Gene therapy with constitutively active focal adhesion kinase (FAK) promotes retinal ganglion cells survival and regeneration after optic nerve crush

Abstract

Purpose : Vision loss in traumatic and ischemic neuropathies and glaucoma is attributed to retinal ganglion cells (RGCs) death and inability to regenerate after injury. We recently demonstrated that laminin-integrin-FAK signaling is disrupted in RGCs in optic neuropathy, and FAK is a key regulator of RGC survival and neurite growth. The purpose of this study is to evaluate the neuroprotective and regenerative effect of gene therapy with constitutively active FAK in an optic nerve crush model.

Methods : We generated AAV2 containing a constitutively active FAK with an HA epitope tag (AAV2-^Y397EFAK-HAtag; AAV2-FAKE). AAV2-FAKE, or AAV2-GFP (control) were intravitreally injected unilaterally in 6 weeks old C57BL/6 mice, followed 2 weeks later by optic nerve crush (ONC). FAKE expression in the retina was determined 2 weeks after gene transfer by immunohistochemistry with a HA tag antibody. Retinas and optic nerves were harvested 4 weeks post-ONC. 2 days prior to the end of the experiment Cholera toxin B (CTB) was injected in the injured eyes to anterogradely label regenerating axons. RGC survival was assessed by quantification of RGCs after immunofluorescence with β-III tubulin antibody in retinal flat-mounts. Axon regeneration was evaluated by quantification of CTB labeled axons in optic nerve sections.

Results : Immunohistochemistry with HA tag antibodies demonstrated that 75% of RGCs in the retina of AAV-FAKE injected mice specifically express the FAKE transgene. FAKE gene transfer significantly increased RGC survival in the injured eyes, administration of AAV2-FAKE protected 70% RGCs (n=10; p<0.05) in the retina 4 weeks post-ONC, compared with the control AAV2-GFP group (n=10). In control animals, a few axons sprouted short distances at the crush site (n=5), whereas many axons extended into and beyond the lesion in AAV-FAKE injected mice (n=5) 4 weeks post-injury.

Conclusions : FAKE was neuroprotective after ONC, preventing loss of RGCs and axons. Potential use of gene therapy with activated FAK might be relevant to treatment of optic neuropathies.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.