Pig Photoreceptors Research Articles

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in mouse and pig photoreceptors

Inherited retinal dystrophies caused by dominant mutations in photoreceptor cell expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor cell expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor cell expressed genes with 1-2 intravitreal injections per year.

28. Mouse and Pig Photoreceptor Transduction Mediated by Triple AAV Vectors

Purpose: The majority of inherited blinding diseases are caused by mutations in genes expressed specifically in photoreceptors (PRs). However the size of several of these genes exceeds the DNA cargo capacity of AAV vectors, to date the safest and most effective gene therapy vectors in the retina. We have previously demonstrated that dual AAV vectors expand AAV PR transfer capacity to about 9 kb. Our aim is to further expand this to around 13.5 kb using a triple AAV vector system. Methods: To assess the percentage of PR transduction mediated by three independent vectors, we initially injected subretinally in 4 week-old C57/BL6 mice single AAV8 vectors encoding either EGFP, dsRed or beta-gal reporter proteins. Then, in order to evaluate the PR transduction efficiency of triple AAV vectors we generated a reporter EGFP-dsRed fusion protein under the control of either the ubiquitous CMV or the PR-specific IRBP(interphotoreceptor retinoid-binding protein) promoters. The corresponding EGFP-dsRed expression cassettes were either included in single or triple AAV8 vectors (Fig1Fig1) which were administered by subretinal injection to C57/BL6 mice or large white pigs. Direct fluorescence, Western blot, and ELISA analysis were used to evaluate transgene expression. Results: In mice injected with three independent AAV8 vectors encoding for the different EGFP, dsRed and beta-gal reporter proteins, the percentage of PR co-transduced was 12%. In mouse and pig eyes injected with triple and single AAV8 vectors encoding EGFP-dsRed, full length protein expression was confirmed by Western Blot analysis independently of the CMV or IRBP promoters used; importantly, 75% of mouse and 65% of pig eyes injected with triple IRBP vectors showed bands of the expected size. Quantification in mice of EGFP-dsRed protein expression from triple AAV8 vectors mediated by either the CMV or the IRBP promoter shows 30% and 1%, respectively, of the levels observed with a single AAV8. Interestingly, in pigs, the levels of EGFP-dsRed protein expression from triple AAV8-IRBP vectors were 40% ratio of those observed with a single AAV8. Conclusions: Our results show that triple AAV vectors, which increase AAV transfer capacity up to 13.5 kb, transduce mouse and pig PR at levels that are 1% and 40% respectively of those obtained with single AAV vectors. This bode well for further testing this platform in animal models of inherited retinal degenerations.View Large Image | Download PowerPoint Slide

Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Effective delivery of large genes to the retina by dual AAV vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

FGF19 Exhibits Neuroprotective Effects on Adult Mammalian Photoreceptors In Vitro

Several fibroblast growth factors (FGFs) exhibit neuroprotective influences against retinal photoreceptor degeneration. The expression of FGF receptor (FGFR) 4 on photoreceptors suggests a specific ligand, FGF-19, might also be beneficial. The authors hence examined the potential role of FGF-19 in this regard. Adult human retinal sections were processed for anti-FGFR-4 immunohistochemistry. Total RNA and proteins were extracted from parallel cultures of human Y79 retinoblastoma and primary adult pig photoreceptors; RNA samples were used for RT-PCR analysis of FGF-19, and proteins were subjected to immunoprecipitation for FGFR-1 and FGFR-4 or to Western blotting of FGF-19. Cultures were incubated with increasing concentrations of FGF-19 before extraction and Western blotting for phosphotyrosine. Photoreceptor cultures were screened for cell survival and processed for immunocytochemistry using anti-neural retina leucine zipper (Nrl) antibody. FGF-19 mRNA was detected in adult pig retinal pigment epithelial cells, and FGF-19 protein was found in cell extracts and conditioned medium prepared from retinal pigment epithelium. The addition of FGF-19 to Y79 retinoblastoma or primary adult pig photoreceptor cultures led to time- and dose-dependent changes in proliferation (for Y79) or survival (for primary photoreceptors). FGF-19 induced the phosphorylation of an FGFR-4-immunoreactive band of approximately 80 kDa and led to the heterodimerization of FGFR-1 and FGFR-4. Y79 and primary photoreceptor cells maintained in serum-supplemented media exhibited Nrl immunoreactivity by Western blotting, which decreased after serum deprivation. The addition of FGF-19 led to the reexpression of Nrl immunoreactivity in both culture models. These data indicate a physiological role for FGF-19 in adult photoreceptor phenotypic maintenance and survival and argue in favor of its use as a neuroprotectant.

Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.

