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1.
Nat Commun ; 12(1): 4934, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400638

RESUMO

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.


Assuntos
Terapia Genética , RNA/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/metabolismo , Interferência de RNA , Splicing de RNA , Retina , Degeneração Retiniana , Rodopsina/genética , Rodopsina/metabolismo
2.
FASEB J ; 35(9): e21802, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383984

RESUMO

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.


Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Homeodomínio/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Luz , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa/métodos , Células Ganglionares da Retina/metabolismo , Visão Ocular/fisiologia
3.
Gene Ther ; 28(5): 265-276, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33750925

RESUMO

Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

4.
CRISPR J ; 3(4): 276-283, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32833533

RESUMO

Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.

5.
Int J Mol Sci ; 21(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842706

RESUMO

Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho-/- mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model.


Assuntos
Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/fisiopatologia , Animais , Distrofias de Cones e Bastonetes/terapia , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/farmacologia , Humanos , Camundongos Transgênicos , Oftalmoscopia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Rodopsina/genética , Opsinas de Bastonetes/genética , Tomografia de Coerência Óptica , Transativadores/genética , Transativadores/farmacologia , Visão Ocular/genética
6.
Transl Vis Sci Technol ; 9(7): 20, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32724727

RESUMO

Purpose: Adeno-associated viral (AAV) gene therapy treatment for Stargardt disease currently requires a dual vector approach owing to the size of the ATP-binding cassette transporter family member gene (ABCA4). The nature of the dual vector system creates the potential for adverse events. Here we have investigated an overlapping adeno-associated viral ABCA4 dual vector system for signs of toxicity in Abca4 -/- mice as a prelude to dual vector first in human clinical trials. Methods: Abca4 -/- mice received a subretinal injection of a 1:1 5':3' dual vector mix; 5' vector only; 3 ' vector only; a GFP reporter vector; or diluent only (sham). All vectors were adeno-associated virus-8 Y733F. Mice were subsequently assessed for signs of toxicity as measured by loss in retinal structure by optical coherence tomography and retinal function by electroretinography up to 6 months after injection. Results: Subretinal delivery of the dual vector system and its comprising parts induced no structural or functional changes relative to paired uninjected eyes beyond those observed in the sham control cohort. Histologic changes were limited to the superior retina where the injection was performed. Electroretinography analysis confirmed the dual vector system inferred no functional changes beyond those observed in the sham control cohort. Conclusions: An optimized overlapping dual vector system for the treatment of Stargardt disease shows no additional signs of toxicity beyond those observed from a sham injection. Translational Relevance: This presentation of safety of a dual vector system for the treatment of Stargardt disease encourages its future use in clinical trial.


Assuntos
Dependovirus , Vetores Genéticos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Dependovirus/genética , Eletrorretinografia , Terapia Genética , Vetores Genéticos/genética , Camundongos , Doença de Stargardt
7.
Int J Mol Sci ; 21(14)2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674481

RESUMO

Effective treatment of retinal diseases with adeno-associated virus (AAV)-mediated gene therapy is highly dependent on the proportion of successfully transduced cells. However, due to inflammatory reactions at high vector doses, adjunctive treatment may be necessary to enhance the therapeutic outcome. Hydroxychloroquine and chloroquine are anti-malarial drugs that have been successfully used in the treatment of autoimmune diseases. Evidence suggests that at high concentrations, hydroxychloroquine and chloroquine can impact viral infection and replication by increasing endosomal and lysosomal pH. This effect has led to investigations into the potential benefits of these drugs in the treatment of viral infections, including human immunodeficiency virus and severe acute respiratory syndrome coronavirus-2. However, at lower concentrations, hydroxychloroquine and chloroquine appear to exert immunomodulatory effects by inhibiting nucleic acid sensors, including toll-like receptor 9 and cyclic GMP-AMP synthase. This dose-dependent effect on their mechanism of action supports observations of increased viral infections associated with lower drug doses. In this review, we explore the immunomodulatory activity of hydroxychloroquine and chloroquine, their impact on viral infections, and their potential to improve the efficacy and safety of retinal gene therapy by reducing AAV-induced immune responses. The safety and practicalities of delivering hydroxychloroquine into the retina will also be discussed.


