Alexander B. Quiambao

Efficient Non-Viral Ocular Gene Transfer with Compacted DNA Nanoparticles

Background The 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 Findings Here 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. Conclusions As 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.

Retention of function without normal disc morphogenesis occurs in cone but not rod photoreceptors

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.

The Cys214→Ser mutation in peripherin/rds causes a loss-of-function phenotype in transgenic mice

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.

Ocular Delivery of Compacted DNA-Nanoparticles Does Not Elicit Toxicity in the Mouse Retina

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.

Native cone photoreceptor cyclic nucleotide-gated channel is a heterotetrameric complex comprising both CNGA3 and CNGB3 : a study using the cone-dominant retina of Nrl-/-mice

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.

Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

ABSTRACT 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.

Molecular Pathogenesis of Achromatopsia Associated with Mutations in the Cone Cyclic Nucleotide-Gated Channel CNGA3 Subunit

Cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are linked to achromatopsia and progressive cone dystrophy in humans. Over 50 mutations have been identified in the CNGA3 subunit. The R277C and R283W substitutions are among the most frequently occurring mutations. This study investigated the defects of these two mutations using a heterologous expression system. The wild type and mutant CNGA3 were expressed in HEK293 cells, the channels expression and cellular localization were examined by immunoblotting and immunofluorecences labeling, and activity of the channel was evaluated by ratiometric [Ca(2+)](i) measurements and by electrophysiological recordings. By using this model system we observed dysfunction of the mutant channels. Co-expression of the mutant channel with the wild type subunit did not affect the wild type channels activity. Immunoflurescence labeling showed apparent cytosol aggregation of the immunoreactivity in cells expressing the mutants. Thus these disease-causing mutations appear to induce loss of function by impairing the channel cellular trafficking and plasma membrane targeting. Therapeutic supplementation of the wild type transgene may help correct the visual disorders caused by these two mutations.

Loss of bipolar cells resulting from the expression of bcl-2 directed by the IRBP promoter

We have recently noted marked reductions in the electroretinographic (ERG) b-wave in HIBA transgenic mice expressing bcl-2 under control of the human IRBP promoter. These electrophysiological results are unexpected as this promoter has been shown to specifically target transgene expression to the rod and cone photoreceptors. Here, we have carried out a series of studies to better understand this result. ERGs were recorded from three lines of HIBA transgenic mice. Mice with higher levels of transgene expression developed progressive photoreceptor degeneration, and an associated reduction in the ERG a-wave. These higher-expressing lines also exhibited a severe reduction in the ERG b-wave that affected both rod- and cone-mediated responses. These mice were mated to L7 transgenic mice, which express beta-galactosidase in bipolar cells. In double transgenic mice, the ERG b-wave reduction was associated with a decrease in the number of bipolar cells in the inner retina. These results indicate that bcl-2, targeted to photoreceptors, can induce bipolar cell degeneration, and indicate that the potential benefit for bcl-2 in treating hereditary retinal disease appears limited.

Once-Daily Cyclosporine-A-MiDROPS for Treatment of Dry Eye Disease

Purpose To determine if a Microemulsion Drug Ocular Penetration System (MiDROPS) formulation of cyclosporine A (CsA) delivers more drug and is more efficacious for treatment of dry eye disease (DED) than the current clinical formulation. Methods Tissue distribution of CsA was quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). To assess tolerability, CsA-MiDROPS (0.1%) was applied to the eyes of rabbits twice per day for 14 days and assessed using ophthalmoscopic examinations. Mice were exposed to desiccating stress for 10 days and received daily topical instillation of the vehicle or test agent. Cornea staining was done to quantify corneal permeability. Histologic quantification of goblet cell (GC) density and CD4+ T-cell infiltration in the conjunctiva was performed. Results Ophthalmic distribution studies indicate significantly increased drug concentration with CsA-MiDROPS compared with Restasis. CsA-MiDROPS is well tolerated with little toxicity in a 2-week tolerability study. In the DED model, both 0.05% and 0.1% CsA-MiDROPS conferred a significant effect and were more effective than Restasis for treating experimental DED when dosed twice per day. As compared with Restasis dosed twice per day, 0.1% CsA-MiDROPS dosed once per day demonstrated superiority. Conclusions CsA-MiDROPS showed superior drug delivery and efficacy compared with other clinical formulations. As this product is simple to produce and needs to be only applied once daily, the clinical development of CsA-MiDROPS will help to reduce societal and patient burdens by lowering drug costs and accelerating/improving the activity of CsA. Translational Relevance MiDROPS has broad application concerning the ophthalmic development of lipophilic small molecule therapeutics.

Inhibition of Stat3 by a Small Molecule Inhibitor Slows Vision Loss in a Rat Model of Diabetic Retinopathy

Purpose Diabetic retinopathy is a leading cause of vision loss. Previous studies have shown signaling pathways mediated by Stat3 (signal transducer and activator of transcription 3) play a primary role in diabetic retinopathy progression. This study tested CLT-005, a small molecule inhibitor of Stat3, for its dose-dependent therapeutic effects on vision loss in a rat model of diabetic retinopathy. Methods Brown Norway rats were administered streptozotocin (STZ) to induce diabetes. CLT-005 was administered daily by oral gavage for 16 weeks at concentrations of 125, 250, or 500 mg/kg, respectively, beginning 4 days post streptozotocin administration. Systemic and ocular drug concentration was quantified with mass spectrometry. Visual function was monitored at 2-week intervals from 6 to 16 weeks using optokinetic tracking to measure visual acuity and contrast sensitivity. The presence and severity of cataracts was visually monitored and correlated to visual acuity. The transcription and translation of multiple angiogenic factors and inflammatory cytokines were measured by real-time polymerase chain reaction and Multiplex immunoassay. Results Streptozotocin-diabetic rats sustain progressive vision loss over 16 weeks, and this loss in visual function is rescued in a dose-dependent manner by CLT-005. This positive therapeutic effect correlates to the positive effects of CLT-005 on vascular leakage and the presence of inflammatory cytokines in the retina. Conclusions The present study indicates that Stat3 inhibition has strong therapeutic potential for the treatment of vision loss in diabetic retinopathy.