The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover blindness therapies. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatiydylcholine containing very long chain polyunsaturated fatty acids and severely-attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation, and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.

Inhibition of vascular endothelial growth factor, a key contributor to the choroidal neovascularization associated with wet age-related macular degeneration, is the mode of action of several approved therapies, including aflibercept, which requires frequent intravitreal injections to provide clinical benefit. Lack of compliance with the dosing schedule may result in recurrence of active wet macular degeneration, leading to irreversible vision impairment. Gene therapy providing sustained anti-vascular endothelial growth factor levels in the retina following a single injection could drastically reduce the treatment burden and improve visual outcomes. ADVM-022, an adeno-associated virus vector encoding aflibercept, is optimized for intravitreal delivery and strong protein expression. Here, we report the long-term expression and efficacy of ADVM-022-derived aflibercept in a laser-induced choroidal neovascularization model in non-human primates. Intravitreal administration of ADVM-022 was well tolerated and resulted in sustained aflibercept levels. In addition, ADVM-022 administration 13 months before lasering prevented the occurrence of clinically relevant choroidal neovascularization lesions, similar to animals that received a bolus of intravitreal aflibercept (standard of care) at the time of lesioning. These results demonstrate that a single intravitreal administration of ADVM-022 may provide a safe and effective long-term treatment option for wet macular degeneration and may ultimately improve patients’ visual outcomes.

Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as an ␣ 2 ␤ 2 ␥ 2 hexameric complex with the ␣-and ␤-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the ␣-and ␤-subunits, whereas the ␥-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the ␣/␤ gene or the ␥ gene, we show that disruption of the ␣/␤ gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the ␥ gene results in only a partial loss of phosphorylation. These findings demonstrate that the ␣/␤-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the ␥-subunit.

Abstract. UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an a 2 b 2 c 2 hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. In humans, mutations in the gene encoding the a/b subunit precursor give rise to mucolipidosis II (MLII), whereas mutations in the gene encoding the c subunit cause the less severe mucolipidosis IIIC (MLIIIC). In this study we describe the phenotypic, histologic, and serum lysosomal enzyme abnormalities in knockout mice lacking the c subunit and compare these findings to those of mice lacking the a/b subunits and humans with MLII and MLIIIC. We found that both lines of mutant mice had elevated levels of serum lysosomal enzymes and cytoplasmic alterations in secretory cells of several exocrine glands; however, lesions in c-subunit deficient (Gnptg 2/2 ) mice were milder and more restricted in distribution than in a/b-subunit deficient (Gnptab 2/2 ) mice. We found that onset, extent, and severity of lesions that developed in these two different knockouts correlated with measured lysosomal enzyme activity; with a more rapid, widespread, and severe storage disease phenotype developing in Gnptab 2/2 mice. In contrast to mice deficient in the a/b subunits, the mice lacking the c subunits were of normal size, lacked cartilage defects, and did not develop retinal degeneration. The milder disease in the c-subunit deficient mice correlated with residual synthesis of the mannose 6-phosphate recognition marker. Of significance, neither strain of mutant mice developed cytoplasmic vacuolar inclusions in fibrocytes or mesenchymal cells (I-cells), the characteristic lesion associated with the prominent skeletal and connective tissue abnormalities in humans with MLII and MLIII. Instead, the predominant lesions in both lines of mice were found in the secretory epithelial cells of several exocrine glands, including the pancreas, and the parotid, submandibular salivary, nasal, lacrimal, bulbourethral, and gastric glands. The absence of retinal and chondrocyte lesions in Gnptg 2/2 mice might be attributed to residual b-glucuronidase activity. We conclude that mice lacking either a/b or c subunits displayed clinical and pathologic features that differed substantially from those reported in humans having mutations in orthologous genes.

