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1.
Int J Mol Sci ; 25(16)2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39201561

RESUMO

Glaucoma, a leading cause of blindness, is a multifactorial condition that leads to progressive loss of retinal ganglion cells (RGCs) and vision. Therapeutic interventions based on reducing ocular hypertension are not always successful. Emerging features of glaucoma include mitochondrial dysfunction and oxidative stress. In the current study, NDI1-based gene therapy, which improves mitochondrial function and reduces reactive oxygen species, was delivered intraocularly via an adeno-associated viral vector (AAV). This AAV-NDI1 therapy protected RGCs from cell death in treated (1552.4 ± 994.0 RGCs/mm2) versus control eyes (1184.4 ± 978.4 RGCs/mm2, p < 0.05) in aged DBA/2J mice, a murine model of glaucoma. The photonegative responses (PhNRs) of RGCs were also improved in treated (6.4 ± 3.3 µV) versus control eyes (5.0 ± 3.1 µV, p < 0.05) in these mice. AAV-NDI1 also provided benefits in glaucomatous human lamina cribrosa (LC) cells by significantly increasing basal and maximal oxygen consumption rates and ATP production in these cells. Similarly, NDI1 therapy significantly protected H2O2-insulted primary porcine LC cells from oxidative stress. This study highlights the potential utility of NDI1 therapies and the benefits of improving mitochondrial function in the treatment of glaucoma.


Assuntos
Dependovirus , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos , Glaucoma , Estresse Oxidativo , Células Ganglionares da Retina , Animais , Dependovirus/genética , Glaucoma/terapia , Glaucoma/metabolismo , Glaucoma/patologia , Camundongos , Terapia Genética/métodos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Humanos , Vetores Genéticos/genética , Mitocôndrias/metabolismo , Camundongos Endogâmicos DBA , Espécies Reativas de Oxigênio/metabolismo , Suínos
2.
Int J Mol Sci ; 24(4)2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36835257

RESUMO

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85-90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.


Assuntos
Terapia Genética , Atrofia Geográfica , Proteínas de Saccharomyces cerevisiae , Idoso , Animais , Humanos , Camundongos , Complexo I de Transporte de Elétrons/metabolismo , Terapia Genética/métodos , Atrofia Geográfica/genética , Atrofia Geográfica/terapia , Mitocôndrias/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
3.
Int J Mol Sci ; 23(3)2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-35163535

RESUMO

The challenge of developing gene therapies for genetic forms of blindness is heightened by the heterogeneity of these conditions. However, mechanistic commonalities indicate key pathways that may be targeted in a gene-independent approach. Mitochondrial dysfunction and axon degeneration are common features of many neurodegenerative conditions including retinal degenerations. Here we explore the neuroprotective effect afforded by the absence of sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), a prodegenerative NADase, in a rotenone-induced mouse model of retinal ganglion cell loss and visual dysfunction. Sarm1 knockout mice retain visual function after rotenone insult, displaying preservation of photopic negative response following rotenone treatment in addition to significantly higher optokinetic response measurements than wild type mice following rotenone. Protection of spatial vision is sustained over time in both sexes and is accompanied by increased RGC survival and additionally preservation of axonal density in optic nerves of Sarm1-/- mice insulted with rotenone. Primary fibroblasts extracted from Sarm1-/- mice demonstrate an increased oxygen consumption rate relative to those from wild type mice, with significantly higher basal, maximal and spare respiratory capacity. Collectively, our data indicate that Sarm1 ablation increases mitochondrial bioenergetics and confers histological and functional protection in vivo in the mouse retina against mitochondrial dysfunction, a hallmark of many neurodegenerative conditions including a variety of ocular disorders.


