Cathryn Broderick

Control of myeloid activity during retinal inflammation

Combating myeloid cell‐mediated destruction of the retina during inflammation or neurodegeneration is dependent on the integrity of homeostatic mechanisms within the tissue that may suppress T cell activation and their subsequent cytokine responses, modulate infiltrating macrophage activation, and facilitate healthy tissue repair. Success is dependent on response of the resident myeloid‐cell populations [microglia (MG)] to activation signals, commonly cytokines, and the control of infiltrating macrophage activation during inflammation, both of which appear highly programmed in normal and inflamed retina. The evidence that tissue CD200 constitutively provides down‐regulatory signals to myeloid‐derived cells via cognate CD200‐CD200 receptor (R) interaction supports inherent tissue control of myeloid cell activation. In the retina, there is extensive neuronal and endothelial expression of CD200. Retinal MG in CD200 knockout mice display normal morphology but unlike the wild‐type mice, are present in increased numbers and express nitric oxide synthase 2, a macrophage activation marker, inferring that loss of CD200 or absent CD200R ligation results in “classical” activation of myeloid cells. Thus, when mice lack CD200, they show increased susceptibility to and accelerated onset of tissue‐specific autoimmunity.

Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.

Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice.

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Lentiviral-vector-mediated expression of murine IL-1 receptor antagonist or IL-10 reduces the severity of endotoxin-induced uveitis

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of interleukin 1 receptor antagonist (IL-1ra) or interleukin (IL)-10 to ameliorate murine endotoxin-induced uveitis (EIU). An human immunodeficiency virus-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12u2009h following disease induction. Lenti.IL-1ra or Lenti.IL-10-treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral-mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.