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1.
Am J Hum Genet ; 107(2): 278-292, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707085

RESUMO

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/genética , Mutação/genética , Alelos , Bestrofinas/genética , Cálcio/metabolismo , Linhagem Celular , Canalopatias/genética , Proteínas do Olho/genética , Edição de Genes/métodos , Terapia Genética/métodos , Genótipo , Células HEK293 , Humanos , Epitélio Pigmentado da Retina/fisiologia
2.
Am J Hum Genet ; 104(2): 310-318, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686507

RESUMO

Pathogenic variants of the KCNJ13 gene are known to cause Leber congenital amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 encodes the Kir7.1 subunit that acts as a tetrameric, inwardly rectifying potassium ion channel in the retinal pigment epithelium (RPE) to maintain ionic homeostasis and allow photoreceptors to encode visual information. We sought to determine whether genetic approaches might be effective in treating blindness arising from pathogenic variants in KCNJ13. We derived human induced pluripotent stem cell (hiPSC)-RPE cells from an individual carrying a homozygous c.158G>A (p.Trp53∗) pathogenic variant of KCNJ13. We performed biochemical and electrophysiology assays to confirm Kir7.1 function. We tested both small-molecule readthrough drug and gene-therapy approaches for this "disease-in-a-dish" approach. We found that the LCA16 hiPSC-RPE cells had normal morphology but did not express a functional Kir7.1 channel and were unable to demonstrate normal physiology. After readthrough drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV, and the Kir7.1 current was restored. Similarly, we rescued Kir7.1 channel function after lentiviral gene delivery to the hiPSC-RPE cells. In both approaches, Kir7.1 was expressed normally, and there was restoration of membrane potential and the Kir7.1 current. Loss-of-function variants of Kir7.1 are one cause of LCA. Using either readthrough therapy or gene augmentation, we rescued Kir7.1 channel function in iPSC-RPE cells derived from an affected individual. This supports the development of precision-medicine approaches for the treatment of clinical LCA16.


Assuntos
Cegueira/congênito , Canalopatias/genética , Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Amaurose Congênita de Leber/genética , Modelos Biológicos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Epitélio Pigmentado da Retina/patologia , Sequência de Bases , Cegueira/genética , Cegueira/patologia , Canalopatias/patologia , Criança , Humanos , Amaurose Congênita de Leber/patologia , Epitélio Pigmentado da Retina/metabolismo
3.
Development ; 146(1)2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30567931

RESUMO

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.


Assuntos
Organoides/citologia , Células-Tronco Pluripotentes/citologia , Retina/citologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/ultraestrutura , Reprodutibilidade dos Testes , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Tomografia de Coerência Óptica
4.
Antiviral Res ; 223: 105823, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38331200

RESUMO

Mucosal immunity has regained its spotlight amidst the ongoing Coronavirus disease 19 (COVID-19) pandemic, with numerous studies highlighting the crucial role of mucosal secretory IgA (SIgA) in protection against Severe acute respiratory syndrome coronavirus-2 or SARS-CoV-2 infections. The observed limitations in the efficacy of currently authorized COVID-19 vaccines in inducing effective mucosal immune responses remind us of the limitations of systemic vaccination in promoting protective mucosal immunity. This resurgence of interest has motivated the development of vaccine platforms capable of enhancing mucosal responses, specifically the SIgA response, and the development of IgA-based therapeutics. Recognizing viral respiratory infections as a global threat, we would like to comprehensively review the existing knowledge on mucosal immunity, with a particular emphasis on SIgA, in the context of SARS-CoV-2, influenza, and Respiratory Syncytial Virus (RSV) infections. This review aims to describe the structural and functional specificities of SIgA, along with its nuanced role in combating influenza, RSV, and SARS-CoV-2 infections. Subsequent sections further elaborate promising vaccine strategies, including mucosal vaccines against Influenza, RSV, and SARS-CoV-2 respiratory viruses, currently undergoing preclinical and clinical development. Additionally, we address the challenges associated with mucosal vaccine development, concluding with a discussion on IgA-based therapeutics as a promising platform for the treatment of viral respiratory infections. This comprehensive review not only synthesizes current insights into mucosal immunity but also identifies critical knowledge gaps, strengthening the way for further advancements in our current understanding and approaches to combat respiratory viral threats.


