Constitutive and AP20187-induced Ret activation in photoreceptors does not protect from light-induced damage.

Purpose Delivery of glial cell-derived neurotrophic factor (GDNF), either as a recombinant protein or by retinal gene transfer results in photoreceptor (PR) neuroprotection in genetic models of retinitis pigmentosa (RP). The mechanism of GDNF action and its direct targets in the retina remain unknown. The goal of the present study was to test the neuroprotective effect of GDNF from light-induced damage, a commonly used stimulus of PR degeneration, and to determine whether protection occurs directly on PRs. Methods Adeno-associated viral vectors (AAV) were developed that expressed either GDNF or a constitutively (RetMen2A) or pharmacologically activated chimeric GDNF receptor (Fv2Ret). Fv2Ret homodimerization and activation are induced by the administration of the small dimerizer drug AP20187. AAV2/2 vectors and the cytomegalovirus (CMV) promoter were used to transduce GDNF in the retina, whereas RetMen2A and Fv2Ret were transduced by AAV2/5 vectors and their expression restricted to PRs by the rhodopsin promoter. In vivo GDNF levels were measured by ELISA, RetMen2A and Fv2Ret expression and activation in vitro and/or in vivo were assessed by Western blot and immunofluorescence analyses. ERG measurements and histologic analyses were performed to assess morphologic and functional rescue, respectively. Results GDNF gene transfer resulted in sustained protein expression in the eye. In addition, the results confirmed in vivo that PR-restricted activation of Ret signaling occurred after either AAV-mediated expression of RetMen2A or AP20187-dependent Fv2Ret activation. However, this or AAV-mediated GDNF retinal gene transfer did not result in functional or morphologic PR protection from light-induced damage. Conclusions The results suggest that the apoptotic pathways responsible for light-induced PR degeneration are not inhibited by GDNF. However, GDNF signaling was shown to be regulated in time and levels in the retina by the AP20187/Fv2Ret system which is therefore available to be tested as gene-based therapeutic strategy in models of PR degeneration responsive to GDNF

712. AAV-Mediated Allele-Specific RNA Interference of a Common Dominant Rhodopsin Mutation Causing Retinitis Pigmentosa

Inherited retinal degenerations are a group of clinically and genetically heterogeneous diseases characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Mutations in the rhodopsin gene are common causes of autosomal dominant retinitis pigmentosa (RP). Among them the P23H amino-acid substitution represents the most frequent rhodopsin mutation in US. Given the gain of function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable

Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo

Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations

Amelioration of both Functional and Morphological Abnormalities in the Retina of a Mouse Model of Ocular Albinism Following AAV-Mediated Gene Transfer

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Non-erythropoietic erythropoietin derivatives protect from light-induced and genetic photoreceptor degeneration

Given the high genetic heterogeneity of inherited retinal degenerations (IRDs), a wide applicable treatment would be desirable to halt/slow progressive photoreceptor (PR) cell loss in a mutation-independent manner. In addition to its erythropoietic activity, erythropoietin (EPO) presents neurotrophic characteristics. We have previously shown that adeno-associated viral (AAV) vector-mediated systemic EPO delivery protects from PR degeneration. However, this is associated with an undesired hematocrit increase that could contribute to PR protection. Non-erythropoietic EPO derivatives (EPO-D) are available which allow us to dissect erythropoiesis's role in PR preservation and may be more versatile and safe than EPO as anti-apoptotic agents. We delivered in animal models of light-induced or genetic retinal degeneration either intramuscularly or subretinally AAV vectors encoding EPO or one of the three selected EPO-D: the mutant S100E, the helix A- and B-derived EPO-mimetic peptides. We observed that (i) systemic expression of S100E induces a significantly lower hematocrit increase than EPO and provides similar protection from PR degeneration, and (ii) intraocular expression of EPO-D protects PR from degeneration in the absence of significant hematocrit increase. On the basis of this, we conclude that erythropoiesis is not required for EPO-mediated PR protection. However, the lower efficacy observed when EPO or S100E is expressed intraocularly rather than systemically suggests that hormone systemic effects contribute to PR protection. Unlike S100E, EPO-mimetic peptides preserve PR only when given locally, suggesting that different EPO-D have a different potency or mode of action. In conclusion, our data show that subretinal delivery of AAV vectors encoding EPO-D protects from light-induced and genetic PR degeneration

