June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Assessment of efficacy of Magnetofection transfection system in delivering genes to RPE cells, a novel transfection method for gene therapy in mouse model.
Author Affiliations & Notes
  • Priyanka Priyadarshani
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Shanu Markand
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Jana T Sellers
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Jeffrey H Boatright
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • J M Nickerson
    Ophthalmology, Emory University, Atlanta, Georgia, United States
  • Footnotes
    Commercial Relationships   Priyanka Priyadarshani, None; Shanu Markand, None; Jana Sellers, None; Jeffrey Boatright, None; J Nickerson, None
  • Footnotes
    Support  Abraham J. & Phyllis Katz Foundation, NIH R01EY014026, R01EY021592, R01EY016470, VA RR&D C1924P I21RX001924, VA RR&D C9246C (Atlanta VA Center of Excellence in Vision and Neurocognitive Rehabilitation), P30EY006360, and an unrestricted grant to the Department of Ophthalmology at Emory University from Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 621. doi:
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      Priyanka Priyadarshani, Shanu Markand, Jana T Sellers, Jeffrey H Boatright, J M Nickerson; Assessment of efficacy of Magnetofection transfection system in delivering genes to RPE cells, a novel transfection method for gene therapy in mouse model.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):621.

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

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Abstract

Purpose : Gene therapy promises to be a viable and effective approach for the treatment of ocular disorders. Delivering nucleic acid at a specific target increases the activity at the desired area and improves precision of gene therapy thus allowing full exploitation of their potential in gene therapy application. However, it can be quite challenging to deliver and localize nucleic acids to the target safely and efficiently. The purpose of this study was to assess the level of transfection using a magnetofection system both in vitro in ARPE 19 cells and in vivo through subretinal injection in the C57BL/6J mouse.

Methods : ARPE 19 cells were transfected with gWiz_GFP plasmid using LipoMag transfection reagent and Lipofectamine 3000 and were analyzed after 3-4 days of incubation. Cells transfected with plasmid without transfection reagent were used as negative control. Three females and two males C57BL/6J mice eye at postnatal day 75-80 were subretinally injected with gWiz_GFP plasmid mixed with PolyMag magnetofection transfection reagent. Right eye of each mouse was subretinally injected with reagents and the left eye was control uninjected eye. Concentration of each transfection reagent was used as per the manufacturer’s protocol. The final mixture complex was injected into the subretinal space. The expression of reporter gene was measured by fluorescence and immunoassay. SD-OCT and flatmount staining was used to analyze nucleic acid distribution and morphological changes after 4 days of subretinal injection.

Results : Confocal image analysis showed 20-50% increase in the transfection efficiency of magnetofection reagents compared to Lipofectamine. No visible transfection was seen negative control. Results from SD-OCT and flatmount staining showed that subretinally injected nucleic acid with the Magnetofection transfection system was retained at the magnetically targeted site.

Conclusions : We conclude that the delivery of nucleic acid with the aid of a paramagnetic transfection system, which can overcome the limitations of transfection efficiency, gene vector inactivation, and toxicity. Magnetofection allows high efficiency transfection of RPE cells in a directional manner from the subretinal space into the apical face of the RPE cells.

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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