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3.
Klin Monbl Augenheilkd ; 241(3): 259-265, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38508214

RESUMO

Retinal dystrophies linked to the RPE65 gene are mostly fast-progressing retinal diseases, with childhood onset of night blindness and progressive visual loss up to the middle adult age. Rare phenotypes linked to this gene are known with congenital stationary night blindness or slowly progressing retinitis pigmentosa, as well as an autosomal dominant c.1430A>G (p.Asp477Gly) variant. This review gives an overview of the current knowledge of the clinical phenotypes, as well as experience with the efficacy and safety of the approved gene augmentation therapy voretigene neparvovec.


Assuntos
Cegueira Noturna , Distrofias Retinianas , Retinite Pigmentosa , Adulto , Criança , Humanos , cis-trans-Isomerases/genética , Terapia Genética , Mutação , Cegueira Noturna/terapia , Fenótipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Retinite Pigmentosa/diagnóstico , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia
4.
Graefes Arch Clin Exp Ophthalmol ; 262(2): 601-607, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37768368

RESUMO

BACKGROUND: Voretigene neparvovec (Luxturna®) is the first approved gene therapy for RPE65-linked Leber congenital amaurosis (LCA). Though individual effects are highly variable, most recipients report improved vision in everyday life. To describe such effects, visual navigation tests are now frequently used in clinical trials. However, it is still unclear how their results should be interpreted compared to conventional parameters of visual function. METHODS: Seven LCA patients underwent a multi-luminance visual navigation test (Ora-VNCTM) before and 3 months after receiving Luxturna gene therapy. Their performance was rated based on the luminance level at which they passed the course. Differences between the first and second test were correlated to changes in visual acuity, full-field stimulus thresholds, chromatic pupil campimetry, and dark-adapted perimetry. RESULTS: A few patients displayed notable improvements in conventional measures of visual function whereas patients with advanced retinal degeneration showed no relevant changes. Independent of these results, almost all participants improved in the visual navigation task by one or more levels. The improvement in the mobility test was best correlated to the change in full-field stimulus thresholds. Other measures of visual functions showed no clear correlation with visual navigation. DISCUSSION: In patients who passed the test's more difficult levels, improved visual navigation can be attributed to the reactivation of rods. However, the performance of patients with low vision seemed to depend much more on confounding factors in the easier levels. In sum, such tests might only be meaningful for patients with better preserved visual functions.


Assuntos
Amaurose Congênita de Leber , cis-trans-Isomerases , Humanos , cis-trans-Isomerases/genética , Visão Ocular , Retina , Acuidade Visual , Amaurose Congênita de Leber/diagnóstico , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Terapia Genética/métodos , Mutação
5.
Anal Chem ; 95(41): 15180-15188, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37811788

RESUMO

Tandem mass tags (TMT) and tribrid mass spectrometers are a powerful combination for high-throughput proteomics with high quantitative accuracy. Increasingly, this technology is being used to map the effects of drugs on the proteome. However, the depth of proteomic profiling is still limited by sensitivity and speed. The new Orbitrap Ascend mass spectrometer was designed to address these limitations with a combination of hardware and software improvements. We evaluated the performance of the Ascend in multiple contexts including deep proteomic profiling. We found that the Ascend exhibited increased sensitivity, yielding higher signal-to-noise ratios than the Orbitrap Eclipse with shorter injection times. As a result, higher numbers of peptides and proteins were identified and quantified, especially with low sample input. TMT measurements had significantly improved signal-to-noise ratios, improving quantitative precision. In a fractionated 16plex sample that profiled proteomic differences across four human cell lines, the Ascend was able to quantify hundreds more proteins than the Eclipse, many of them low-abundant proteins, and the Ascend was able to quantify >8000 proteins in 30% less instrument time. We used the Ascend to analyze 8881 proteins in HCT116 cancer cells treated with covalent sulfolane/sulfolene inhibitors of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a phosphorylation-specific peptidyl-prolyl cis-trans isomerase implicated in several cancers. We characterized these PIN1 inhibitors' effects on the proteome and identified discrepancies among the different compounds, which will facilitate a better understanding of the structure-activity relationship of this class of compounds. The Ascend was able to quantify statistically significant, potentially therapeutically relevant changes in proteins that the Eclipse could not detect.