Assuntos
Proteínas do Domínio Armadillo/genética , Proteínas do Citoesqueleto/genética , Fibroblastos/metabolismo , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/fisiologia , Rotenona/efeitos adversos , Animais , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Fibroblastos/citologia , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Consumo de Oxigênio , Cultura Primária de Células , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/genética
4.
Hum Mol Genet ; 26(R1): R2-R11, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28510639

RESUMO

While individually classed as rare diseases, hereditary retinal degenerations (IRDs) are the major cause of registered visual handicap in the developed world. Given their hereditary nature, some degree of intergenic heterogeneity was expected, with genes segregating in autosomal dominant, recessive, X-linked recessive, and more rarely in digenic or mitochondrial modes. Today, it is recognized that IRDs, as a group, represent one of the most genetically diverse of hereditary conditions - at least 260 genes having been implicated, with 70 genes identified in the most common IRD, retinitis pigmentosa (RP). However, targeted sequencing studies of exons from known IRD genes have resulted in the identification of candidate mutations in only approximately 60% of IRD cases. Given recent advances in the development of gene-based medicines, characterization of IRD patient cohorts for known IRD genes and elucidation of the molecular pathologies of disease in those remaining unresolved cases has become an endeavor of the highest priority. Here, we provide an outline of progress in this area.


Assuntos
Degeneração Retiniana/genética , Sequência Conservada , Éxons , Proteínas do Olho/genética , Humanos , Mutação , Linhagem , Distrofias Retinianas/genética , Retinose Pigmentar/genética , Análise de Sequência de DNA
5.
Adv Exp Med Biol ; 1185: 275-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884624

RESUMO

With 329 genes known to be involved in inherited retinal degenerations (IRDs), focus has shifted to generic targets for therapeutics, targets that could provide benefit irrespective of the underlying genetic condition. As one of the most energy-demanding tissues, the retina is acutely sensitive to dysfunction of its energy metabolism. Recent discoveries have shed light on the complex interconnectivity and interdependence of retinal cells on their choice metabolic pathways, highlighting a number of potential targets that could benefit cells in a mutation-independent manner. Some of the latest research on retinal metabolism and mitophagy in photoreceptors and retinal pigment epithelium is discussed, as is how these insights could potentially be used in the design of new therapies.


Assuntos
Metabolismo Energético , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/fisiologia , Degeneração Retiniana , Epitélio Pigmentado da Retina/fisiologia , Humanos , Mitofagia
6.
Vis Neurosci ; 31(4-5): 289-307, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24949856

RESUMO

Significant advances have been made over the last decade or two in the elucidation of the molecular pathogenesis of inherited ocular disorders. In particular, remarkable successes have been achieved in exploration of gene-based medicines for these conditions, both in preclinical and in clinical studies. Progress in the development of gene therapies targeted toward correcting the primary genetic defect or focused on modulating secondary effects associated with retinal pathologies are discussed in the review. Likewise, the recent utilization of genes encoding light-sensing molecules to provide new functions to residual retinal cells in the degenerating retina is discussed. While a great deal has been learned over the last two decades, the next decade should result in an increasing number of preclinical studies progressing to human clinical trial, an exciting prospect for patients, those active in research and development and bystanders alike.


Assuntos
Terapia Genética , Doenças Retinianas/genética , Doenças Retinianas/terapia , Animais , Humanos
7.
Pharmaceutics ; 15(2)2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36839646

RESUMO

AAV gene therapy for ocular disease has become a reality with the market authorisation of LuxturnaTM for RPE65-linked inherited retinal degenerations and many AAV gene therapies currently undergoing phase III clinical trials. Many ocular disorders have a mitochondrial involvement from primary mitochondrial disorders such as Leber hereditary optic neuropathy (LHON), predominantly due to mutations in genes encoding subunits of complex I, to Mendelian and multifactorial ocular conditions such as dominant optic atrophy, glaucoma and age-related macular degeneration. In this study, we have optimised the nuclear yeast gene, NADH-quinone oxidoreductase (NDI1), which encodes a single subunit complex I equivalent, creating a candidate gene therapy to improve mitochondrial function, independent of the genetic mutation driving disease. Optimisation of NDI1 (ophNdi1) substantially increased expression in vivo, protected RGCs and increased visual function, as assessed by optokinetic and photonegative response, in a rotenone-induced murine model. In addition, ophNdi1 increased cellular oxidative phosphorylation and ATP production and protected cells from rotenone insult to a significantly greater extent than wild type NDI1. Significantly, ophNdi1 treatment of complex I deficient patient-derived fibroblasts increased oxygen consumption and ATP production rates, demonstrating the potential of ophNdi1 as a candidate therapy for ocular disorders where mitochondrial deficits comprise an important feature.

