Kidney cell line that expresses the HA protein of IAV. Vectors were then purified through 30% sucrose. IAV-based vectors expressing miR-124 were applied to primary human trabecular meshwork (TM) cell cultures at a 1:1 ratio and miRNA expression was evaluated by Q-PCR, in situ hybridization and Northern blot (NB). To evaluate the ability of these vectors to infect cells from different ocular tissues, mixed rat retina cell cultures were treated with 10E7 MOI/mL expressing GFP, and mice eyes were inoculated intravitreally with 10E6 MOI/mL of the same vector. GFP expression was evaluated by fluorescent microscopy.

 
Results
 

IAV-based vectors at a 1:1 ratio of vector to TM cells demonstrated robust delivery of miR-124 as measured by Q-PCR, RNA in situ hybridization, and NB. Similarly, this vector generated high levels of expression of the reporter gene GFP in retinal ganglion cells in culture (fig.1a). Intravitreal injection of IAV-based vectors expressing GFP in mice eyes resulted in high levels of GFP expression in TM, iris (fig.1b); retinal ganglion cell layer (RGL), inner nuclear layer (INL), and outer nuclear layer (ONL) in the retina (fig.1c); as well as in the lamina cribosa (LC) and meningeal cells (MC) of the optic nerve (fig.1d).

 
Conclusions
 

IAV-based vectors are effective for in vivo delivery of miRNAs to multiple cell types relevant to the pathophysiology of glaucoma. Future studies with these vectors expressing specific miRNAs relevant to glaucoma pathogenesis will help to evaluate the safety and efficacy of these vectors as potential therapeutic agents in glaucoma.

  
Keywords: 538 gene transfer/gene therapy • 688 retina • 735 trabecular meshwork  
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