Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors.

PURPOSE: To develop a gene therapy system that specifically targets transgene expression to S-cones of the mammalian retina, the authors coupled recombinant AAV-mediated delivery with the use of a human blue-opsin (HB) promoter to drive expression. METHODS: Two regions of the HB promoter sequence, HB569 and HB996, were amplified from human DNA, cloned into an AAV vector cassette upstream of the green fluorescent protein (GFP) gene, and packaged into AAV2 and AAV5 capsids. Eyes of postnatal day (P) 40 to P48 Sprague-Dawley rats were subretinally injected with 2 muL vector. Animals were humanely killed 2 to 3 weeks or 20 months after injection, and the pattern and persistence of GFP expression were analyzed in the treated retinas by immunohistochemistry, Western blotting, and RT-PCR. RESULTS: AAV5.HB.GFP vectors targeted photoreceptor transduction with an efficiency 20-fold higher than analogous serotype 2 vector. Both AAV5.HB.GFP vectors exhibited similar transduction efficiencies with patterns of GFP expression that did not vary depending on the size of the HB promoter used. Transgene expression was exclusively localized to photoreceptors of retinas treated with either vector. Furthermore, GFP expression was observed for at least 20 months. Dual GFP immunostaining with S- or M-opsin antibodies and GFP/PNA labeling revealed that cones coexpressing S-opsin/GFP or M-opsin/GFP constituted 37.5% +/- 8% and 13.5% +/- 3% of the GFP-positive photoreceptors, respectively, whereas rods constituted 49% +/- 5% of the GFP-positive photoreceptors. Because cones constitute approximately 1% of adult rat retinal photoreceptors, it was estimated that the relative transduction efficiency of AAV5.HB.GFP vectors was approximately 100:1 for cones versus rods. CONCLUSIONS: AAV5.HB.GFP vector injected into the subretinal space of Sprague-Dawley rats targeted gene expression to photoreceptor cells with an efficiency approximately 20-fold higher than that for AAV2.HB.GFP. Transgene expression regulated by the human blue cone-promoter persisted at least for 20 months. Cones coexpressing S-opsin and the GFP transgene appeared to prevail, confirming that in addition to having properties of the AAV serotype, the promoter choice is key to fine-tuning transgene delivery and expression in specific retinal cells. The system described here may be effective in a therapeutic setting in which strong S-cone transgene expression is required.

Identification of a novel FcgammaRIII receptor that is up-regulated in fetal wound healing.

The mid-gestation fetus is able to heal skin wounds rapidly and without scarring, an ability that is lost as development proceeds. The aim of this study was to identify novel genes involved in this process. We established an ex vivo wound model from embryonic rats and showed that over 72 hours, embryonic day 17 wounds reepithelialized and closed whereas day 19 wounds did not. To investigate the molecular basis of this phenomenon we analyzed changes in gene expression using differential display polymerase chain reaction. We characterized one transcript that was strongly up-regulated in the healing response of wounded, day 17 skin. It encodes a protein of 249 amino acids with striking similarity to the human low-affinity receptor for the Fc portion of IgG (FcgammaRIII), suggesting that it is a novel member of the FcgammaR family, which we named FcgammaRIII-X. A wound-healing timecourse shows that FcgammaRIII-X was up-regulated in healing, wounded day 17 skin but not in nonhealing, wounded day 19 skin and that its up-regulation was accelerated in skin with multiple wounds. We suggest that up-regulation of FcgammaRIII-X may contribute to scarless healing of fetal skin.

Does recombinant adeno-associated virus-vectored proximal region of mouse rhodopsin promoter support only rod-type specific expression in vivo?

PURPOSE: We have previously found that the -385 to +86 portion of the mouse rod opsin promoter (mOP500) can limit recombinant adeno-associated virus (rAAV)-mediated transgene expression to photoreceptor cells when delivered subretinally. However, the photoreceptor (PR) subtype-specificity of expression remains unclear. Here, we evaluated whether the presence of certain cis-elements in this proximal promoter, such as the rod-specific, neural retina leucine zipper protein (NRL) response element (NRE), can render it a driver of rod-specific expression. METHODS: Subretinal injections of a serotype 5 rAAV vector carrying the green fluorescent protein (GFP) cDNA, driven by mOP500, were administered to male Sprague-Dawley rats at postnatal day (P) 40-48. Two weeks to eight months later, the distribution of GFP-expressing cells in the retina was characterized by GFP-, cone-specific alpha-transducin-immuno-, and peanut agglutinin-lectin histochemistry and by morphological criteria. The same viral suspension was also injected sub-retinally into rhodopsin-knockout rho (-/-) mice either at P18 or P78, and retinas were analyzed by immunohistochemistry and PNA lectin histochemistry two weeks later. RESULTS: GFP reactivity was found exclusively in the outer nuclear layer (ONL) of rat retinas two weeks after treatment, with abundant reporter gene expression observed in both rods and cones. GFP-positive cones, defined by their typical morphology and the co-linearity of PNA-lectin labeling with GFP-immunoreactivity, were found in all regions of the transduced retinas. GFP-positive cones constituted up to 6% of the total GFP-positive photoreceptors. By eight months post-injection, a low level of GFP-reactivity was additionally observed in the inner nuclear layer (INL) and ganglion cell layer. Photoreceptor-specific GFP expression was also seen in the rho (-/-) mice at both ages tested. In pups injected at P18, costaining with PNA-lectin revealed that up to 15% of the GFP-positive photoreceptors were cones. Despite only a single row of photoreceptors remaining in these knockout mice by P90, numerous GFP-positive cones were still present. CONCLUSIONS: Subretinal delivery of rAAV5 harboring a reporter gene driven by mOP500 results in passenger gene expression in both rod and cones, indicating that this promoter is photoreceptor-specific but not rod-specific. The lack of photoreceptor subtype-specificity suggests that although cones do not express the NRL and NR2E3 trans-factors considered necessary for activation of mOP500, other general transcription factors in cones may compensate.

Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis.

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a protein responsible for isomerization of all-trans-retinaldehyde to its photoactive 11-cis-retinaldehyde and is essential for the visual cycle. RPE65 mutations can cause severe, early onset retinal diseases such as Leber congenital amaurosis (LCA). A naturally occurring rodent model of LCA with a recessive nonsense Rpe65 mutation, the rd12 mouse, displays a profoundly diminished rod electroretinogram (ERG), an absence of 11-cis-retinaldehyde and rhodopsin, an overaccumulation of retinyl esters in retinal pigmented epithelial (RPE) cells, and photoreceptor degeneration. rd12 mice were injected subretinally at postnatal day 14 with rAAV5-CBA-hRPE65 vector. RPE65 expression was found over large areas of RPE soon after treatment. This led to improved rhodopsin levels with ERG signals restored to near normal. Retinyl ester levels were maintained at near normal, and fundus and retinal morphology remained normal. All parameters of restored retinal health remained stable for at least 7 months. The Morris water maze behavioral test was modified to test rod function under very dim light; rd12 mice treated in one eye performed similar to normally sighted C57BL/6J mice, while untreated rd12 mice performed very poorly, demonstrating that gene therapy can restore normal vision-dependent behavior in a congenitally blind animal.

Adeno-associated virus-mediated expression of vascular endothelial growth factor peptides inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy.

Vascular endothelial growth factor (VEGF) has been demonstrated to be a key stimulator of retinal neovascularization (NV), the most common cause of severe and progressive vision loss. In this study, we used a mouse model of oxygen-induced retinopathy (OIR) to explore the potential of gene expression and secretion of short VEGF peptides as a treatment. Peptide-encoding fragments of exons 6 and 7 of the VEGF gene were cloned into a recombinant adeno-associated virus (rAAV) vector. Expression of each peptide in vector-injected eyes was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. Intravitreal injection of each rAAV vector inhibited retinal NV by 71-83% (p < 0.001) compared with contralateral control eyes in the OIR mouse. Injection and expression of these peptides did not seem to affect the normal appearance of the retina. The results demonstrated that exon 6- and 7-derived VEGF peptides effectively inhibited oxygen-induced retinal NV. Therefore, these VEGF peptides have potential in the treatment of angiogenesis-associated retinal diseases in humans.

Differential effect of wounding on actin and its associated proteins, paxillin and gelsolin, in fetal skin explants.

Skin from the embryonic day 17 rat retains the ability to epithelialize an excisional wound when isolated in serum-supplemented suspension culture. This ability is lost by embryonic day 19. We have investigated this effect of gestational age on fetal epithelial wound closure by correlating the involvement of filamentous actin (F-actin) and its associated proteins, paxillin and gelsolin, in the wound margins of embryonic day 17 and 19 rat skins, with the ability to close a full thickness excisional wound. Using fluorescent-phalloidin histochemistry and scanning confocal microscopy, actin polymerization was observed some five to six cells back from the margin of wounds in the embryonic day 17 skin as early as 3 h postwounding. As the wounds closed over the following 48-72 h, the actin further condensed around the epithelial margin before dispersing after wound closure. In contrast, no organization of actin was seen in the epithelial margin of wounds in skin from the embryonic day 19 embryos. Instead, actin filaments were observed surrounding the dermal wound margins. Chemical or mechanical disruption of the actin in wounded embryonic day 17 skins prevented epithelial closure, although wound repair was independent of cell division. In particular, incising the wound margin 24 h after wounding resulted in the "springing-open" of the embryonic day 17 wound but not the embryonic day 19 wound, reflecting the development of tension in the embryonic day 17 wound margin. Expression of paxillin mRNA was upregulated following wounding at embryonic day 17 but not at embryonic day 19. Paxillin was also observed to colocalize with actin in embryonic day 17 wounds, but not embryonic day 19 wounds, indicating a potential role for paxillin in epithelial repair of the fetal wound. In contrast, gelsolin mRNA was upregulated in embryonic day 19 fetal skin but not at embryonic day 17 and gelsolin protein was observed surrounding actin filaments at embryonic day 19 but not embryonic day 17. These results demonstrate a change in the mechanism of wound epithelialization at the same gestational age that fetal wounds change from scar-free to scar-forming wound repair.