8.
Mol Ther ; 19(4): 642-9, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21224835

RESUMO

For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3-4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.


Assuntos
Terapia Genética/métodos , Retinose Pigmentar/terapia , Animais , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase
9.
Mol Ther Methods Clin Dev ; 25: 236-249, 2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35474956

RESUMO

Recombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches; however, there is a need to further improve the efficacy, tropism, and safety of these vectors. In this study, using a CMV-EGFP expression cassette, we characterize the retinal utility of AAV-PHP.eB, a serotype recently developed by in vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in the high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50-70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate the pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina.

10.
J Neurochem ; 112(4): 991-1004, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20002519

RESUMO

Long-term memory is formed by alterations in glutamate-dependent excitatory synaptic transmission, which is in turn regulated by synaptosomal protein of 25 kDa (SNAP-25), a key component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex essential for exocytosis of neurotransmitter-filled synaptic vesicles. Both reduced and excessive SNAP-25 activity has been implicated in various disease states that involve cognitive dysfunctions such as attention deficit hyperactivity disorder, schizophrenia and Alzheimer's disease. Here, we over-express SNAP-25 in the adult rat dorsal hippocampus by infusion of a recombinant adeno-associated virus vector, to evaluate the consequence of late adolescent-adult dysfunction of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein in the absence of developmental disruption. We report a specific and significant increase in the levels of extracellular glutamate detectable by microdialysis and a reduction in paired-pulse facilitation in the hippocampus. In addition, SNAP-25 over-expression produced cognitive deficits, delaying acquisition of a spatial map in the water maze and impairing contextual fear conditioning, both tasks known to be dorsal hippocampal dependent. The high background transmission state and pre-synaptic dysfunction likely result in interference with requisite synapse selection during spatial and fear memory consolidation. Together these studies provide the first evidence that excess SNAP-25 activity, restricted to the adult period, is sufficient to mediate significant deficits in the memory formation process.


Assuntos
Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Transtornos da Memória , Plasticidade Neuronal/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Aprendizagem da Esquiva/fisiologia , Biofísica/métodos , Linhagem Celular Transformada , Condicionamento Clássico/fisiologia , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Comportamento Exploratório/fisiologia , Citometria de Fluxo/métodos , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Microdiálise/métodos , Inibição Neural/fisiologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/genética , Transdução Genética/métodos , Transfecção/métodos
11.
Hum Mol Genet ; 17(14): 2084-100, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18385099

RESUMO

Mutations within the inosine 5'-monophosphate dehydrogenase 1 (IMPDH1) gene cause the RP10 form of autosomal dominant retinitis pigmentosa (adRP), an early-onset retinopathy resulting in extensive visual handicap owing to progressive death of photoreceptors. Apart from the prevalence of RP10, estimated to account for 5-10% of cases of adRP in United States and Europe, two observations render this form of RP an attractive target for gene therapy. First, we show that while recombinant adeno-associated viral (AAV)-mediated expression of mutant human IMPDH1 protein in the mouse retina results in an aggressive retinopathy modelling the human counterpart, expression of a normal human IMPDH1 gene under similar conditions has no observable pathological effect on retinal function, indicating that over-expression of a therapeutic replacement gene may be relatively well tolerated. Secondly, complete absence of IMPDH1 protein in mice with a targeted disruption of the gene results in relatively mild retinal dysfunction, suggesting that significant therapeutic benefit may be derived even from the suppression-only component of an RNAi-based gene therapy. We show that AAV-mediated co-expression in the murine retina of a mutant human IMPDH1 gene together with short hairpin RNAs (shRNA) validated in vitro and in vivo, targeting both human and mouse IMPDH1, substantially suppresses the negative pathological effects of mutant IMPDH1, at a point where, in the absence of shRNA, expression of mutant protein in the RP10 model essentially ablates all photoreceptors in transfected areas of the retina. These data strongly suggest that an RNAi-mediated approach to therapy for RP10 holds considerable promise for human subjects.


