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PubMed1991

Induction of immunoreactive proliferating cell nuclear antigen (PCNA) in goldfish retina following intravitreal injection with tunicamycin.

Effects of a photoreceptor-specific biotoxin, tunicamycin (TM), injected intravitreally into the goldfish eye at one side, were explored on electroretinograms (ERGs) and proliferating cell nuclear antigen-immunoreactive (PCNA-ir) nuclei, representing the mitotic activity of rod precursors, in the retina at both sides. The eye-cup preparations were made for ERG recording, and the retinas were isolated and processed as cryosections or wholemounts by a routine immunohistochemical method for visinin (cones), opsin (rods), tyrosine hydroxylase (dopaminergic cells) and proliferating cell nuclear antigen (PCNA), at various intervals after intravitreal injection with TM (1.0 micrograms/eye). On some thin sections, autoradiographic study was combined following intravitreal injection with [3H]thymidine (TdR, 0.1 microCi/eye). The dose of TM used heavily destroyed cones and rods only in the treated retinas 2-15 days after injection, the photoreceptors being renewed for further 15-20 days. Approximately in parallel, ERGs were largely impaired 2-10 days after TM injection and recovered for 10-20 days. However, intravitreal TM altered the distribution and density of PCNA-ir nuclei in both treated and untreated retinas. The density of PCNA-ir nuclei reduced at first (on days 1 and 2), and then clustered and rapidly increased on days 3-5 and maintained at high levels with diffuse distribution over the whole area, particularly in the treated retinas, up to 60 days after TM injection; the maximum peak of 3.7 and 20 times the initial level was seen on day 20 in the outer nuclear layer (ONL) and inner nuclear layer (INL), respectively. PCNA-ir nuclei were found to be abundant in the ONL even after the photoreceptors and ERGs had been restored in the treated retinas on day 20, suggesting a kind of overproduction of retinal cells. The autoradiographic study provided comparable results to those obtained with PCNA immunohistochemistry. The mechanism by which damage to the treated retina causes rod precursor cells to proliferate in the untreated retina remains unresolved.

Animals↗
PubMed2004

Expression of photoreceptor-specific nuclear receptor NR2E3 in rod photoreceptors of fetal human retina.

PURPOSE: To study the physiological function of NR2E3 and possible molecular mechanisms underlying enhanced short-wavelength cone syndrome (ESCS) pathogenesis in developing human retina, and to compare its expression to that of Neural Retina Leucine zipper (NRL), a transcription factor essential for rod differentiation. METHODS: Expression of NR2E3, a photoreceptor-specific orphan nuclear receptor, was examined in human retinas between fetal weeks (Fwk) 9 to 22 by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Both NR2E3 and NRL expression patterns were followed by immunocytochemistry. The human retina develops in a central to peripheral pattern, in which a protein may take weeks to be expressed throughout the entire retina. This allowed a detailed temporal analysis of NR2E3 and NRL expression. RESULTS: NR2E3 expression was detected shortly after the appearance of NRL in putative immature rods on the foveal edge at Fwk 11.7. Expression of both markers was maintained in rod opsin expressing fetal photoreceptors. NR2E3 expression was not detected in either long/medium- or short-wavelength cones. Its absence from cones was also supported by the position of labeled nuclei deep in the outer nuclear layer, and by the absence of NR2E3 from the fovea. CONCLUSIONS: A role for NR2E3 in the rod developmental pathway is suggested. The closely related expression patterns of NRL and NR2E3 supported an interactive function, where both transcription factors determine the rod fate and suppress immature rods from adopting the S-cone fate.

Basic-Leucine Zipper Transcription Factors↗
PubMed2002

HEK293S cells have functional retinoid processing machinery.

Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins. After rhodopsin bleaching in cells loaded with 11-cis-retinal, ERC signals recover in minutes and recurrently over a period of hours by simple dark adaptation, with no added chromophore. The purpose of this study is to investigate the source of ERC signal recovery in these cells. Giant HEK293S cells expressing normal wild-type (WT)-human rod opsin (HEK293S) were regenerated by solubilized 11-cis-retinal, all-trans-retinal, or Vitamin A in darkness. ERCs were elicited by flash photolysis and measured by whole-cell recording. Visible flashes initially elicit bimodal (R(1), R(2)) ERC signals in WT-HEK293S cells loaded with 11-cis-retinal for 40 min or overnight. In contrast, cells regenerated for 40 min with all-trans-retinal or Vitamin A had negative ERCs (R(1)-like) or none at all. After these were placed in the dark overnight, ERCs with outward R(2) signals were recorded the following day. This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells. These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.