Assuntos
Cloroquina/uso terapêutico , Terapia Genética , Hidroxicloroquina/uso terapêutico , Doenças Retinianas/terapia , Viroses/tratamento farmacológico , Animais , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Dependovirus/genética , Humanos , Hidroxicloroquina/farmacologia , Imunomodulação/efeitos dos fármacos , Doenças Retinianas/patologia , SARS-CoV-2
8.
Hum Gene Ther ; 31(13-14): 730-742, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32394751

RESUMO

Mutations in the rhodopsin gene may cause photoreceptor degeneration in autosomal dominant retinitis pigmentosa (ADRP) by dominant negative or toxic gain-of-function mechanisms. Controversy exists as to the mechanism by which the widely studied P23H mutation induces rod cell dysfunction and death. Inherited disease caused by dominant negative mutations may be amenable to treatment using wild-type gene augmentation. Indeed, prior studies in the RHOP23H, Rho+/- transgenic mouse model of ADRP have suggested that a therapeutic benefit may be achieved when wild-type rhodopsin is overexpressed following subretinal delivery of a recombinant adeno-associated viral (AAV) vector. In this study, we investigated the effect of wild-type rhodopsin supplementation on the rate of retinal degeneration in the more clinically relevant RhoP23H/+ knock-in mouse model of ADRP. Four AAVs carrying the human rhodopsin coding sequence were first designed and compared for efficacy in the rhodopsin knockout mouse. All four vectors were capable of driving expression of the human transgene in the knockout retina with the protein being appropriately trafficked to de novo rod outer segments. The most efficient of these vectors was injected at one of two doses into the subretinal space of RhoP23H/+ mice and the effect on retinal structure and function determined longitudinally by spectral-domain optical coherence tomography and electroretinography, respectively, over a 3-month period. Although significant overexpression of rhodopsin protein was achieved in this model, no beneficial effect on retinal structure or function was observed at either dose. Lack of therapeutic efficacy in this model may be attributable to the relative rapidity of degeneration in the RhoP23H/+ mouse relative to the human disease, over- or under dosing at the level of individual photoreceptors, late timing of the intervention, or a possible predominant toxic gain-of-function mechanism of degeneration.

9.
PLoS Genet ; 16(4): e1008583, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236127

RESUMO

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.


Assuntos
Olho/anatomia & histologia , Olho/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Eletrorretinografia , Anormalidades do Olho/genética , Feminino , Deleção de Genes , Mutação com Perda de Função , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão/genética , Ligação Proteica , Transporte Proteico , Epitélio Pigmentado da Retina/anormalidades , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
10.
J Ocul Pharmacol Ther ; 36(4): 257-266, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32027217

RESUMO

Purpose: Age-related macular degeneration leads to retinal pigment epithelium (RPE) cell death and loss of central vision. In vivo studies have shown that the RPE layer has an innate, but limited, ability to repopulate atrophic areas. We aimed to establish a semiautomated, in vitro, wound healing assay workflow for targeted screening of compounds able to influence RPE wound healing. Methods: The ARPE-19 phenotype was evaluated using bright-field microscopy, immunocytochemistry, and quantitative real-time polymerase chain reaction. ARPE-19 monolayers were simultaneously scratched in a 96-well format and treated with Hoechst-33342 and an array of compounds. Initial wound dimensions and wound healing were subsequently evaluated using the EVOS FL Auto 2.0 imaging platform combined with automated image analyses. Results: Long-term cultured ARPE-19 cells displayed a more in vivo RPE-like phenotype compared with recently seeded or short-term cultured cells. No statistical difference of initial scratch width was observed between short-term and long-term cultured cells, but more wells were excluded from analyses in total in the latter case due to scratch width, scratch smoothness, and imaging errors. Furthermore, the previous time spent in continuous culture had an effect on the observation of an altered wound healing response to different treatment conditions. Conclusions: We have established a semiautomated, 96-well format, in vitro wound healing assay with a reproducible workflow. This would enable screening of a significant number of compounds and greatly advances the potential of identifying novel therapeutics that may enhance the innate ability of RPE cells to repopulate atrophic areas.