Assuntos
Terapia Genética , IMP Desidrogenase/genética , RNA Interferente Pequeno/genética , Retinose Pigmentar/terapia , Animais , Sequência de Bases , Dependovirus/genética , Regulação para Baixo , Genes Dominantes , Vetores Genéticos/genética , Células HeLa , Humanos , IMP Desidrogenase/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Dados de Sequência Molecular , Mutação , Interferência de RNA , RNA Interferente Pequeno/química , Transdução Genética
12.
Mol Ther ; 17(4): 593-9, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19174761

RESUMO

Mutational heterogeneity represents one of the greatest barriers impeding the progress toward the clinic of gene therapies for many dominantly inherited disorders. A general strategy of gene suppression in conjunction with replacement has been proposed to overcome this mutational heterogeneity. In the current study, various aspects of this strategy are explored for a dominant form of the retinal degeneration, retinitis pigmentosa (RP), caused by mutations in the rhodopsin gene (RHO-adRP). While > 200 mutations have been identified in rhodopsin (RHO), in principle, suppression and replacement may be employed to provide a single mutation-independent therapeutic for this form of the disorder. In the study we demonstrate in a transgenic mouse simulating human RHO-adRP that RNA interference-based suppression, together with gene replacement utilizing the endogenous mouse gene as the replacement, provides significant benefit as evaluated by electroretinography (ERG). Moreover, this is mirrored histologically by preservation of photoreceptors. AAV-based vectors were utilized for in vivo delivery of the therapy to the target cell type, the photoreceptors. The results demonstrate that RNAi-based mutation-independent suppression and replacement can provide benefit for RHO-adRP and promote the therapeutic approach as potentially beneficial for other autosomal dominantly inherited disorders.


Assuntos
Dependovirus/genética , Genes Dominantes , Terapia Genética , Retina/fisiopatologia , Retinose Pigmentar/terapia , Animais , Citometria de Fluxo , Camundongos , Interferência de RNA , Retinose Pigmentar/fisiopatologia
13.
Front Neurosci ; 14: 656, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32655363

RESUMO

Mutations in tubby like protein 1 gene (TULP1) are causative of early-onset recessive inherited retinal degenerations (IRDs); similarly, the Tulp1-/- mouse is also characterized by a rapid IRD. Tulp1 mRNA and protein expression was analyzed in wild type mouse retinas and expression data sets (NCBI) during early postnatal development. Comparative histology was undertaken in Tulp1-/-, rhodopsin-/- (Rho-/-) and retinal degeneration slow-/- (Rds-/-) mouse retinas. Bioinformatic analysis of predicted TULP1 interactors and IRD genes was performed. Peak expression of Tulp1 in healthy mouse retinas was detected at p8; of note, TULP1 was detected in both the outer and inner retina. Bioinformatic analysis indicated Tulp1 expression in retinal progenitor, photoreceptor and non-photoreceptor cells. While common features of photoreceptor degeneration were detected in Tulp1-/-, Rho-/-, and Rds-/- retinas, other alterations in bipolar, amacrine and ganglion cells were specific to Tulp1-/- mice. Additionally, predicted TULP1 interactors differed in various retinal cell types and new functions for TULP1 were suggested. A pilot bioinformatic analysis indicated that in a similar fashion to Tulp1, many other IRD genes were expressed in both inner and outer retinal cells at p4-p7. Our data indicate that expression of Tulp1 extends to multiple retinal cell types; lack of TULP1 may lead to primary degeneration not only of photoreceptor but also non-photoreceptor cells. Predicted interactors suggest widespread retinal functions for TULP1. Early and widespread expression of TULP1 and some other IRD genes in both the inner and outer retina highlights potential hurdles in the development of treatments for these IRDs.

