RESUMO
Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-ß1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.
Assuntos
Doenças Retinianas , Neovascularização Retiniana , Animais , Humanos , Camundongos , Ratos , Citocinas/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Lactonas/uso terapêutico , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , NF-kappa B , Oxigênio , Doenças Retinianas/patologia , Neovascularização Retiniana/metabolismoRESUMO
Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports a potential role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes.
Assuntos
Estudo de Associação Genômica Ampla , Degeneração Macular , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epitélio Pigmentado da Retina/metabolismo , Degeneração Macular/metabolismo , Estresse Oxidativo , Epitélio/metabolismoRESUMO
Angiogenesis plays a critical role in both physiological responses and disease pathogenesis. Excessive angiogenesis can promote neoplastic diseases and retinopathies, while inadequate angiogenesis can lead to aberrant perfusion and impaired wound healing. Transforming growth factor ß activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is a key modulator involved in a range of cellular functions including the immune responses, cell survival and death. TAK1 is activated in response to various stimuli such as proinflammatory cytokines, hypoxia, and oxidative stress. Emerging evidence has recently suggested that TAK1 is intimately involved in angiogenesis and mediates pathogenic processes related to angiogenesis. Several detailed mechanisms by which TAK1 regulates pathological angiogenesis have been clarified, and potential therapeutics targeting TAK1 have emerged. In this review, we summarize recent studies of TAK1 in angiogenesis and discuss the crosstalk between TAK1 and signaling pathways involved in pathological angiogenesis. We also discuss the approaches for selectively targeting TAK1 and highlight the rationales of therapeutic strategies based on TAK1 inhibition for the treatment of pathological angiogenesis.
Assuntos
Sistemas de Liberação de Medicamentos , MAP Quinase Quinase Quinases/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Transdução de Sinais , Animais , Humanos , Neovascularização Patológica/patologiaRESUMO
Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
Assuntos
Terapia Genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Animais , Hipóxia Celular , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Injeções Intravítreas , Domínios Proteicos , Ratos Sprague-Dawley , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Transgenes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.
Assuntos
Oftalmopatias/patologia , Oftalmopatias/terapia , Terapia Genética , Neovascularização Patológica/terapia , Animais , Sistemas CRISPR-Cas , Ensaios Clínicos como Assunto , Gerenciamento Clínico , Suscetibilidade a Doenças , Oftalmopatias/etiologia , Edição de Genes , Terapia Genética/métodos , Humanos , Neovascularização Patológica/genética , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE OF REVIEW: Diabetic retinopathy (DR), a leading cause of visual impairment in the developed country, is characterized by vascular lesions and neuronal damage of the retina. Treatment options for this condition are currently limited. The advent of therapy targeting vascular endothelial growth factor (VEGF) demonstrated significant benefits to patients with DR. However, this treatment is limited by its short half-life and requirement for frequent invasive intravitreal injections. In addition, many patients failed to achieve clinically significant improvement in visual function. Gene therapy has the potential to provide an alternative treatment for DR with distinct advantages, such as longer therapeutic effect, less injection frequency, ability to intervene at disease onset, and potentially fewer side effects. RECENT FINDINGS: Strategies for gene therapy in DR, stemming from the current understanding of the disease pathogenesis, focus on the inhibition of neovascularization and protection of neurovascular degeneration in the retina. Studies with promising results have mainly focussed on animal models due to efficacy and safety concerns, despite a number of successful preclinical studies using adeno-associated virus-mediated transduction to treat both vascular dysfunction and neuronal degeneration. With the optimization of delivery vectors, transgene regulation, and outcome measure, gene therapy will potentially become available for patients with DR. This review provides an update on the current strategies utilized in DR gene therapy research. Several barriers to the clinical application of gene therapy for DR are highlighted, and future directions for this research are proposed.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Animais , Retinopatia Diabética/genética , Retinopatia Diabética/terapia , Terapia Genética , Humanos , Neovascularização Patológica , Retina , Fator A de Crescimento do Endotélio VascularRESUMO