Assuntos
Proteoma , Proteômica , Humanos , Proteoma/metabolismo , Espectrometria de Massas , Células HCT116 , cis-trans-Isomerases , Peptidilprolil Isomerase de Interação com NIMA
6.
PeerJ ; 11: e15702, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37547722

RESUMO

Purpose: This study aimed to develop a prediction model to classify RPE65-mediated inherited retinal disease (IRDs) based on protein secondary structure and to analyze phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. Methods: Pathogenic or likely pathogenic missense variants of RPE65 were obtained from UniProt, ClinVar, and HGMD databases. The three-dimensional structure of RPE65 was retrieved from the Protein Data Bank (PDB) and modified with Pymol software. A novel prediction model was developed using LASSO regression and multivariate logistic regression to identify RPE65-associated IRDs. A total of 21 Chinese probands with RPE65 variants were collected to analyze phenotype-protein structure correlations of RPE65 missense variants. Results: The study found that both pathogenic and population missense variants were associated with structural features of RPE65. Pathogenic variants were linked to sheet, ß-sheet, strands, ß-hairpins, Fe2+ (iron center), and active site cavity, while population variants were related to helix, loop, helices, and helix-helix interactions. The novel prediction model showed accuracy and confidence in predicting the disease type of RPE65 variants (AUC = 0.7531). The study identified 25 missense variants in Chinese patients, accounting for 72.4% of total mutations. A significant correlation was observed between clinical characteristics of RPE65-associated IRDs and changes in amino acid type, specifically for missense variants of F8 (H68Y, P419S). Conclusion: The study developed a novel prediction model based on the protein structure of RPE65 and investigated phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. The findings provide insights into the precise diagnosis of RPE65-mutated IRDs.


Assuntos
Amaurose Congênita de Leber , Doenças Retinianas , cis-trans-Isomerases , Humanos , Amaurose Congênita de Leber/genética , Linhagem , Retina/patologia , Doenças Retinianas/genética , Mutação de Sentido Incorreto , cis-trans-Isomerases/genética , Fenótipo , Conformação Proteica
7.
Adv Exp Med Biol ; 1415: 415-419, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440066

RESUMO

The retina pigmented epithelium 65 kDa protein (RPE65) is an essential enzyme in the visual cycle that regenerates the 11-cis-retinal chromophore obligatory for vision. Mutations in RPE65 are associated with blinding diseases. D477G (C.1430G > A) is the only known RPE65 variant to cause autosomal dominant retinitis pigmentosa (adRP). Previously, we reported that the heterozygous D477G knock-in (WT/KI) mice exposed to dim light intensity demonstrated delayed chromophore regeneration rates and slowed recovery of photoreceptor sensitivity following photobleaching. However, visual function and retinal architecture were indistinguishable from the wild-type (WT) mice. In this study, when maintained under the physiological day-light intensity (2 K lux), the WT/KI heterozygous mice displayed retina degeneration and reduced electroretinography (ERG) amplitude, recapitulating that observed in human patients. Our findings indicated the importance of the light environment in the mechanism of RPE65 D477G pathogenicity.


Assuntos
Degeneração Retiniana , cis-trans-Isomerases , Humanos , Camundongos , Animais , Modelos Animais de Doenças , cis-trans-Isomerases/genética , Retina/metabolismo , Mutação , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Epitélio
8.
Adv Exp Med Biol ; 1415: 533-537, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440083

RESUMO

The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.


Assuntos
Epitélio Pigmentado da Retina , Retinoides , Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , cis-trans-Isomerases
9.
Hum Gene Ther ; 34(13-14): 639-648, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37014074