14.
Front Neurosci ; 14: 891, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32973439

RESUMO

With marketing approval of the first ocular gene therapy, and other gene therapies in clinical trial, treatments for inherited retinal degenerations (IRDs) have become a reality. Biallelic mutations in the tubby like protein 1 gene (TULP1) are causative of IRDs in humans; a mouse knock-out model (Tulp1-/-) is characterized by a similar disease phenotype. We developed a Tulp1 supplementation therapy for Tulp1-/- mice. Utilizing subretinal AAV2/5 delivery at postnatal day (p)2-3 and rhodopsin-kinase promoter (GRK1P) we targeted Tulp1 to photoreceptor cells exploring three doses, 2.2E9, 3.7E8, and 1.2E8 vgs. Tulp1 mRNA and TULP1 protein were assessed by RT-qPCR, western blot and immunocytochemistry, and visual function by electroretinography. Our results indicate that TULP1 was expressed in photoreceptors; achieved levels of Tulp1 mRNA and protein were similar to wild type levels at p20. However, the thickness of the outer nuclear layer (ONL) did not improve in treated Tulp1-/- mice. There was a small and transient electroretinography benefit in the treated retinas at 4 weeks of age (not observed by 6 weeks) when using 3.7E8 vg dose. Dark-adapted mixed rod and cone a- and b-wave amplitudes were 24.3 ± 13.5 µV and 52.2 ± 31.7 µV in treated Tulp1-/- mice, which were significantly different (p < 0.001, t-test), from those detected in untreated eyes (7.1 ± 7.0 µV and 9.4 ± 15.1 µV, respectively). Our results indicate that Tulp1 supplementation in photoreceptors may not be sufficient to provide robust benefit in Tulp1-/- mice. As such, further studies are required to fine tune the Tulp1 supplementation therapy, which, in principle, should rescue the Tulp1-/- phenotype.

15.
Front Neurosci ; 14: 571479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33324145

RESUMO

Optic Atrophy 1 (OPA1) is a mitochondrially targeted GTPase that plays a pivotal role in mitochondrial health, with mutations causing severe mitochondrial dysfunction and typically associated with Dominant Optic Atrophy (DOA), a progressive blinding disease involving retinal ganglion cell loss and optic nerve damage. In the current study, we investigate the use of codon-optimized versions of OPA1 isoform 1 and 7 as potential therapeutic interventions in a range of in vitro and in vivo models of mitochondrial dysfunction. We demonstrate that both isoforms perform equally well in ameliorating mitochondrial dysfunction in OPA1 knockout mouse embryonic fibroblast cells but that OPA1 expression levels require tight regulation for optimal benefit. Of note, we demonstrate for the first time that both OPA1 isoform 1 and 7 can be used independently to protect spatial visual function in a murine model of retinal ganglion cell degeneration caused by mitochondrial dysfunction, as well as providing benefit to mitochondrial bioenergetics in DOA patient derived fibroblast cells. These results highlight the potential value of OPA1-based gene therapy interventions.

16.
Sci Rep ; 10(1): 16515, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020509

RESUMO

Retinal ganglion cells (RGCs) are known to be involved in several ocular disorders, including glaucoma and Leber hereditary optic neuropathy (LHON), and hence represent target cells for gene therapies directed towards these diseases. Restricting gene therapeutics to the target cell type in many situations may be preferable compared to ubiquitous transgene expression, stimulating researchers to identify RGC-specific promoters, particularly promoter sequences that may also be appropriate in size to fit readily into recombinant adeno associated viral (AAV) vectors, the vector of choice for many ocular gene therapies. In the current study we analysed EGFP expression driven by various sequences of the putative human NEFH promoter in order to define sequences required for preferential expression in RGCs. EGFP expression profiles from four different potential NEFH promoter constructs were compared in vivo in mice using retinal histology and mRNA expression analysis. Notably, two efficient promoter sequences, one comprising just 199 bp, are presented in the study.


Assuntos
Proteínas de Neurofilamentos/genética , Regiões Promotoras Genéticas/genética , Células Ganglionares da Retina/metabolismo , Animais , Pareamento de Bases , Dependovirus/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Terapia Genética , Vetores Genéticos , Glaucoma/patologia , Humanos , Camundongos , Camundongos da Linhagem 129 , Proteínas de Neurofilamentos/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , Retina/patologia , Células Ganglionares da Retina/fisiologia , Transgenes
17.
Stem Cell Reports ; 15(1): 67-79, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32531192

RESUMO

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.


