RESUMEN
Young mammals possess a limited regenerative capacity in some tissues, which is lost upon maturation. We investigated whether cellular senescence might play a role in such loss during liver regeneration. We found that following partial hepatectomy, the senescence-associated genes p21, p16Ink4a, and p19Arf become dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response. However, we show that treatment with a senescence-inhibiting drug improves regeneration, by disrupting aberrantly prolonged p21 expression. This work suggests that senescence may initially develop from heterogeneous cellular responses, and that senotherapeutic drugs might be useful in promoting organ regeneration.
Asunto(s)
Compuestos de Bifenilo/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/fisiología , Nitrofenoles/farmacología , Regeneración/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Piperazinas/farmacologíaRESUMEN
Cell therapy approaches hold great potential for treating retinopathies, which are currently incurable. This study addresses the problem of inadequate migration and integration of transplanted cells into the host retina. To this end, we have identified the chemokines that were most upregulated during retinal degeneration and that could chemoattract mesenchymal stem cells (MSCs). The results were observed using a pharmacological model of ganglion/amacrine cell degeneration and a genetic model of retinitis pigmentosa, from both mice and human retinae. Remarkably, MSCs overexpressing Ccr5 and Cxcr6, which are receptors bound by a subset of the identified chemokines, displayed improved migration after transplantation in the degenerating retina. They also led to enhanced rescue of cell death and to preservation of electrophysiological function. Overall, we show that chemokines released from the degenerating retinae can drive migration of transplanted stem cells, and that overexpression of chemokine receptors can improve cell therapy-based regenerative approaches.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Receptores CCR5/genética , Receptores CXCR6/genética , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Animales , Biomarcadores , Movimiento Celular , Susceptibilidad a Enfermedades , Expresión Génica , Humanos , Ratones , Receptores CCR5/metabolismo , Receptores CXCR6/metabolismo , Degeneración Retiniana/patologíaRESUMEN
Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Factores de Transcripción , Animales , Ratones , Blastocisto/metabolismo , Blastocisto/citología , Nucléolo Celular/metabolismo , Desarrollo Embrionario/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Uniones Estrechas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Wnt/metabolismo , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Eritropoyetina/farmacología , Luz/efectos adversos , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/terapia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Dependovirus , Eritropoyesis , Eritropoyetina/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Hematócrito , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Modelos Animales , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Periferinas , Ratas , Ratas Endogámicas Lew , Degeneración Retiniana/genéticaRESUMEN
Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4(-/-) mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.
Asunto(s)
Dependovirus , Técnicas de Transferencia de Gen , Vectores Genéticos , Retina , Serotipificación , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Dependovirus/genética , Dependovirus/metabolismo , Dineínas/genética , Dineínas/metabolismo , Electrorretinografía , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Retina/citología , Retina/metabolismoRESUMEN
Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.
Asunto(s)
Albinismo Oculocutáneo/metabolismo , Albinismo Oculocutáneo/terapia , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Melaninas/metabolismo , Monofenol Monooxigenasa/fisiología , Retina/metabolismo , Albinismo Oculocutáneo/patología , Albinismo Oculocutáneo/ultraestructura , Animales , Electrofisiología , Humanos , Iris/metabolismo , Iris/patología , Iris/ultraestructura , Melanocitos/metabolismo , Melanocitos/patología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Monofenol Monooxigenasa/genética , Retina/patología , Retina/ultraestructura , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
RESUMEN
Cells with high ploidy content are common in mammalian extraembryonic and adult tissues. Cell-to-cell fusion generates polyploid cells during mammalian development and tissue regeneration. However, whether increased ploidy can be occasionally tolerated in embryonic lineages still remains largely unknown. Here, we show that pluripotent, fusion-derived tetraploid cells, when injected in a recipient mouse blastocyst, can generate diploid cells upon ploidy reduction. The generated diploid cells form part of the adult tissues in mouse chimeras. Parental chromosomes in pluripotent tetraploid cells are segregated through tripolar mitosis both randomly and nonrandomly and without aneuploidy. Tetraploid-derived diploid cells show a differentiated phenotype. Overall, we discovered an unexpected process of controlled genome reduction in pluripotent tetraploid cells. This mechanism can ultimately generate diploid cells during mouse embryo development and should also be considered for cell fusion-mediated tissue regeneration approaches.