Assuntos
COVID-19 , Vacinas contra Influenza , Influenza Humana , Infecções por Vírus Respiratório Sincicial , Humanos , Imunoglobulina A Secretora , Vacinas contra COVID-19 , COVID-19/prevenção & controle , SARS-CoV-2
5.
Nucleic Acids Res ; 39(19): 8378-91, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21749977

RESUMO

There is a close relationship between histone acetylation and ATP-dependent chromatin remodeling that is not fully understood. We show that acetylation of histone H3 tails affects SWI/SNF (mating type switching/ sucrose non fermenting) and RSC (remodels structure of chromatin) remodeling in several distinct ways. Acetylation of the histone H3 N-terminal tail facilitated recruitment and nucleosome mobilization by the ATP-dependent chromatin remodelers SWI/SNF and RSC. Tetra-acetylated H3, but not tetra-acetylated H4 tails, increased the affinity of RSC and SWI/SNF for nucleosomes while also changing the subunits of SWI/SNF that interact with the H3 tail. The enhanced recruitment of SWI/SNF due to H3 acetylation is bromodomain dependent, but is not further enhanced by additional bromodomains found in RSC. The combined effect of H3 acetylation and transcription activators is greater than either separately which suggests they act in parallel to recruit SWI/SNF. Besides enhancing recruitment, H3 acetylation increased nucleosome mobilization and H2A/H2B displacement by RSC and SWI/SNF in a bromodomain dependent manner and to a lesser extent enhanced ATP hydrolysis independent of bromodomains. H3 and H4 acetylation did not stimulate disassembly of adjacent nucleosomes in short arrays by SWI/SNF or RSC. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.


Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Nucleossomos/metabolismo , Transativadores/metabolismo , Xenopus laevis
6.
J Clin Invest ; 133(19)2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37561581

RESUMO

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.


Assuntos
Canalopatias , Células-Tronco Pluripotentes Induzidas , Camundongos , Animais , Humanos , Criança , Edição de Genes , Canalopatias/genética , RNA Guia de Sistemas CRISPR-Cas , Retina , Epitélio Pigmentado da Retina , Mutação , RNA Mensageiro
7.
PLoS Pathog ; 6(12): e1001224, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21187891

RESUMO

Identifying the major routes of disease transmission and reservoirs of infection are needed to increase our understanding of disease dynamics and improve disease control. Despite this, transmission events are rarely observed directly. Here we had the unique opportunity to study natural transmission of Bordetella bronchiseptica--a directly transmitted respiratory pathogen with a wide mammalian host range, including sporadic infection of humans--within a commercial rabbitry to evaluate the relative effects of sex and age on the transmission dynamics therein. We did this by developing an a priori set of hypotheses outlining how natural B. bronchiseptica infections may be transmitted between rabbits. We discriminated between these hypotheses by using force-of-infection estimates coupled with random effects binomial regression analysis of B. bronchiseptica age-prevalence data from within our rabbit population. Force-of-infection analysis allowed us to quantify the apparent prevalence of B. bronchiseptica while correcting for age structure. To determine whether transmission is largely within social groups (in this case litter), or from an external group, we used random-effect binomial regression to evaluate the importance of social mixing in disease spread. Between these two approaches our results support young weanlings--as opposed to, for example, breeder or maternal cohorts--as the age cohort primarily responsible for B. bronchiseptica transmission. Thus age-prevalence data, which is relatively easy to gather in clinical or agricultural settings, can be used to evaluate contact patterns and infer the likely age-cohort responsible for transmission of directly transmitted infections. These insights shed light on the dynamics of disease spread and allow an assessment to be made of the best methods for effective long-term disease control.