Aniline Compounds↗
PubMed1998

Transgene expression in Xenopus rods.

The photoreceptors of the vertebrate retina express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod- and cone-specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219-226). The mechanisms that regulate gene expression in a rod/cone-specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99-126, Wiley-Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173-3183) can be used to study cell-specific expression in the retina. We have used a 5.5 kb 5' upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179-3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina and pineal, which is apparent by 4 days post-nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.

Animals↗
PubMed2002

Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31.

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology.

Alternative Splicing↗
PubMed2005

Ribozyme knockdown of the gamma-subunit of rod cGMP phosphodiesterase alters the ERG and retinal morphology in wild-type mice.

PURPOSE: To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the gamma-subunit of the rod cGMP phosphodiesterase (PDEgamma) mRNA in the retina of wild-type mice. METHODS: Two hammerhead ribozymes, HRz35 and HRz42, were designed to target the PDEgamma gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDEgamma gene were monitored by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblot assay. RESULTS: The ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K(cat) of 0.48 minute(-1) and a K(m) of 980 nM, and HRz42 showed a K(cat) of 0.17 minute(-1) and a K(m) of 971 nM. Both ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and full-length mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a- and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDEgamma mRNA and protein paralleled the ERG results. CONCLUSIONS: Ribozyme-mediated somatic knockdown of wild-type PDEgamma mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Degeneration of photoreceptors in rhodopsin mutants of Drosophila.

Five different, well-characterized mutants of the R1-6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1-6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1-6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1-6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1-6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1-6 rhodopsin in the ninaE mutants.

Aging↗
PubMed1998

Erx, a novel retina-specific homeodomain transcription factor, can interact with Ret 1/PCEI sites.

Our previous studies on the transcriptional regulation of rod opsin gene expression had defined a strikingly conserved element, Ret 1/PCEI, present in the upstream regulatory regions of opsin and other photoreceptor-specific genes. This element interacts with a 40 kDa, developmentally regulated, retina-specific protein. In this study we report the cloning of the novel retina-specific homeodomain protein Erx. Erx contains a homeodomain that is 79% homologous to that of Drosophila empty spiracles. This 40 kDa protein can interact with the Ret 1 element in electrophoretic mobility shift assays. Mutation of key residues in Ret 1 eliminates all Erx binding. Transient transfection of Y79 retinobalstoma cells with Erx leads to significant transcriptional activation of a reporter gene via Ret 1 elements. We conclude that Erx is the Ret 1 binding activity. This is the first example of a Q50 homeodomain protein expressed in retinal photoreceptors.

Amino Acid Sequence↗
PubMed1999

An immunohistochemical study of an autosomal dominant feline rod/cone dysplasia (Rdy cats).

An autosomal dominant, early onset feline model of rod/cone dysplasia has been described. The clinical features, light and electron microscopy, and the electrophysiology were documented. We have now examined in more detail the histopathological and immunohistochemical changes during the early phase of the disease using antibodies against opsin, synaptophysin, glial fibrillary acidic protein (GFAP) and an epithelial marker (MNF118). We have also demonstrated programmed cell death by a modified TUNEL (Terminal deoxynucleotidyl transferase, Uridine triphosphate, Nick End Labelling) technique. In the Rdy cats, there was significant photoreceptor degeneration between 5 and 17 weeks of age. The TUNEL-labeled cell and pyknotic cell counts in the outer nuclear layer peaked at around 9 weeks of age. Accumulation of opsin in the entire outer nuclear layer of the retina was noted with opsin-immunolabeled rod neurite sprouting. There was a reduction in synaptophysin immunoreactivity in the outer plexiform layer. The Muller cells were activated and expressed GFAP. No significant change of immunolabeling of MNF118 was found. These findings closely parallel those seen in human RP.

