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

Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy

We recently reported that the Ins2Akita mouse is a good model for late-onset diabetic retinopathy. Here, we investigated the effect of miR200-b, a potential anti-angiogenic factor, on VEGF receptor 2 (VEGFR-2) expression and to determine the underlying angiogenic response in mouse endothelial cells, and in retinas from aged Ins2Akita mice. MiR200-b and its native flanking sequences were amplified and cloned into a pCAG-eGFP vector directed by the ubiquitous CAG promoter (namely pCAG-miR200-b-IRES-eGFP). The plasmid was compacted by CK30PEG10K into DNA nanoparticles (NPs) for in vivo delivery. Murine endothelial cell line, SVEC4-10, was first transfected with the plasmid. The mRNA levels of VEGF and VEGFR-2 were quantified by qRT-PCR and showed significant reduction in message expression compared with lipofectamine-transfected cells. Transfection of miR200-b suppressed the migration of SVEC4-10 cells. There was a significant inverse correlation between the level of expression of miR200-b and VEGFR-2. Intravitreal injection of miR200-b DNA NPs significantly reduced protein levels of VEGFR-2 as revealed by western blot and markedly suppressed angiogenesis as evaluated by fundus imaging in aged Ins2Akita mice even after 3 months of post-injection. These findings suggest that NP-mediated miR200-b delivery has negatively regulated VEGFR-2 expression in vivo

Defective phototransductive disk membrane morphogenesis in transgenic mice expressing opsin with a mutated N-terminal domain

Retinitis pigmentosa is a heterogeneous group of inherited retinal disorders in which the photoreceptor cells degenerate. A line of transgenic mice expresses a mutant opsin gene that encodes three missense mutations near the amino terminus, including P23H, which is the basis for a common form of dominant retinitis pigmentosa. By studying the photoreceptor cells of these mice and their normal littermates, we found that: (1) opsin was routed correctly, (2) the concentration of opsin in the disk membranes appeared normal by freeze fracture analysis, (3) the amount of disk membrane shedding was normal, but (4) the basal disks of the outer segments were disorganized, indicating defective disk membrane morphogenesis. Defective disk membrane morphogenesis appears to result in the formation of fewer mature disks, thus accounting for observed gradual shortening of the photoreceptor outer segments with age. We suggest that abnormal disk membrane morphogenesis is the primary cellular defect that leads to blindness, and that it arises from the inability of nascent disk membranes, containing normal and mutant opsin, to interact normally with each other

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

Defects in the Outer Limiting Membrane Are Associated with Rosette Development in the Nrl−/− Retina

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl−/− retina, rods are converted into functional cone-like cells. The Nrl−/− retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl−/− retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl−/− retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl−/− retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM

Differential Developmental Deficits in Retinal Function in the Absence of either Protein Tyrosine Sulfotransferase-1 or -2

To investigate the role(s) of protein-tyrosine sulfation in the retina and to determine the differential role(s) of tyrosylprotein sulfotransferases (TPST) 1 and 2 in vision, retinal function and structure were examined in mice lacking TPST-1 or TPST-2. Despite the normal histologic retinal appearance in both Tpst1−/− and Tpst2−/− mice, retinal function was compromised during early development. However, Tpst1−/− retinas became electrophysiologically normal by postnatal day 90 while Tpst2−/− mice did not functionally normalize with age. Ultrastructurally, the absence of TPST-1 or TPST-2 caused minor reductions in neuronal plexus. These results demonstrate the functional importance of protein-tyrosine sulfation for proper development of the retina and suggest that the different phenotypes resulting from elimination of either TPST-1 or -2 may reflect differential expression patterns or levels of the enzymes. Furthermore, single knock-out mice of either TPST-1 or -2 did not phenocopy mice with double-knockout of both TPSTs, suggesting that the functions of the TPSTs are at least partially redundant, which points to the functional importance of these enzymes in the retina

A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds+/− mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme (ANR-11-IDEX-0004-0), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

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