July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
Study into the ability of viral and non-viral vectors to overcome vitreous and ILM in a vitreoretinal explant
Author Affiliations & Notes
  • Karen Peynshaert
    Ghent University, Ghent, Belgium
  • Joke Devoldere
    Ghent University, Ghent, Belgium
  • An-Katrien Minnaert
    Ghent University, Ghent, Belgium
  • Deniz Dalkara
    Insitut de la Vision, Paris, France
  • Hanen Khabou
    Insitut de la Vision, Paris, France
  • Stefaan De Smedt
    Ghent University, Ghent, Belgium
  • Katrien Remaut
    Ghent University, Ghent, Belgium
  • Footnotes
    Commercial Relationships   Karen Peynshaert, None; Joke Devoldere, None; An-Katrien Minnaert, None; Deniz Dalkara, None; Hanen Khabou, None; Stefaan De Smedt, None; Katrien Remaut, None
  • Footnotes
    Support  This project was financed by a research fund granted by the agency for Innovation by Science and Technology (IWT), and financially supported by Funds for Research in Ophthalmology (FRO)
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5708. doi:https://doi.org/
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      Karen Peynshaert, Joke Devoldere, An-Katrien Minnaert, Deniz Dalkara, Hanen Khabou, Stefaan De Smedt, Katrien Remaut; Study into the ability of viral and non-viral vectors to overcome vitreous and ILM in a vitreoretinal explant. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5708. doi: https://doi.org/.

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

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Abstract

Purpose : The vitreoretinal (VR) interface, comprising peripheral vitreous and the ILM, is a delivery barrier that greatly restricts the retinal entry of intravitreally (IVT) injected therapeutic carriers. To examine the barrier role of this interface, we developed a bovine vitreoretinal (VR) explant that keeps the vitreous attached to the retina during dissection and explant culture. In this way, drug carriers can be IVT injected ex vivo after which their extent of retinal penetration and their retinal entry route can be identified. After validation of this model with polystyrene beads we now aim to apply the model as a set-up to evaluate the penetration of promising non-viral and viral carriers after IVT injection. Carriers investigated involve AAV2 with and w/o 7m8 capsid variant, small lipid nanocapsules (LNCs) and lipid-based vector MessengerMAX (mMAX).

Methods : LNCs of sizes 25 and 50 nm are synthetized following the solvent-free phase inversion method, AAV2 vectors are provided by D. Dalkara, mMAX is purchased from Invitrogen. The size and zeta potential of the lipid-based carriers are measured by Dynamic Light Scattering (DLS). After dissection of a VR explant, DiD-labeled lipid carriers or unlabeled viral vectors are injected into the vitreous and incubated for 48 hours at 37°C (5% CO2). Next, explant cryosections are prepared followed by staining with antibodies against Collagen IV (for the ILM) and against the AAV2 capsid. Finally, the ability of the particles to overcome the vitreous and ILM is evaluated by confocal microscopy.

Results : DLS analysis shows that LNCs have a narrow size distribution (~25 and ~50 nm) and a neutral to slightly negative surface charge. mMAX particles have a charge of ~-27 mV and a size of ~135 nm. While mMAX particles had great difficulty overcoming the ILM (Fig.), the smaller LNCs entered the retina more effectively. We successfully visualized the AAV vectors (~25 nm) in the model, yet the influence of the 7m8 capsid variant on AAV passage through the ILM has to be determined.

Conclusions : Our VR model serves as a straightforward set-up to evaluate the potential of promising therapeutic carriers, viral and non-viral, to overcome the vitreous and ILM.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Retinal section of a VR explant injected with mMAX particles (green). ILM stained for Collagen IV (Red), which also stains retinal blood vessels (*); Nuclei are stained with Hoechst (blue).Scale:25 µm.

Retinal section of a VR explant injected with mMAX particles (green). ILM stained for Collagen IV (Red), which also stains retinal blood vessels (*); Nuclei are stained with Hoechst (blue).Scale:25 µm.

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