Wnt/β-Catenin Signaling Triggers Neuron Reprogramming and Regeneration in the Mouse Retina

Cell-fusion-mediated somatic-cell reprogramming can be induced in culture; however, whether this process occurs in mammalian tissues remains enigmatic. Here, we show that upon activation of Wnt/β-catenin signaling, mouse retinal neurons can be transiently reprogrammed in vivo back to a precursor stage. This occurs after their spontaneous fusion with transplanted hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that retinal damage is essential for cell-hybrid formation in vivo. Newly formed hybrids can proliferate, commit to differentiation toward a neuroectodermal lineage, and finally develop into terminally differentiated neurons. This results in partial regeneration of the damaged retinal tissue, with functional rescue. Following retinal damage and induction of Wnt/β-catenin signaling, cell-fusion-mediated reprogramming also occurs after endogenous recruitment of bone-marrow-derived cells in the eyes. Our data demonstrate that in vivo reprogramming of terminally differentiated retinal neurons after their fusion with HSPCs is a potential mechanism for tissue regeneration

β-catenin fluctuates in mouse ESCs and is essential for Nanog-mediated/nreprogramming of somatic cells to pluripotency

The Wnt/β-catenin pathway and Nanog are key regulators of embryonic stem cell (ESC) pluripotency and the reprogramming of somatic cells. Here, we demonstrate that the repression of Dkk1 by Nanog, which leads indirectly to β-catenin activation, is essential for reprogramming after fusion of ESCs overexpressing Nanog. In addition, β-catenin is necessary in Nanog-dependent conversion of preinduced pluripotent stem cells (pre-iPSCs) into iPSCs. The activation of β-catenin by Nanog causes fluctuations of β-catenin in ESCs cultured in serum plus leukemia inhibitory factor (serum+LIF) medium, in which protein levels of key pluripotency factors are heterogeneous. In 2i+LIF medium, which favors propagation of ESCs in a ground state of pluripotency with many pluripotency genes losing mosaic expression, we show Nanog-independent β-catenin fluctuations. Overall, we demonstrate Nanog and β-catenin cooperation in establishing naive pluripotency during the reprogramming process and their correlated heterogeneity in ESCs primed toward differentiation.This work was funded by an ERC grant (242630-RERE) (M.P.C.), an HFSP grant (M.P.C.), the Ministerio de Ciencia e Inovación SAF2011-28580 (M.P.C.), Fundacio La Marató de TV3 (M.P.C.), AXA Research Fund (M.P.C.), an EMBO Long-Term Fellowship (L.M.), and Wolfson Foundation and BBSRC Alert 13 capital grant (BB/L014181/1) (Bristol University

Endogenous mobilization of bone-marrow cells into the murine retina induces fusion-mediated reprogramming of müller glia cells

Müller glial cells (MGCs) represent the most plastic cell type found in the retina. Following injury, zebrafish and avian MGCs can efficiently re-enter the cell cycle, proliferate and generate new functional neurons. The regenerative potential of mammalian MGCs, however, is very limited. Here, we showed that N-methyl-d-aspartate (NMDA) damage stimulates murine MGCs to re-enter the cell cycle and de-differentiate back to a progenitor-like stage. These events are dependent on the recruitment of endogenous bone marrow cells (BMCs), which, in turn, is regulated by the stromal cell-derived factor 1 (SDF1)-C-X-C motif chemokine receptor type 4 (CXCR4) pathway. BMCs mobilized into the damaged retina can fuse with resident MGCs, and the resulting hybrids undergo reprogramming followed by re-differentiation into cells expressing markers of ganglion and amacrine neurons. Our findings constitute an important proof-of-principle that mammalian MGCs retain their regenerative potential, and that such potential can be activated via cell fusion with recruited BMCs. In this perspective, our study could contribute to the development of therapeutic strategies based on the enhancement of mammalian endogenous repair capabilities