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

Gene Ther. 2006 Sep;13(18):1328-41. doi: 10.1038/sj.gt.3302791. Epub 2006 May 18.

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 betaIII-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 betaIII-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Axotomy
  • Cell Survival
  • Ciliary Neurotrophic Factor / analysis
  • Ciliary Neurotrophic Factor / genetics*
  • Ciliary Neurotrophic Factor / metabolism
  • Dependovirus / genetics*
  • Female
  • Gene Expression
  • Genetic Therapy / methods*
  • Genetic Vectors / administration & dosage*
  • Genetic Vectors / genetics
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Immunohistochemistry
  • Injections
  • Nerve Regeneration
  • Optic Nerve Injuries / metabolism
  • Optic Nerve Injuries / pathology
  • Optic Nerve Injuries / therapy*
  • Rats
  • Rats, Wistar
  • Retinal Ganglion Cells / metabolism
  • Retinal Ganglion Cells / pathology
  • Retinal Ganglion Cells / virology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transduction, Genetic / methods*
  • Vitreous Body

Substances

  • Ciliary Neurotrophic Factor
  • Green Fluorescent Proteins