11.
Mol Ther ; 28(3): 830-844, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32027843

RESUMO

The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.


Assuntos
DNA/administração & dosagem , Terapia Genética , Células-Tronco Neurais/metabolismo , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Transplante de Células-Tronco , Animais , Diferenciação Celular , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Camundongos , Camundongos Knockout , Plasmídeos/genética , Rodopsina/genética , Transplante de Células-Tronco/métodos , Transdução Genética , Transgenes
12.
Nat Med ; 26(3): 354-359, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32094925

RESUMO

Retinal gene therapy has shown great promise in treating retinitis pigmentosa (RP), a primary photoreceptor degeneration that leads to severe sight loss in young people. In the present study, we report the first-in-human phase 1/2, dose-escalation clinical trial for X-linked RP caused by mutations in the RP GTPase regulator (RPGR) gene in 18 patients over up to 6 months of follow-up (https://clinicaltrials.gov/: NCT03116113). The primary outcome of the study was safety, and secondary outcomes included visual acuity, microperimetry and central retinal thickness. Apart from steroid-responsive subretinal inflammation in patients at the higher doses, there were no notable safety concerns after subretinal delivery of an adeno-associated viral vector encoding codon-optimized human RPGR (AAV8-coRPGR), meeting the pre-specified primary endpoint. Visual field improvements beginning at 1 month and maintained to the last point of follow-up were observed in six patients.


Assuntos
Proteínas do Olho/genética , Proteínas do Olho/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Mutação/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Adulto , Humanos , Pessoa de Meia-Idade , Retina/patologia , Retina/fisiopatologia , Retinite Pigmentosa/fisiopatologia , Adulto Jovem
13.
Int J Mol Sci ; 21(3)2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991730

RESUMO

RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.


Assuntos
Edição de Genes , Terapia Genética , Edição de RNA , Retina/metabolismo , Transgenes , Adenosina Desaminase/metabolismo , Animais , Sistemas CRISPR-Cas , Imunofluorescência , Marcação de Genes , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Humanos , Proteínas de Ligação a RNA/metabolismo , Retina/patologia , Doenças Retinianas/genética , Doenças Retinianas/terapia
14.
Mol Ther Methods Clin Dev ; 17: 99-106, 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31890744

RESUMO

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high- and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