Asunto(s)
Embrión de Mamíferos/fisiología , Desarrollo Embrionario/fisiología , Animales , Blastocisto/fisiología , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Quimera/genética , Quimera/fisiología , Cromosomas/genética , Diploidia , Genoma/genética , Ratones , PloidiasRESUMEN
Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged in vitro culture conditions. Several factors control mESC pluripotency, including Wnt/ß-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/ß-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/ß-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Epigénesis Genética , Homeostasis , Células Madre Embrionarias de Ratones/metabolismo , Vía de Señalización Wnt , Animales , Línea Celular , Metilación de ADN , Ratones , Células Madre Embrionarias de Ratones/citologíaRESUMEN
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.
Asunto(s)
Células de la Médula Ósea/citología , Reprogramación Celular , Neuroglía/citología , Retina/citología , Células Amacrinas/citología , Células Amacrinas/efectos de los fármacos , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Desdiferenciación Celular/efectos de los fármacos , Fusión Celular , Proliferación Celular/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Ratones Transgénicos , N-Metilaspartato/toxicidad , Neuroglía/efectos de los fármacos , Receptores CXCR4/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Inmunosupresores/farmacología , Luz , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Degeneración Retiniana/prevención & control , Tacrolimus/análogos & derivados , Animales , Western Blotting , Dependovirus/genética , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Neoplasia Endocrina Múltiple Tipo 2a/genética , Fosforilación , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Plásmidos/genética , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Tacrolimus/farmacología , TransfecciónRESUMEN
Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration.
Asunto(s)
Reprogramación Celular , Células Ependimogliales/citología , Neuroglía/citología , Células Fotorreceptoras/citología , Retina/crecimiento & desarrollo , Células Madre/citología , Animales , Diferenciación Celular , Fusión Celular , Linaje de la Célula , Proliferación Celular , Electrorretinografía , Femenino , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Células Fotorreceptoras/patología , Retina/citología , Degeneración Retiniana/patología , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Parkinson's disease is a common neurodegenerative disorder, which is due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and for which no definitive cure is currently available. Cellular functions in mouse and human tissues can be restored after fusion of bone marrow (BM)-derived cells with a variety of somatic cells. Here, after transplantation of hematopoietic stem and progenitor cells (HSPCs) in the SNpc of two different mouse models of Parkinson's disease, we significantly ameliorated the dopaminergic neuron loss and function. We show fusion of transplanted HSPCs with neurons and with glial cells in the ventral midbrain of Parkinson's disease mice. Interestingly, the hybrids can undergo reprogramming in vivo and survived up to 4weeks after transplantation, while acquiring features of mature astroglia. These newly generated astroglia produced Wnt1 and were essential for functional rescue of the dopaminergic neurons. Our data suggest that glial-derived hybrids produced upon fusion of transplanted HSPCs in the SNpc can rescue the Parkinson's disease phenotype via a niche-mediated effect, and can be exploited as an efficient cell-therapy approach.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Recuento de Células , Fusión Celular , Supervivencia Celular , Reprogramación Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/patología , Células Híbridas , Masculino , Ratones , Neuroglía/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Sustancia Negra/metabolismo , Sustancia Negra/patología , Vía de Señalización WntRESUMEN
The Wnt/ß-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 ß-catenin activation, is essential for reprogramming after fusion of ESCs overexpressing Nanog. In addition, ß-catenin is necessary in Nanog-dependent conversion of preinduced pluripotent stem cells (pre-iPSCs) into iPSCs. The activation of ß-catenin by Nanog causes fluctuations of ß-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 ß-catenin fluctuations. Overall, we demonstrate Nanog and ß-catenin cooperation in establishing naive pluripotency during the reprogramming process and their correlated heterogeneity in ESCs primed toward differentiation.
Asunto(s)
Reprogramación Celular , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , beta Catenina/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factor Inhibidor de Leucemia/farmacología , Ratones , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/genéticaRESUMEN
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/ß-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/ß-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.