RESUMO

The use of AAV-RPE65 vectors for gene supplementation has achieved spectacular success as a treatment for individuals with autosomal recessive retinal disease caused by biallelic mutations in the visual cycle gene RPE65. However, the efficacy of this approach in treating autosomal dominant retinitis pigmentosa (adRP) associated with a monoallelic mutation encoding a rare D477G RPE65 variant has not been studied. Although lacking a severe phenotype, we now find that knock-in mice heterozygous for D477G RPE65 (D477G KI mice) can be used to evaluate outcomes of AAV-RPE65 gene supplementation. Total RPE65 protein levels, which are decreased in heterozygous D477G KI mice, were doubled following subretinal delivery of rAAV2/5.hRPE65p.hRPE65. In addition, rates of recovery of the chromophore 11-cis retinal after bleaching were significantly increased in eyes that received AAV-RPE65, consistent with increased RPE65 isomerase activity. While dark-adapted chromophore levels and a-wave amplitudes were not affected, b-wave recovery rates were modestly improved. The present findings establish that gene supplementation enhances 11-cis retinal synthesis in heterozygous D477G KI mice and complement previous studies showing that chromophore therapy results in improved vision in individuals with adRP associated with D477G RPE65.


Assuntos
Retina , Retinite Pigmentosa , Animais , Camundongos , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Mutação , Retina/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Retinite Pigmentosa/metabolismo
10.
Biochemistry ; 62(9): 1429-1432, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37057907

RESUMO

Retinal G-protein-coupled receptor (RGR) plays a crucial role in the visual system of vertebrates as a retinal photoisomerase, which isomerizes all-trans-retinal to 11-cis-retinal to maintain the photosensitivity of visual rhodopsins. Despite the previous characterization of bovine RGR, little is known about the spectral properties of RGR from other species. In addition, photoreactivity of the 11-cis-retinal-binding form remains unclear. In this study, we revealed that human and chicken RGRs form blue-absorbing pigments similar to bovine RGR. Furthermore, the spectroscopic and biochemical analyses revealed that bovine and chicken RGRs are bistable rhodopsins displaying a reversible photoreaction. These findings provide insight into the behavior of RGR as a retinal photoisomerase and aid in understanding its role in the visual system.


Assuntos
Retina , Retinaldeído , Animais , Bovinos , Humanos , Retinaldeído/química , Receptores Acoplados a Proteínas G , cis-trans-Isomerases , Proteínas do Olho/química , Rodopsina
11.
Plant J ; 113(5): 986-1003, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36602437

RESUMO

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , beta Caroteno/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , Regulação da Expressão Gênica de Plantas , Isomerases/genética , Isomerases/metabolismo
12.
J Enzyme Inhib Med Chem ; 38(1): 2162047, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36629452

RESUMO

hRPE65 is a fundamental enzyme of the retinoid visual cycle, and many missense mutations affecting its expression or function are associated with a wide range of diseases. Many hRPE65 missense mutations lack a clear pathogenicity classification or are labelled as VUS. In this context, we recently developed a protocol based on µs-long molecular dynamics simulations to study the potential pathogenic effect of hRPE65 missense mutations. In the present work, the structure-based protocol was integrated with a hRPE65-tailored consensus bioinformatics strategy, named ConPath, that showed high performance in predicting known pathogenic/benign hRPE65 missense mutations. The combined strategy was used to perform a multi-level evaluation of the potential pathogenicity of 13 different hRPE65 VUS, which were classified based on their likelihood of pathogenic effect. The obtained results provide information that may support the reclassification of these VUS and help clinicians evaluate the eligibility for gene therapy of patients diagnosed with such variants.


Assuntos
Mutação de Sentido Incorreto , cis-trans-Isomerases , Humanos , cis-trans-Isomerases/genética , Biologia Computacional
13.
Retina ; 43(9): 1608-1611, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33394965

RESUMO

PURPOSE: To report surgical observations formulated during the first 120 cases of subretinal gene therapy in patients with inherited retinal degenerations (IRDs). METHODS: A two-surgeon team compiled surgical observations and formulated surgical pearls based on the consecutive cases of subretinal viral vector injection in patients enrolled in clinical trials focusing on choroideremia, achromatopsia, and RP GTPase regulator associated retinitis pigmentosa, as well as patients with retinal pigment epithelium-specific-65-kDa (RPE65) associated Leber congenital amaurosis receiving Food and Drug Administration-approved voretigene neparvovec-rzyl therapy. RESULTS: One hundred twenty subretinal surgeries were performed by a two-surgeon team. Key anatomical features pertinent to surgical management were noted and are described in this article. Surgical decision making for successful subretinal administration of viral vectors and management of potential surgical challenges were formulated. CONCLUSION: Lessons learned during subretinal gene therapy cases may be helpful to other surgeons entering clinical trials or performing postapproval gene therapy administration. Surgical pearls outlined in this article may also be helpful for other targeted subretinal therapies, such as cellular transplantation or retinal prosthesis implantation.