15.
JAMA Ophthalmol ; 138(2): 128-135, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31855248

RESUMO

Importance: Gene therapy is a promising treatment for choroideremia, an X-linked retinal degeneration. The required minimum level of gene expression to ameliorate degeneration rate is unknown. This can be interrogated by exploring the association between messenger RNA (mRNA) levels and phenotype in mildly affected patients with choroideremia. Objective: To analyze CHM mRNA splicing outcomes in 2 unrelated patients with the same c.940+3delA CHM splice site variant identified as mildly affected from a previous study of patients with choroideremia. Design, Setting, and Participants: In this retrospective observational case series, 2 patients with c.940+3delA CHM variants treated at a single tertiary referral center were studied. In addition, a third patient with a c.940+2T>A variant that disrupts the canonical dinucleotide sequence at the same donor site served as a positive control. Data were collected from October 2013 to July 2018. Main Outcomes and Measures: Central area of residual fundus autofluorescence was used as a biomarker for disease progression. CHM transcript splicing was assessed by both end point and quantitative polymerase chain reaction. Rab escort protein 1 (REP1) expression was assessed by immunoblot. Results: The 2 mildly affected patients with c.940+3delA variants had large areas of residual autofluorescence for their age and longer degeneration half-lives compared with the previous cohort of patients with choroideremia. The control patient with a c.940+2T>A variant had a residual autofluorescence area within the range expected for his age. Both patients with the c.940+3delA variant expressed residual levels of full-length CHM mRNA transcripts relative to the predominant truncated transcript (mean [SEM] residual level: patient 1, 2.3% [0.3]; patient 2, 4.7% [0.2]), equivalent to approximately less than 1% of the level of full-length CHM expressed in nonaffected individuals. Full-length CHM expression was undetectable in the control patient. REP1 expression was less than the threshold for detection both in patients 1 and 2 and the control patient compared with wild-type controls. Conclusions and Relevance: These results demonstrate the first genotype-phenotype association in choroideremia. A +3 deletion in intron 7 is sufficient to cause choroideremia in a milder form. If replicated with gene therapy, these findings would suggest that relatively low expression (less than 1%) of the wild-type levels of mRNA would be sufficient to slow disease progression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/terapia , Terapia Genética , RNA Mensageiro/análise , Adolescente , Criança , Coroideremia/genética , Estudos de Associação Genética , Humanos , Masculino , Fenótipo , Estudos Retrospectivos
16.
Exp Eye Res ; 190: 107895, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816293

RESUMO

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho-/-) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls. Mean grey value significantly decreased over time in the Rho-/- and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho-/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT). Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss.


Assuntos
Modelos Animais de Doenças , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/patologia , Animais , Expressão Gênica/fisiologia , Vetores Genéticos , Injeções Intraoculares , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oftalmoscopia , Parvovirinae/genética , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinite Pigmentosa/metabolismo , Rodopsina/genética , Coloração e Rotulagem , Tomografia de Coerência Óptica
17.
Genes (Basel) ; 10(10)2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548516

RESUMO

Advances in molecular research have culminated in the development of novel gene-based therapies for inherited retinal diseases. We have recently witnessed several groundbreaking clinical studies that ultimately led to approval of Luxturna, the first gene therapy for an inherited retinal disease. In parallel, international research community has been engaged in conducting gene therapy trials for another more common inherited retinal disease known as choroideremia and with phase III clinical trials now underway, approval of this therapy is poised to follow suit. This chapter discusses new insights into clinical phenotyping and molecular genetic testing in choroideremia with review of molecular mechanisms implicated in its pathogenesis. We provide an update on current gene therapy trials and discuss potential inclusion of female carries in future clinical studies. Alternative molecular therapies are discussed including suitability of CRISPR gene editing, small molecule nonsense suppression therapy and vision restoration strategies in late stage choroideremia.


Assuntos
Coroideremia/genética , Coroideremia/terapia , Terapia Genética , Animais , Genótipo , Humanos , Fenótipo
18.
Mol Ther Methods Clin Dev ; 14: 77-89, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31309129

RESUMO

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 µM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy.

19.
Invest Ophthalmol Vis Sci ; 60(7): 2733-2742, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31247114

RESUMO

Purpose: The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit. Methods: Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts. Results: Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group. Conclusions: Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.


Assuntos
Luz , Mutação , Fototerapia/métodos , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Rodopsina/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Eletrorretinografia , Filtração , Técnicas de Genotipagem , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/patologia , Polimorfismo de Nucleotídeo Único , Ondas de Rádio , Retina/fisiopatologia , Retinite Pigmentosa/fisiopatologia , cis-trans-Isomerases/genética
20.
Hum Gene Ther Methods ; 30(2): 44-52, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30963787

RESUMO

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.


Assuntos
Bestrofinas/fisiologia , Parvovirinae/genética , Bestrofinas/genética , Vetores Genéticos , Células HEK293 , Humanos , Transdução Genética
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