Animals↗
PubMed1997

Molecular evolution of the cottoid fish endemic to Lake Baikal deduced from nuclear DNA evidence.

Lake Baikal in Eastern Siberia contains a remarkable flock of 29 species of teleost fishes of the suborder Cottoidei (sculpins, bullheads) that are endemic to the lake and its associated rivers and occupy all depth habitats down to over 1500 m. The species are divided into three families, the Cottidae with 7 species, the Abyssocottidae with 20 species, and the Comephoridae with 2 species. Nucleotide sequences of the rod opsin gene from 12 of these species, plus a non-Baikal marine species, have been used to examine the evolutionary relations and the divergence time of the flock. Phylogenetic trees, generated by neighbor-joining and maximum parsimony, indicate that the unique Comephoridae family with its viviparity and unusual appearance is closely related to the Cottidae and Abyssocottidae, whereas the genus Cottocomephorus, at present placed in the Cottidae, was the first to diverge from the ancestral species and forms a separate lineage. The major adaptation to deep water would appear to be of relatively recent origin, and there is evidence that the ancestral species occupied a shallow-water-marine or brackish habitat. Estimates of antiquity obtained from synonymous substitutions place the origin of the species flock at around 4.9 million years ago.

Animals↗
PubMed1992

An immunohistochemical study of regenerating newt retinas.

Light-microscopical examination was carried out to investigate the emergence and development of several classes of immunoreactive cells in regenerating retinas of the adult newt (Triturus pyrrhogaster) after total retinal ablation. Immunoreactive proliferating cell nuclear antigen (ir-PCNA, a marker for replicating cells) was present in nuclei of all neuroblasts in the early mono-layered to several-layered stages (15-20 days after retinal ablation; days 15-20), but was lost progressively in an intermediate-to-central/peripheral order as cells and layers increased (days 20-25). Cells, which had lost ir-PCNA, began to separate to form the outer nuclear, inner nuclear and ganglion cell layers around days 25-30 (the cell separation stage). Finally, the location of ir-PCNA was restricted to a band of neuroblast cells at the retinal margin (days 30-35) as seen in intact adult retinas. Visinin-immunoreactive (ir) cells, mainly destined to be cones, appeared first singly or as clusters at the most distal layer in the intermediate region of retinas multi-layered with PCNA-ir neuroblasts, which was followed by appearance of opsin-ir rod outer segments and tyrosine hydroxylase-ir amacrine cells around the cell separation stage. Shortly later, cells respectively immunoreactive to glutamic acid decarboxylase, neuropeptide Y, serotonin, glucagon, glutamine synthetase, glial fibrillary acidic protein, substance P and protein kinase C were found to emerge also in an intermediate-to-central/peripheral sequence. Some of the glucagon-ir cells appeared to be of an interplexiform type.

Animals↗
PubMed2004

Adult human retinal pigment epithelial cells capable of differentiating into neurons.

We investigated the ability of adult human RPE cells to differentiate into neurons. Two cell lines were evaluated. The cells were cultured in medium with 8% serum, transferred to a neural stem cell maintenance culture, and induced to differentiate with retinoic acid. The cells were immunocytochemically examined at each step. The cells showed epithelial-like morphology with 8% serum and all were immunoreactive for beta-III tubulin. After transfer to the stem cell maintenance culture, they changed morphologically and became immunoreactive for MAP5 and neurofilament200 after inducement with retinoic acid. The ratio of MAP5 positive cells was higher in the young adult RPE cell line. No GFAP or rod-opsin immunoreactive cells were observed. Adult human RPE cells even from old person are capable of differentiating into neurons, although the ratio of mature neurons was greater in the young than in the old cell line in this condition.

Adult↗
PubMed2005

Treatment of experimental autoimmune uveoretinitis with poly(lactic acid) nanoparticles encapsulating betamethasone phosphate.