Assuntos
Amaurose Congênita de Leber , Degeneração Retiniana , Retinite Pigmentosa , Humanos , Retina , Terapia Genética , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Degeneração Retiniana/terapia , cis-trans-Isomerases/genética
14.
Br J Ophthalmol ; 107(3): 299-301, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-35835501

RESUMO

We report a series of three young patients (ages: 22 months, 2 years, and 5 years) who developed subretinal deposits at post-operative week one following subretinal voretigene neparvovec-rzyl treatment for RPE65-mediated retinal dystrophy. In the 5-year-old, subretinal deposits were also observed in the inferior periphery of both eyes. All three patients experienced improved visual function with treatment, and both the macular and inferior subretinal deposits have improved or resolved over the follow-up period. These findings may inform the delivery parameters and safety profile of AAV-based gene therapy as the number of retinal gene therapy trials continues to grow.


Assuntos
Retina , Distrofias Retinianas , Humanos , Lactente , Pré-Escolar , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Terapia Genética , Visão Ocular , cis-trans-Isomerases/genética
15.
Ophthalmic Genet ; 44(3): 276-280, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-35904185

RESUMO

BACKGROUND: It is of utmost importance to define the molecular diagnosis of patients with retinitis pigmentosa (RP) due to existing targeted therapeutic option: voretigene neparvovec.We provide clinical evidence for pathogenicity reclassification of variants of uncertain significance (VUSs) RPE65 c.1580A>G (p.His527Arg). MATERIALS AND METHODS: A case report of a 10-year-old boy with progressive vision loss. The patient manifested disease highly suggestive of RPE65 retinal dystrophy: nyctalopia, fairly good central vision, severely depressed full-field electroretinography responses and complete loss of peripheral fundus aut ofluorescence. RESULTS: Invitae Inherited Retinal Disorders Panel identified likely pathogenic mutation RPE65 c.499G>T (p.Asp167Tyr) and RPE65 c.1580A>G (p.His527Arg), variant of uncertain significance. Segregation analysis confirmed that these variants are in trans. CONCLUSIONS: We conclude that the variant RPE65 c.1580A>G (p.His527Arg) has contributed to the pathologic phenotype, demonstrating its significance clearly in the case presented, and should be reclassified according to the criteria of evidence as pathogenic. Therefore, patients with this specific variant in homozygous or compound heterozygous form would likely benefit from genetic treatment based on recombinant adeno-associated virus vector, providing a working RPE65 gene to act in place of a mutated RPE65 gene.


Assuntos
Distrofias Retinianas , Retinite Pigmentosa , Humanos , cis-trans-Isomerases/genética , Mutação , Distrofias Retinianas/genética , Retinite Pigmentosa/diagnóstico , Retinite Pigmentosa/genética
16.
Life Sci Alliance ; 6(1)2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36265895

RESUMO

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.


Assuntos
Cisteína , Proteínas do Olho , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Lipoilação , Conformação Proteica em alfa-Hélice , cis-trans-Isomerases
17.
Artigo em Inglês | MEDLINE | ID: mdl-36167727

RESUMO

In the 5 years following U.S. Food and Drug Administration (FDA) approval of the first gene therapy reagent approved to treat a genetic disease, voretigene neparvovec-rzyl (Luxturna), retinal disease clinics, hospital pharmacies, operating rooms, and even health insurance entities around the world have incorporated gene therapy as a standard procedure. The success of Luxturna has helped pave the way to establish a template for developing other gene therapy reagents that promise to restore sight or halt the progression of photoreceptor cell loss in both inherited and acquired retinal diseases. Here we review lessons learned from development of a gene therapy drug for RPE65 disease and how these lessons may expedite the development of additional treatments for previously untreatable blinding conditions.