Assuntos
Infecções por Bordetella/transmissão , Bordetella bronchiseptica , Surtos de Doenças , Fatores Etários , Animais , Humanos , Prevalência , Coelhos , Análise de Regressão , Fatores Sexuais , Meio Social
8.
Stem Cell Reports ; 17(11): 2409-2420, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36206764

RESUMO

Interphotoreceptor matrix proteoglycan 2 (IMPG2) mutations cause a severe form of early-onset retinitis pigmentosa (RP) with macular involvement. IMPG2 is expressed by photoreceptors and incorporated into the matrix that surrounds the inner and outer segments (OS) of rods and cones, but the mechanism of IMPG2-RP remains unclear. Loss of Impg2 function in mice produces a mild, late-onset photoreceptor phenotype without the characteristic OS loss that occurs in human patients. We generated retinal organoids (ROs) from patient-derived induced pluripotent stem (iPS) cells and gene-edited embryonic stem cells to model human IMPG2-RP in vitro. All ROs harboring IMPG2 mutations lacked an OS layer, in contrast to isogenic controls. Subsequent protein analyses revealed that this phenotype arises due to a loss of IMPG2 expression or its inability to undergo normal post-translational modifications. We hypothesized that loss of IMPG2 function destabilizes the interphotoreceptor matrix and renders the OS vulnerable to physical stressors, which is accentuated in the tissue culture environment. In support of this mechanism, transplantation of IMPG2 mutant ROs into the protected subretinal space of immunocompromised rodents restored OS production. Beyond providing a robust platform to study IMPG2-RP, this human RO model system may serve a broader role in honing strategies to treat advanced photoreceptor-based diseases.


Assuntos
Organoides , Retinose Pigmentar , Humanos , Camundongos , Animais , Organoides/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas do Olho/genética , Proteoglicanas/genética , Retinose Pigmentar/genética , Retina/metabolismo , Mutação , Células Fotorreceptoras Retinianas Cones/metabolismo , Fenótipo
9.
J Biol Chem ; 285(22): 16572-81, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20351095

RESUMO

In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To determine whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide cross-linking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand association but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Additionally, we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermolecular self-association.


Assuntos
Histonas/química , Nucleossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Centrifugação , Cromatina/química , Reagentes de Ligações Cruzadas/química , DNA Ribossômico/química , Dissulfetos/química , Conformação de Ácido Nucleico , Nucleossomos/química , Ligação Proteica , Xenopus
10.
Stem Cell Reports ; 10(2): 642-654, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29307579

RESUMO

Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.


Assuntos
Acetiltransferases/genética , Diferenciação Celular/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas , Acetiltransferases/química , Sistemas CRISPR-Cas/genética , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Genoma Humano/genética , Humanos
11.
Prog Retin Eye Res ; 58: 70-88, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28111324

RESUMO

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.


Assuntos
Bestrofinas/genética , DNA/genética , Oftalmopatias Hereditárias , Regulação da Expressão Gênica , Terapia Genética/métodos , Células Fotorreceptoras Retinianas Cones/patologia , Doenças Retinianas , Epitélio Pigmentado da Retina/patologia , Animais , Bestrofinas/biossíntese , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/terapia , Humanos , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Doenças Retinianas/terapia , Epitélio Pigmentado da Retina/metabolismo
12.
Invest Ophthalmol Vis Sci ; 57(5): ORSFf1-9, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27116663

RESUMO

As applications of human pluripotent stem cells (hPSCs) continue to be refined and pursued, it is important to keep in mind that the strengths and weaknesses of this technology lie with its developmental origins. The remarkable capacity of differentiating hPSCs to recapitulate cell and tissue genesis has provided a model system to study stages of human development that were not previously amenable to investigation and experimentation. Furthermore, demonstration of developmentally appropriate, stepwise differentiation of hPSCs to specific cell types offers support for their authenticity and their suitability for use in disease modeling and cell replacement therapies. However, limitations to farming cells and tissues in an artificial culture environment, as well as the length of time required for most cells to mature, are some of the many issues to consider before using hPSCs to study or treat a particular disease. Given the overarching need to understand and modulate the dynamics of lineage-specific differentiation in stem cell cultures, this review will first examine the capacity of hPSCs to serve as models of retinal development. Thereafter, we will discuss efforts to model retinal disorders with hPSCs and present challenges that face investigators who aspire to use such systems to study disease pathophysiology and/or screen for therapeutics. We also refer readers to recent publications that provide additional insight and details on these rapidly evolving topics.


Assuntos
Células-Tronco Pluripotentes/citologia , Retina/crescimento & desenvolvimento , Doenças Retinianas , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Humanos , Modelos Biológicos , Retina/embriologia , Doenças Retinianas/metabolismo , Doenças Retinianas/fisiopatologia
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