Several standard-of-care therapies for the treatment of retinal disease, including aflibercept, inhibit vascular endothelial growth factor (VEGFA). The main shortcoming of these therapies is potential undertreatment due to a lack of compliance resulting from the need for repeated injections. Gene therapy may provide sustained levels of anti-VEGFA proteins in the retina following a single injection. In this nonhuman primate study, we explored whether ADVM-022, a recombinant adeno-associated virus (AAV) vector designed to express aflibercept, could induce anti-VEGFA protein levels comparable with those observed following a single-bolus intravitreal (IVT) injection of the standard-of-care aflibercept recombinant protein. The results demonstrated that intraocular levels of aflibercept measured at 56 days after a single IVT injection of ADVM-022 were equivalent to those in the aflibercept recombinant protein-injected animals measured 21–32 days post-administration. ADVM-022-injected animals exhibited signs of an initial self-limiting inflammatory response, but overall all doses were well tolerated. ADVM-022 administration did not result in systemic exposure to aflibercept at any dose evaluated. These results demonstrated that a single IVT injection of ADVM-022 resulted in safe and efficacious aflibercept levels in the therapeutic range, suggesting the potential of a gene therapy approach for long-term treatment of retinal disease with anti-VEGF therapy.

The GCR1 gene product is required for maximal transcription of many yeast genes including genes encoding glycolytic enzymes. Transcription of the yeast enolase gene EN02 is reduced 50-fold in strains carrying a gcrl null mutation. cis-acting sequences that are sufficient for GCRI-dependent regulation ofEN02 expression were identified by using an enhancerless CYC1 promoter which is not normally dependent on GCR1 for expression. A 60-bp EN02 sequence that was sufficient to provide high-level, GCRI-dependent transcriptional activation of the CYCi promoter was identified. This 60-bp element could be subdivided into a 30-bp sequence containing a novel RAPl-binding site and a GCR1-binding site which did not activate CYCi transcription and a 30-bp sequence containing a novel enhancer element that conferred moderate levels of GCRI-independent transcriptional activation. (12,24,30,36,39,45,53,55,58). RAPl-binding sites were mapped within UAS elements of the glycolytic genes PGK1 (13), PYKI (43), PDC1 (11), TPI1 (49), EN01 (7), EN02 (7); also see below), and TDH3 (5 mRNA are reduced 20-to 50-fold in strains carrying a gcrl null mutation, suggesting that GCR1 modulates transcription of these genes. Several genes whose expression depends on the GCR1 gene product contain a nucleotide sequence motif (CTICC), often located adjacent to a RAPl-binding site (5, 10,11,13,48,49). It was shown for the EN01, PYK1, PGK1, and TDH3 genes that the CTTCC sequence is required for maximal RAPI-dependent UAS element activity (5, 10, 13) (the CTTCC sequence is also required for full RAPl-dependent activation of the RNR2 gene; however, the GCR1 dependence of this gene remains to be determined [36]). The GCR1 protein has recently been shown to bind specifically to C1TTlCC and CATCC sequences (4, 35) within the 5' regulatory region of several yeast glycolytic genes, suggesting that the GCR1 protein may function with RAP1 to activate glycolytic gene expression.Transcription of the yeast enolase genes EN01 and EN02 is regulated by multiple UAS elements (7,17,18). We showed previously that deletions within the regulatory regions of the EN01 and EN02 genes rendered expression of these genes independent of GCRI (reference 31 and unpublished results). This suggests that GCR1-dependent activation of these genes is not direct but, rather, that the GCR1 gene product must function to overcome an inhibitor of transcription. This also suggests that regulation by GCR1 involves at least two cis-acting elements, one positive and one negative.Here we report the identification and characterization of EN02 sequences that are required for GCR1-dependent activation of transcription. We show that GCR1-dependent activation of transcription involves interactions among multiple cis-acting sites and trans-acting factors. The role of the GCR1 gene product in the activation mechanism is discussed. MATERIALS AND METHODSStrains, growth conditions, and yeast transformation. Saccharomyces cerevisiae S173-6B (a leu2-3 leu2-112 his3-1 trpl-289 ura3-52) (41) was used as the wil...

These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.