Assuntos
Distrofias Retinianas , Humanos , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , cis-trans-Isomerases/genética , Vetores Genéticos , Terapia Genética/métodos
18.
Invest Ophthalmol Vis Sci ; 63(13): 19, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36534385

RESUMO

Purpose: Both photodamage and aberrant visual cycle contribute to disease progress of many retinal degenerative disorders, whereas the signaling pathways causing photoreceptor death remain unclear. Here we investigated the effects of intense photo-stress on (1) necrosome activation in wild-type and RPE65-null mice, (2) interaction of p62/Sequestosome-1 with the necrosome proteins, and (3) the effects of rapamycin on photodamage-induced necrosome activation and retinal degeneration in wild-type mice. Methods: Dark-adapted rd12 mice and 129S2/Sv mice with or without rapamycin treatment were exposed to 15,000 lux light for different times. Expression levels and subcellular localization of proteins were determined through immunoblot and immunohistochemical analyses. Cone sheaths were stained with peanut agglutinin. Correlation between photoreceptor degeneration and receptor-interacting protein kinase-1 (RIPK1) expression was assessed with Spearman's correlation analysis. Protein-protein interaction was analyzed by immunoprecipitation. Results: Intense light caused rod and cone degeneration accompanied by a significant increase in RIPK1-RIPK3 expressions, mixed lineage kinase domain-like protein phosphorylation, damage-associated molecular patterns protein release, and inflammatory responses in wild-type mouse retina. The same intense light did not induce the necrosome activation in rd12 retina, but it did in rd12 mice that received 9-cis-retinal supply. RIPK1 expression levels are positively correlated with the degrees of rod and cone degeneration. Photodamage upregulated expression and interaction of the p62 autophagosome cargo protein with the necrosome proteins, whereas rapamycin treatment attenuated the light-induced necrosome activation and photoreceptor degeneration. Conclusions: Necrosome activation contributed to photodamage-induced rod and cone degeneration. The visual cycle and autophagy are the important therapeutic targets to alleviate light-induced retinal necroptosis.


Assuntos
Proteínas do Olho , Degeneração Retiniana , Sirolimo , cis-trans-Isomerases , Animais , Camundongos , cis-trans-Isomerases/metabolismo , Proteínas do Olho/metabolismo , Camundongos Knockout , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Sirolimo/farmacologia
19.
Orv Hetil ; 163(48): 1923-1931, 2022 Nov 27.
Artigo em Húngaro | MEDLINE | ID: mdl-36436058

RESUMO

INTRODUCTION: Leber's congenital amaurosis is a genetically determined disease belonging to the group of hereditary retinal dystrophies that leads to significant visual impairment in childhood. The disease initially causes a concentric narrowing of the visual field and, with time, loss of central vision. The RPE65 gene mutation-related retinal dystrophy is the first ophthalmic disease for which gene therapy is available using voretigene neparvovec (Luxturna®, Novartis Pharmaceuticals AG, Basel, Switzerland). OBJECTIVE: To present the treatment outcomes of Hungarian patients who were the first to receive voretigene neparvovec gene therapy for the RPE65 biallelic gene mutation. METHOD: Two patients with RPE65 biallelic gene mutations confirmed by genetic testing received voretigene neparvovec gene therapy in one eye each. Before treatment and during the follow-up period, we assessed the best corrected visual acuity, the central retinal thickness, the degree of visual field defects and performed electrophysiological studies. RESULTS: Both the best corrected visual acuity (+3 letters in the older sibling and +10 letters in the younger sibling) and the degree of visual field narrowing improved in both patients. The change in visual function resulted in a significant improvement in the quality of life of our patients. CONCLUSION: Postoperative outcomes of our patients correlate with the results of clinical trials. Orv Hetil. 2022; 163(48): 1923-1931.


Assuntos
Distrofias Retinianas , cis-trans-Isomerases , Humanos , cis-trans-Isomerases/genética , Qualidade de Vida , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Terapia Genética/métodos , Mutação
20.
Commun Biol ; 5(1): 1006, 2022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-36198910

RESUMO

Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N2. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis-trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N2 fixation in planta. Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops.


Assuntos
Molibdoferredoxina , Oryza , Fertilizantes , Molibdoferredoxina/genética , Molibdoferredoxina/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo , Oryza/genética , Oryza/metabolismo , Oxirredutases , cis-trans-Isomerases/metabolismo
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