Asunto(s)
Neuronas/fisiología , Regeneración/fisiología , Retina/fisiología , Células Madre/fisiología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular/fisiología , Fusión Celular , Ratones , N-Metilaspartato , Neuronas/citología , Neuronas/metabolismo , Retina/citología , Retina/efectos de los fármacos , Retina/metabolismo , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Madre/citología , Células Madre/metabolismo , Transcriptoma , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1(-/-)) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1(-/-) photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1(-/-) retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1(-/-) photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1(-/-) retina is effective.
Asunto(s)
Terapia Genética/métodos , Miosinas/deficiencia , Retina/fisiopatología , Degeneración Retiniana/terapia , Adulto , Animales , Western Blotting , Dependovirus/genética , Modelos Animales de Enfermedad , Electrorretinografía , Ojo/metabolismo , Ojo/fisiopatología , Ojo/ultraestructura , Femenino , Vectores Genéticos/genética , Células HEK293 , Humanos , Masculino , Melanosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Microscopía Electrónica , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Retina/metabolismo , Retina/ultraestructura , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Síndromes de Usher/genética , Síndromes de Usher/fisiopatología , Síndromes de Usher/terapiaRESUMEN
PURPOSE: Mutations in the PDE6B gene cause recessive, severe retinitis pigmentosa (RP). PDE6B encodes the ß subunit of the rod-specific phosphodiesterase (ßPDE), which, when absent, results in toxic levels of intracellular Ca(2+) and photoreceptor cell death. Ca(2+) blockers, such as nilvadipine, as well as light restriction, slow photoreceptor degeneration in animal models of ßPDE deficiencies. The goal of the study was to evaluate the efficacy of AAV2/5- or AAV2/8-mediated gene replacement in combination with nilvadipine and/or with light restriction in the rd10 mouse bearing homozygous pde6b mutations. METHODS: AAV vectors encoding either ßPDE or EGFP were subretinally administered at postnatal day (P)2. Nilvadipine was administered from P7 to P28. For light restriction, pregnant rd10 mice were kept in a dark environment until their pups were 28 days old. All functional and histologic analyses were performed at P35. RESULTS: Significant morphologic photoreceptor protection was observed after subretinal administration of AAV vectors encoding EGFP. This protection further increased after administration of AAV2/8 or -2/5 encoding for ßPDE and was not associated with significant functional improvement. Photoreceptor protection was higher after AAV2/8- than after AAV2/5-mediated delivery and was not significantly augmented by additional drug therapy and/or light restriction. The protective effect was lost after P35. CONCLUSIONS: In conclusion, more efficient gene transfer tools than those used in this study, as well as a better understanding of the disease pathogenesis, should be explored to increase the effect of gene replacement and to design gene-based strategies that block the apoptotic pathways activated by ßPDE deficiency.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Terapia Genética/métodos , Nifedipino/análogos & derivados , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Animales , Células COS , Bloqueadores de los Canales de Calcio/farmacología , Chlorocebus aethiops , Terapia Combinada , Oscuridad , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/genética , Homocigoto , Inyecciones Intravítreas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Nifedipino/farmacología , Células Fotorreceptoras de Vertebrados/patología , Embarazo , Retinitis Pigmentosa/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Terapia Genética/métodos , Proteínas Represoras/metabolismo , Retinitis Pigmentosa/terapia , Rodopsina/biosíntesis , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oftalmoscopía , Proteínas Represoras/genética , Retinitis Pigmentosa/genética , Rodopsina/genéticaRESUMEN
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.
Asunto(s)
Silenciador del Gen , Mutación/genética , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Rodopsina/genética , Alelos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Dependovirus/genética , Vectores Genéticos/genética , Ratones , Modelos Animales , Datos de Secuencia Molecular , Prolina/genética , Prolina/metabolismo , ARN Interferente Pequeño/genética , Ratas , Degeneración Retiniana/metabolismo , Rodopsina/metabolismoRESUMEN
X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1(-/-)) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1(-/-) mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1(-/-) mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1(-/-) mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.