BackgroundThe eye is an excellent candidate for gene therapy as it is immune privileged and much of the disease-causing genetics are well understood. Towards this goal, we evaluated the efficiency of compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissues. The compacted DNA nanoparticles examined here have been shown to be safe and effective in a human clinical trial, have no theoretical limitation on plasmid size, do not provoke immune responses, and can be highly concentrated.Methods and FindingsHere we show that these nanoparticles can be targeted to different tissues within the eye by varying the site of injection. Almost all cell types of the eye were capable of transfection by the nanoparticle and produced robust levels of gene expression that were dose-dependent. Most impressively, subretinal delivery of these nanoparticles transfected nearly all of the photoreceptor population and produced expression levels almost equal to that of rod opsin, the highest expressed gene in the retina.ConclusionsAs no deleterious effects on retinal function were observed, this treatment strategy appears to be clinically viable and provides a highly efficient non-viral technology to safely deliver and express nucleic acids in the retina and other ocular tissues.

It is commonly assumed that photoreceptor (PR) outer segment (OS) morphogenesis is reliant upon the presence of peripherin/rds, hereafter termed Rds. In this study, we demonstrate a differential requirement of Rds during rod and cone OS morphogenesis. In the absence of this PR-specific protein, rods do not form OSs and enter apoptosis, whereas cone PRs develop atypical OSs and are viable. Such OSs consist of dysmorphic membranous structures devoid of lamellae. These tubular OSs lack any stacked lamellae and have reduced phototransduction efficiency. The loss of Rds only appears to affect the shape of the OS, as the inner segment and connecting cilium remain intact. Furthermore, these structures fail to associate with the specialized extracellular matrix that surrounds cones, suggesting that Rds itself or normal OS formation is required for this interaction. This study provides novel insight into the distinct role of Rds in the OS development of rods and cones.

AbstracL We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (A1- Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198. To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOTI by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence.Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-W'mtersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen.

P/rds (peripherin/retinal degeneration slow) is a photoreceptor-specific membrane glycoprotein necessary for outer segment disc morphogenesis. Mutations in P/rds are associated with different blinding diseases. A C214S (Cys214-->Ser) missense mutation has been shown to be the cause for a late-onset form of ADRP (autosomal dominant retinitis pigmentosa) in humans. In the present study, we generated transgenic mice expressing P/rds with the C214S mutation and crossed them into rds mutant mice to elucidate the mechanism underlying the pathology of ADRP. Although an ample amount of transgene message was formed in C214S retinas from all transgenic lines, only a trace amount of the mutant protein was detected by Western blotting and immunoprecipitation. C214S mice on the wild-type or rds+/- backgrounds exhibited no signs of negative effects of the mutation on retinal structure or function, suggesting a loss-of-function phenotype. This phenotype is further supported by the absence of outer segment formation in the C214S mice on the rds-/- background. In contrast, expression of C214S protein in the inner retinal cells of transgenic mice or in COS cells resulted in the formation of a substantial amount of mutant protein, signifying a possible photoreceptor-specific regulation of P/rds. These results provide evidence that the loss-of-function phenotype seen in C214S transgenic mice shows a disease progression that correlates with ADRP patients carrying the same mutation, indicating that the C214S mutation on one allele of P/rds results in haploinsufficiency.

These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.

Subretinal delivery of polyethylene glycol-substituted lysine peptide (CK30PEG)-compacted DNA nanoparticles results in efficient gene expression in retinal cells. This work evaluates the ocular safety of compacted DNA nanoparticles. CK30PEG-compacted nanoparticles containing an EGFP expression plasmid were subretinally injected in adult mice (1 µl at 0.3, 1.0 and 3.0 µg/µl). Retinas were examined for signs of inflammation at 1, 2, 4 and 7 days post-injection. Neither infiltration of polymorphonuclear neutrophils or lymphocytes was detected in retinas. In addition, elevation of macrophage marker F4/80 or myeloid marker myeloperoxidase was not detected in the injected eyes. The chemokine KC mRNA increased 3–4 fold in eyes injected with either nanoparticles or saline at 1 day post-injection, but returned to control levels at 2 days post-injection. No elevation of KC protein was observed in these mice. The monocyte chemotactic protein-1, increased 3–4 fold at 1 day post-injection for both nanoparticle and saline injected eyes, but also returned to control levels at 2 days. No elevations of tumor necrosis factor alpha mRNA or protein were detected. These investigations show no signs of local inflammatory responses associated with subretinal injection of compacted DNA nanoparticles, indicating that the retina may be a suitable target for clinical nanoparticle-based interventions.

Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients. S erum retinol-binding protein 4 (RBP4) is the sole specific transport protein for vitamin A (retinol) in the blood (1-4).The eye is the organ most dependent on the RBP4-mediated delivery of retinol to maintain optimal function, as RBP4 loss of function in either mice or humans impairs retinal function, leading to visual impairment, while other organ systems remain intact (5-7). However, in the past decade numerous clinical studies have linked increased serum levels of RBP4 to disease, including obesity (8, 9), insulin resistance (8-13), type 2 diabetes (9, 13), and cardiovascular disease (hypertension, atherosclerosis, stroke) (14-18). This appears to be more than a spurious correlation, since recent studies have demonstrated that RBP4 is an adipokine (adipose-derived cytokine) with retinoid-independent, proinflammatory activity that contributes to the development of insulin resistance (13,(19)(20)(21). Moreover, a human single nucleotide polymorphism that increases RBP4 promoter activity confers a 2-fold increase for the risk of type 2 diabetes (22, 23).Several studies have provided mechanistic insights into RBP4-induced insulin resistance. Mice with genetic or pharmacologic elevation of RBP4 levels develop insulin resistance (13), whereas lowering of RBP4 levels improves insulin sensitivity in mice (13,24). RBP4 inhibits insulin signaling in adipocytes indirectly by activating proinflammatory cytokine production in macrophages through retinol-independent and Toll-like receptor 4 (TLR4)-and c-Jun N-terminal kinase (JNK)-dependent ...

Cone vision mediated by photoreceptor cyclic nucleotide‐gated (CNG) channel activation is essential for central and color vision and visual acuity. Mutations in genes encoding the cone CNG channel subunits, CNGA3 and CNGB3, have been linked to various forms of achromatopsia and progressive cone dystrophy in humans. This study investigates the biochemical components of native cone CNG channels, using the cone‐dominant retina in mice deficient in the transcription factor neural retina leucine zipper (Nrl). Abundant expression of CNGA3 and CNGB3 but no rod CNG channel expression was detected in Nrl−/− retina by western blotting and immunolabeling. Localization of cone CNG channel in both blue (S)‐ and red/green (M)‐cones was shown by double immunolabeling using antibodies against the channel subunits and against the S‐ and M‐opsins. Immunolabeling also showed co‐localization of CNGA3 and CNGB3 in the mouse retina. Co‐immunoprecipitation demonstrated the direct interaction between CNGA3 and CNGB3. Chemical cross‐linking readily generated products at sizes consistent with oligomers of the channel complexes ranging from dimeric to tetrameric complexes, in a concentration‐ and time‐dependent pattern. Thus this work provides the first biochemical evidence showing the inter‐subunit interaction between CNGA3 and CNGB3 and the presence of heterotetrameric complexes of the native cone CNG channel in retina. No association between CNGA3 and the cone Na+/Ca2+‐K+ exchanger (NCKX2) was shown by co‐immunoprecipitation and chemical cross‐linking. This may implicate a distinct modulatory mechanism for Ca2+ homeostasis in cones compared to rods.