June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Successful AAV2 readministration to porcine retinal ganglion cells and serum neutralizing antibody response
Author Affiliations & Notes
  • Ashley Nies
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • Kathleen Heng
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • BaoXiang Li
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • Rain Wen
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • Albert Y. Wu
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • Jeffrey L Goldberg
    Byers Eye Institute, Spencer Center for Vision Research, California, United States
  • Footnotes
    Commercial Relationships   Ashley Nies None; Kathleen Heng None; BaoXiang Li None; Rain Wen None; Albert Wu None; Jeffrey Goldberg None
  • Footnotes
    Support  Stanford University Human Biology Research Exploration program, Medical Technologies Enterprise Consortium, Gilbert Family Foundation, National Eye Institute (P30-EY026877), and Research to Prevent Blindness, Inc
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2828 – A0344. doi:
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      Ashley Nies, Kathleen Heng, BaoXiang Li, Rain Wen, Albert Y. Wu, Jeffrey L Goldberg; Successful AAV2 readministration to porcine retinal ganglion cells and serum neutralizing antibody response. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2828 – A0344.

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

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Abstract

Purpose : Adeno-associated virus serotype 2 (AAV2) is a promising gene therapy platform for glaucoma and other optic neuropathies. However, pre-existing serum neutralizing antibodies (Nab) may significantly inhibit effective transduction and gene transfer. To investigate the effects of serum Nab on viral transduction in a large animal model, we delivered AAV2 to retinal ganglion cells (RGCs) in the porcine visual system at two timepoints and assessed transduction while monitoring serum Nab over time.

Methods : Yucatan minipigs (5.5-6 months old) were pre-screened for serum Nab titers, and animals with a positive titer were excluded. On day 0, AAV2-CMV-eGFP was intravitreally injected into the nasal and temporal aspects of the right eye, while the left eye served as a negative control. On day 14, the right eye was re-injected with AAV2-CAG-TdTomato in a similar fashion. On day 28, eyes were harvested and fixed in 4% PFA. The retinas were dissected, stained by immunofluorescence for RBPMS, flatmounted, imaged by confocal microscopy, and analyzed. To determine Nab titers, pig serum was collected on days 0, 3, 7, 14, and 28. Serial dilutions of pig serum were incubated with AAV2-Luciferase and subsequently with human embryonic kidney (HEK293) cells. Luciferase activity was measured and used to establish anti-AAV2 Nab titers.

Results : RGCs were transduced by viral administrations at both timepoints (AAV2-CMV-eGFP, day 0; AAV2-CAG-TdTomato, day 14) as evidenced by colocalization of eGFP and TdTomato with RBPMS immunofluorescence. In the negative control eye, the absence of eGFP and TdTomato expression was confirmed. Transduction efficiency was highest at the temporal and nasal retina near injection sites. Serum anti-AAV2 Nab levels increased over time beginning at day 7.

Conclusions : AAV2 can be successfully readministered over a short interval to porcine RGCs despite systemic production of anti-AAV2 Nab. Nab were produced in response to AAV2 exposure and increased over the study time course.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Transduction of porcine RGCs after administration and readministration of AAV2. (A-D) Retinal flatmount harvested at day 28 after AAV2-CMV-eGFP exposure on day 0 and AAV2-CAG-TdTomato exposure on day 14. (E-H) Contralateral negative control. Scale bar, 200 μm.

Transduction of porcine RGCs after administration and readministration of AAV2. (A-D) Retinal flatmount harvested at day 28 after AAV2-CMV-eGFP exposure on day 0 and AAV2-CAG-TdTomato exposure on day 14. (E-H) Contralateral negative control. Scale bar, 200 μm.

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