Assuntos
Proteínas de Ligação ao GTP/genética , Células-Tronco Pluripotentes Induzidas/patologia , Proteínas de Membrana/genética , Modelos Biológicos , Organoides/patologia , Retina/patologia , Retinose Pigmentar/genética , Morte Celular , Sobrevivência Celular , Dependovirus , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Organoides/ultraestrutura , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/patologia
18.
Exp Eye Res ; 87(6): 529-34, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18834879

RESUMO

Perturbed microRNA (miR) expression is a feature of, and may play a fundamental role in, certain disease states such as different forms of cancer. Retinitis pigmentosa (RP) a group of inherited retinal degenerations is characterised by a progressive loss of photoreceptor cells and consequent visual handicap. We have previously reported an altered pan-retinal expression of miR-96, -183, -1 and -133 in a P347S-Rhodopsin transgenic mouse model of RP. As many different mutations in Rhodopsin and other genes such as RDS/Peripherin can lead to RP, it was of interest to explore whether the characterized retinal miR expression signature was observed in three other mouse models of RP linked to rhodopsin and rds/peripherin. Therefore, pan-retinal expression of miR-96, -182, -183, -1, -133 and -142 was analysed using quantitative real-time RT-PCR. A common signature of altered miR expression was found; expression of miR-96, -182 and -183 decreased by 14.1-53.2%, while expression of miR-1, -133 and -142 was up-regulated by 186.1-538.5%. Significantly, the detected pan-retinal miR signature was mirrored by similar miR expression profiles in FACS-isolated rod photoreceptors from these mice. In an attempt to understand the function of these miRs, corresponding target genes were predicted using computational means. Many 'enriched' targets (with binding sites for at least two of the above miRs) were found to be regulatory molecules and members of intracellular signalling circuits. However, further studies are required to highlight which of the large number of in silico predicted targets are actually controlled by these miRs.


Assuntos
MicroRNAs/metabolismo , Retinose Pigmentar/genética , Animais , Modelos Animais de Doenças , Expressão Gênica , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Periferinas , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rodopsina/genética , Rodopsina/metabolismo
19.
Vision Res ; 48(3): 386-91, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17920651

RESUMO

Mutational heterogeneity in genes causative of dominantly inherited disorders represents a significant barrier for development of therapies directed towards correction of the primary genetic defect. To circumvent the mutational heterogeneity present in rhodopsin- (RHO-) linked autosomal dominant Retinitis Pigmentosa (adRP), a strategy involving suppression and replacement of RHO has been adopted. RNA interference- (RNAi-) mediated suppression of RHO has been explored as has the generation of an RNAi-resistant replacement gene using the degeneracy of the genetic code. Additionally, the functional equivalence of codon-modified replacement genes has been demonstrated in a transgenic animal (RHO-M). Suppression and replacement, while exemplified by adRP, may also be relevant to many other dominantly inherited diseases with the hallmark of mutational heterogeneity.


Assuntos
Modelos Animais de Doenças , Terapia Genética/métodos , Retinose Pigmentar/terapia , Rodopsina/genética , Animais , Células Cultivadas , Eletrorretinografia , Expressão Gênica , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Rodopsina/biossíntese
20.
Artigo em Inglês | MEDLINE | ID: mdl-29348794

RESUMO

MicroRNAs are important determinants of gene expression via post-transcriptional control of the protein levels of their mRNA targets. MicroRNA-134 (miR-134) has emerged as an important brain-specific microRNA which has been implicated in the control of dendritic spine morphology, neuronal differentiation and apoptosis. Here we show that Tubby-like protein 1 (Tulp1) is a target of miR-134. Tulp1 protein showed a similar cellular distribution pattern in the hippocampus to miR-134 and displayed an inverse expression pattern in the mouse retina. Bioinformatics analyses identified a conserved miR-134 binding site in the 3' untranslated region of both mouse and human Tulp1 and luciferase reporter assays confirmed miR-134 targets Tulp1 in vitro. Induction of prolonged seizures in mice resulted in upregulation of miR-134 and downregulation of protein levels of Tulp1 which were reversed in animals injected with locked nucleic acid-modified antagomirs targeting miR-134. Finally, knockdown of Tulp1 in human neurons caused an increase in vulnerability to excitotoxicity. These data identify Tulp1/TULP1 as a novel target of miR-134, which may contribute to underlying pathomechanisms in epilepsy.

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