We have developed nanoparticles (NPs), which are capable of targeting a specific lesion and gradually releasing the agent at the site over a prolonged time period after a single intravenous administration. In this study, we evaluated the effects of intravenously administered poly(lactic acid) nanoparticles encapsulating betamethasone phosphate (BP-PLA NPs) on experimental autoimmune uveoretinitis (EAU) in Lewis rats. To determine the localization of NPs within the retina and choroid of rats with EAU, rhodamine (Rh)-encapsulated PLA NPs were injected intravenously and visualized by confocal microscopy. After the disease onset of EAU induced by S-antigen peptide in Lewis rats, either BP-PLA NPs, BP, or saline was injected intravenously, and the eyes were obtained 7 days following treatment and the histological score was determined. The clinical course of EAU was examined using pathological findings and the expression of the glial fibrillary acidic protein, rod opsin, and the surface markers of inflammatory cells (ED1 and pan T-cell) were immunohistochemically determined. Furthermore, T-cell proliferation and delayed-type hypersensitivity (DTH) to S-antigen were assessed. Intravenously injected Rh-PLA NPs accumulated in the retina and choroid of rats with EAU within 3 hr and remained over the succeeding 7-day-period. Furthermore, systemically administered BP-PLA NPs reduced the clinical scores of rats with EAU in 1 day, which were maintained for 2 weeks and decreased the histological scores. In addition, the ocular infiltration of activated T-cells and macrophages in addition to the hypertrophy of Müller cells were markedly reduced with this treatment. Meanwhile, T-cell proliferation and DTH of BP-PLA NPs-treated rats against S-antigen peptide were not significantly different from those of saline-treated rats. Systemically administered BP-PLA NPs inhibit the development of EAU due to the targeting and the sustained release of steroids in situ. The results of these studies suggest that the systemic administration of BP-PLA NPs may lead to a new therapeutic strategy in controlling intraocular inflammation.

Animals↗
PubMed2005

Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy.

Retinitis pigmentosa (RP) is a group of retinal degenerative diseases that are characterised primarily by the loss of rod photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal-dominant RP (ADRP). Here, we propose a new classification for rhodopsin mutations based on their biochemical and cellular properties. Several different potential gain-of-function mechanisms for rhodopsin ADRP are described and discussed. Possible dominant-negative mechanisms, which affect the processing, translocation or degradation of wild-type rhodopsin, are also considered. Understanding the molecular and cellular consequences of rod-opsin mutations and the underlying disease mechanisms in ADRP are essential to develop future therapies for this class of retinal dystrophies.

Amino Acid Sequence↗
PubMed2000

Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

Animals↗
PubMed1997

Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

Animals↗
PubMed2003

Cone photoreceptor recovery after experimental detachment and reattachment: an immunocytochemical, morphological, and electrophysiological study.

PURPOSE: To compare the morphologic and functional recovery of the retina after detachment and reattachment in an animal with a cone-dominant retina, the ground squirrel. METHODS: Ground squirrel (Spermophilus beecheyi) retinas were detached for 1 day and reattached for 7, 35, or 96 days (n = 2, each time point). Flicker ERGs were recorded 1 day after the detachment and at various times after reattachment. Contrast-response functions were measured for isochromatic modulation and for selective modulation of short-wavelength-sensitive (S) and middle-wavelength-sensitive (M) cones. At the end of the experiment, retinas were prepared for light microscopy or immunocytochemical staining with antibodies to rod opsin, S and M cone opsins, cytochrome oxidase, synaptophysin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), interphotoreceptor-binding protein (IRBP), and peanut agglutinin lectin (PNA). Photoreceptor density maps were created from wholemount preparations labeled with biotinylated PNA and anti-S cone opsin. Cell counts of photoreceptor nuclei and cone outer segments (OS) were compared with flicker ERG data. Cell death was examined by the TUNEL method. RESULTS: Reattachment stopped photoreceptor cell death and reversed the disruption of interphotoreceptor matrix as well as the redistribution of Müller cell proteins. It also activated some astrocytes based on anti-GFAP staining. S- and M-cone OS showed a gradual recovery in length after reattachment, and this recovery continued to the longest time points examined. ERG contrast gains also recovered after reattachment, but these reached asymptotic levels by approximately a week after reattachment. There were significant correlations between outer nuclear layer (ONL) cell counts and ERG contrast gains. No differences were noted in the indices of recovery of M and S cones. CONCLUSIONS: The ERG can be used to follow specifically the changes in the retina that occur after retinal detachment and reattachment.

Animals↗
PubMed2006

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.

Animals↗