Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP + cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Mü ller cells. Mü ller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.

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We have examined whether transplanted freeze-thawed peripheral nerve (PN) sheaths repopulated ex vivo with purified adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Cultured adult SCs were derived from donor rats or from the host animals themselves. We also transplanted PN sheaths filled with neonatal SCs or donor adult olfactory ensheathing glia (OEG). 100,000 cells were injected into 1.5-cm lengths of freeze-thawed PN. After 2 days in culture, repopulated PN segments were grafted onto the transected optic nerve of adult Fischer rats. Three weeks later, 6% fluorogold (FG) was applied to distal PN. Retrogradely labeled RGCs were counted in retinal wholemounts and PN grafts were processed for immunohistochemistry. As expected, there was no RGC axon regeneration in cell-free grafts. Regrowth was also absent in neonatal SC- and adult OEG-filled grafts, which contained only small numbers of surviving donor cells. Many cells were, however, seen in adult SC repopulated PN grafts, intermingled with pan-neurofilament(+) and GAP-43(+) fibers. SCs were aligned along the grafts and were S-100(+), p75(+). Ultrastructurally, SCs were associated with myelinated and unmyelinated axons. Hundreds of FG-labeled RGCs were seen in retinas of rats with congeneic or allogeneic PN sheaths repopulated with either donor or autologous (host-derived) adult SCs. Intraocular CNTF injections significantly increased the number of regenerating RGCs in donor and autologous adult SC groups. The use of chimeric grafts to bridge CNS tissue defects could provide a clinical alternative to using multiple PN autografts, the harvesting of which would exacerbate peripheral dysfunction in already injured patients.

Recombinant adeno-associated viral (rAAV) vectors expressing neurotrophic genes reduce neuronal death and promote axonal regeneration in central nervous system (CNS) injury models. Currently, however, use of rAAV to treat clinical neurotrauma is problematic because there is a delay in the onset of transgene expression. Using the adult rat retina and optic nerve (ON), we have tested whether rAAV gene therapy administered at the time of injury combined with short-term pharmacotherapy has synergistic effects that enhance neuronal survival and regeneration. The ON was transected and a 1.5 cm segment of autologous peripheral nerve (PN) was grafted onto the cut end. At this time, bicistronic rAAV2 encoding ciliary neurotrophic factor (CNTF) and green fluorescent protein (rAAV2-CNTF-GFP) was injected into the injured eye. To provide interim support for axotomized retinal ganglion cells (RGCs) during vector integration and therapeutic transgene expression, rCNTF protein and a cyclic adenosine monophosphate (cAMP) analogue (CPT-cAMP) were injected intravitreally 3 and 10 days postoperatively. For comparison, another rAAV2-CNTF-GFP group received two intravitreal saline injections 3 and 10 days after the PN-ON surgery. A further PN graft group received only postoperative intravitreal injections of rCNTF plus CPT-cAMP. After 4 weeks, regenerating RGCs were retrogradely labelled by applying fluorogold to the distal end of each PN graft. Compared to saline-injected animals, both RGC survival and axonal regrowth were significantly higher in the rCNTF and CPT-cAMP injected rAAV2-CNTF-GFP group; approximately one third of the RGC population survived axotomy, and 27% of these regrew an axon. These values were also higher than those obtained in rats that received only rCNTF plus CPT-cAMP injections. Therefore, we show for the first time that rAAV-mediated gene delivery at the time of, or just after, neurotrauma is most successful when combined with temporary post-injury trophic support, and is potentially a viable treatment strategy for patients after acute CNS injury.