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AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells

Abstract

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of βIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450±358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200±4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many βIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135±367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (±2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (±1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

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Acknowledgements

This work was supported by NHMRC Grants (254507, 211924) to ARH and QC, the WA Neurotrauma Research Program, and an RD Wright NHMRC Fellowship to GWP. SGL received a UWA Postgraduate Award and further scholarship support from the Western Australian Institute for Medical Research. We thank Dr Kleinschmidt (Heidelberg) for the gift of the AAV helper plasmid pDG, and Dr Sendtner (Wurzburg) for the gift of the CNTF plasmid. Our thanks also to Erich Ehlert, William Hendricks and Simone Niclou (Netherlands Institute for Neuroscience) for technical support and assistance with plasmid cloning and viral production.

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Leaver, S., Cui, Q., Plant, G. et al. AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells. Gene Ther 13, 1328–1341 (2006). https://doi.org/10.1038/sj.gt.3302791

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