July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Corneal fluorescein distribution following intrastromal injection using a purpose-designed precise corneal injection (PCI) needle
Author Affiliations & Notes
  • Allison Blanchard
    Clinical Science, North Carolina State University, Raleigh, North Carolina, United States
  • Megan Cullen
    Clinical Science, North Carolina State University, Raleigh, North Carolina, United States
  • Elizabeth Crabtree
    Clinical Science, North Carolina State University, Raleigh, North Carolina, United States
  • Jacklyn H Salmon
    Clinical Science, North Carolina State University, Raleigh, North Carolina, United States
  • Liujiang Song
    Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, United States
    Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States
  • Matthew Hirsch
    Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, United States
    Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States
  • Brian C Gilger
    Clinical Science, North Carolina State University, Raleigh, North Carolina, United States
    Theia Medical, Inc., Raleigh, North Carolina, United States
  • Footnotes
    Commercial Relationships   Allison Blanchard, None; Megan Cullen, None; Elizabeth Crabtree, None; Jacklyn Salmon, None; Liujiang Song, None; Matthew Hirsch, None; Brian Gilger, Theia Medical, Inc. (P)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2918. doi:https://doi.org/
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      Allison Blanchard, Megan Cullen, Elizabeth Crabtree, Jacklyn H Salmon, Liujiang Song, Matthew Hirsch, Brian C Gilger; Corneal fluorescein distribution following intrastromal injection using a purpose-designed precise corneal injection (PCI) needle. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2918. doi: https://doi.org/.

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

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Abstract

Purpose : Advanced corneal treatments, such as gene therapy, require accurate and consistent dosing therefore, new administration techniques are needed. The purpose of this study was to evaluate the depth and distribution of fluorescein or AAV-GFP (gene delivery) after intrastromal injection using a purpose-designed precise corneal injection (PCI) needle.

Methods : This study was performed using porcine cadaver eyes ex vivo. Corneas were injected in the axial cornea with volumes of 10, 25, or 50µL of 0.01% fluorescein using a PCI needle. Color images, high frequency ultrasound (HFU), and IVIS imaging were obtained at 1, 3, and 24 hrs after injection. Fluorescein distribution was measured on photographs (pixel counts) and IVIS imaging (fluorescent intensity at regions of interest [ROI]). Additionally, to determine depth and precision of PCI needles, eyes were injected with 10µL of 0.01% fluorescein using 330, 460, or 600µM lengths and measured using HFU. Finally, eyes were injected using a PCI needle with 50µL of PBS or AAV8-GFP (1e9 viral genomes) diluted in 10, 25, or 50µL of saline. IVIS imaging was performed for 7 days following injection to evaluate GFP expression.

Results : After injection with the PCI needle, corneal thickness increased significantly with volume (p<0.04). Percent cornea infiltrated revealed a significant injection volume to area relationship (p<0.0001). Depth of injection on HFU was significantly (p<0.004) related to the length of the needle. Interestingly, the fixed dose of AAV-GFP in the low volume (10µL) resulted in earlier detection of GFP fluorescence is a smaller area while the higher volume group demonstrated later GFP onset yet over a broader corneal area.

Conclusions : Corneal intrastromal injection using PCI needles provided precise depth of injection. Higher volume resulted in higher distribution. Fluorescein diffused peripherally to allow nearly complete stromal exposure by 24 hrs. AAV8-GFP was administered precisely to the cornea using the PCI needle with a higher vg dose resulting in earlier GFP stromal transduction near the site of injection. The same dose administered in a larger volume resulted in slower transduction over a larger corneal area demonstrating an important relationship between vector dose, volume, transduction intensity and dissemination. Further development of the PCI needle for gene therapy is warranted.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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