To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

A comparison of the size of photoreceptor cells from the retina of the domestic pig as determined by light and scanning electron microscopy

While gathering data on the visual pigments of numerous species, many light micrographs have been taken of the photoreceptor cells containing the visual pigment after spectral recordings have been made from these cells. These micrographs are used to view the cells and obtain measurements of their size. Usually the morphology of the photoreceptor cells is retained adequately so that such measurements can be taken. Occasionally it is necessary to partially fix the retinal tissue which aids in the maintenance of photoreceptor morphology while allowing visual pigment spectral recordings to be made. We have found that primate retinal tissue, as well as some other mammalian tissue, disintegrates rapidly. Although partial fixation allows spectral recordings to be made before the micrographs are taken, the treatment does not always adequately preserve cell morphology for quality micrographs to be obtained. In these cases, visual pigment recordings are made from pieces of unfixed and partially fixed retinal tissue; an additional piece of the same retinal material is well-fixed, embedded and thick-sectioned for light microscopy. Preparing retinal material for sectioning is lengthy and time consuming so an alternative tissue preparation technique was sought. Material can be processed for the scanning electron microscope (SEM) more rapidly than for sectioning, however severe tissue shrinkage occurs during this process. It was found that although shrinkage does occur in the retinal tissue prepared for SEM, the relative proportions of the photoreceptor cells are maintained extremely well. Using the critical point drying method (CPD) pig retinal tissue was prepared for SEM. Scanning electron micrographs of the pig photoreceptors were taken for cell measurement. Since these micrographs could be made at higher magnification than is available by light microscopy, a more detailed view of the pig photoreceptor cells was obtained. Cell measurements made from the light and the scanning electron micrographs indicate that an approximate shrinkage of 50% occurs in the SEM prepared material.

Localization of ATPases in retinal receptor cells.

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K- NPPase ) activity of the Na-K-ATPase complex and the Ca-ATPase activity were studied ultracytochemically in the inner and outer segments of guinea pig photoreceptor cells by means of newly developed methods. The K- NPPase activity was demonstrated on the plasmalemma of the inner segments, whereas the Ca-ATPase activity was restricted to the matrix side of the inner membrane of the mitochondria which were accumulated in the inner segments. In contrast to the enzyme activities in the inner segments no such activity could be detected on the plasma and disk membranes of the outer segments. The functional meaning of the enzyme activities is discussed.

Aspartate aminotransferase-like immunoreactivity as a marker for aspartate/glutamate in guinea pig photoreceptors.

Aspartate aminotransferase-like immunoreactivity as a marker for aspartate/glutamate in guinea pig photoreceptors

ULTRACYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT <i>P</i>-NITROPHENYLPHOSPHATASE ACTIVITY IN THE GUINEA PIG RETINA

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase activity was studied cytochemically in photoreceptor cells of the normal adult guinea pig retina with a newly developed one step lead citrate method (Mayahara et al., 1980). In guinea pig photoreceptor cells fixed for 5min in a mixture of 2% paraformaldehyde and 0.25% glutalaldehyde, electron dense reaction products were observed on plasma membranes of the inner segments and more proximal portion, but not on membranes of the outer segments. The most intensive reaction products were evident in the pre-synaptic membranes in the outer plexiform layer. It is also noteworthy that this activity was positive on the apical villi of the Muller cells, which were spread out distal to the outer limiting membrane into interstices between the inner segments. This reaction was almost completely abolished by 10mM ouabain or elimination of K. This activity was also substrate-dependent and completely inhibited by PCMBS or preheating. In contrast to K-dependent p-nitrophenylphosphatase activity, Mg-ATPase and non-specific alkaline phosphatase activity were not observed on every part of the plasma membranes of the photoreceptor cells.

The structure and distribution of the cross-striated fibril and associated membranes in guinea pig photoreceptors.

Examination of longitudinally and transversely sectioned photoreceptor cells of the guinea pig retina revealed an aggregate of thin filaments forming a single cross-striated fibril coursing through the full length of the non-receptor portion of the cell. The fibril begins as the ciliary rootlet from the region of the basal body of the connecting cilium. From the basal body it passes between the mitochondria of the ellipsoid and along the Golgi zone of the myoid region of the inner segment, narrowing from an irregularly shaped bundle to a ribbon-shaped aggregate. The fibril separates into discrete strands, each curving along the nucleus, reuniting into a single bundle to pass down the cell's axon, and terminating deep within the synaptic terminal. The fibril is flanked by two separate membranous saccules, each continuous along nearly its full length. The fibril's extensive course in the guinea pig and its association with continuous membranes necessitates a reexamination of earlier proposals for the function of cross-striated filamentous structures in photoreceptor cells.