Mark Hassall1,2, Liu Guei‐Sheung3, Mammone Teresa2, Wood John2, Jack Ao2, Alex Hewitt3,4, Robert Casson2, Jamie Craig1, Glyn Chidlow
Purpose: Intravitreal injection is used for gene delivery to retinal ganglion cells, but has low efficiencies of transduction. The aim of the present study was to optimise recombinant adenovirus‐associated vector (rAAV)‐mediated gene transduction of retinal ganglion cells by co‐administration with proteolytic enzymes.
Method: Female Sprague‐Dawley rats (n = 4) received a 5 μL intravitreal injection containing 5e+9 genome copies (gc) rAAV. The rAAV contained a bistronic cassette expressing green fluorescent protein (rAAV2/2.CAG.NGB.HA.IRES.GFP.pA). Further groups received co‐administration of Pronase E (n = 6) or Heparinase III + Hyaluronidase (n = 8). The fellow eye received a vehicle injection. Retinal function was analysed at 2 weeks by scotopic electroretinography (ERG). At 3 weeks, GFP expression was assessed in vivo using a confocal scanning laser ophthalmoscope (cSLO). Animals were then euthanized for whole mount retinas.
Results: The mean in vivo GFP expression on cSLO fundal images in rAAV + Pronase E eyes (360° of fundus ±0°) was significantly greater than rAAV + Hep/Hyal (70° ± 35°;P < 0.001) or rAAV alone (200° ± 47°;P < 0.001) eyes. Analysis of retinal whole mounts supported this pattern of increased transduction with Pronase E. The ERG b‐wave of PBS‐injected eyes (1195 ± 59 μV) was not different from rAAV‐only injected eyes (1246 ± 101 μV; P = 0.46), rAAV + Pronase E eyes (1004 ± 105 μV; P = 0.18) or rAAV + Hep/Hyal eyes (1150 ± 64 μV; P = 0.33). Conclusion: Retinal ganglion cell transduction by rAAV was enhanced by co‐administration of Pronase E. Importantly, this effect was not associated with increased retinal toxicity, as assessed by electroretinography. The use of pronase E should be considered for delivery of candidate neuroprotective genes to retinal ganglion cells.