Choroidal neovascularization (CNV) is a severe complication of the wet form of age-related macular degeneration (AMD). Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of different ocular neovascular diseases. To identify the function and therapeutic potential of lncRNAs in CNV, we assessed lncRNAs and mRNA expression profile in a mouse model of laser-induced CNV by microarray analysis. The results of altered lncRNAs were validated by qRT-PCR. Bioinformatics analyses, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were performed to clarify the potential biological functions and signaling pathways with which altered genes are most closely related. Moreover, to identify the interaction of lncRNAs and mRNAs, we constructed a coding-non-coding gene co-expression (CNC) network. By microarray analysis, we identified 716 altered lncRNAs and 821 altered mRNAs in CNV mice compared to controls. A CNC network profile based on 7 validated altered lncRNAs (uc009ewo.1, AK148935, uc029sdr.1, ENSMUST00000132340, AK030988, uc007mds.1, ENSMUST00000180519) as well as 282 interacted and altered mRNAs, and were connected by 713 edges. GO and KEGG analyses suggested that altered mRNAs, as well as those lncRNA-interacted mRNAs were enriched in immune system process and chemokine signaling pathway. Thus, lncRNAs are significantly altered in this mouse model of CNV and are involved in immunological regulation, suggesting that lncRNAs may play a critical role in the pathogenesis of CNV. Thus, dysregulated lncRNAs and their target genes might be promising therapeutic targets to suppress CNV in AMD.
Assuntos
Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , RNA Longo não Codificante/metabolismo , Transdução de Sinais/fisiologia , Animais , Neovascularização de Coroide/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Retinal neovascularization (RNV) is characterized in retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO), which leads to severe vision loss and even blindness. To reveal the altered transfer RNA-derived small RNA (tsRNA)s in RNV, and to investigate the underlying mechanisms of the altered tsRNAs involved in RNV, we carried out a small RNA sequencing to profile tsRNA expressions in the retinas of mice with oxygen-induced retinopathy (OIR) and control mice. A total of 45 tsRNAs were significantly changed (fold change ≥ 1.5 and P < 0.05) in the retinas of OIR mice compared with controls. Validation by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in four selected tsRNAs was consistent with the results of small RNA sequencing. Bioinformatics analyses identified 153 altered target genes of the four validated tsRNAs. These altered target genes were largely enriched in developmental process, cell periphery and protein binding, as well as Th1 and Th2 cell differentiation pathway. Our study suggests tsRNAs play key roles in the pathogenesis of RNV, indicating their therapeutic potential to treat patients with RNV. Moreover, small RNA sequencing is a useful tool to identify changes in tsRNA expression, an important indicator of the progress of retinal diseases.
Assuntos
Retinopatia Diabética/genética , RNA de Transferência/genética , Neovascularização Retiniana/genética , Oclusão da Veia Retiniana/genética , Retinopatia da Prematuridade/genética , Animais , Cegueira/genética , Cegueira/patologia , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Oxigênio/toxicidade , RNA Mensageiro/genética , RNA de Transferência/classificação , Retina/crescimento & desenvolvimento , Retina/patologia , Neovascularização Retiniana/patologia , Oclusão da Veia Retiniana/patologia , Retinopatia da Prematuridade/induzido quimicamente , Retinopatia da Prematuridade/patologia , Análise de Sequência de RNA , Células Th1/metabolismo , Células Th1/patologia , Células Th2/metabolismo , Células Th2/patologia , Transcriptoma/genéticaRESUMO
Objective: Retinal neovascularization is a severe complication of many ocular diseases. To clarify the possible functions and therapeutic potential of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in retinal neovascularization, we assessed their expression profile in a mouse model of oxygen-induced retinopathy (OIR). Methods: Microarray analysis was performed to identify altered lncRNA and mRNA expressions between OIR and control mice. The microarray results were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to determine biological functions and signaling pathways of the altered or interacted mRNAs. A coding-non-coding gene co-expression (CNC) network was constructed to identify the interaction of lncRNAs and mRNAs. Results: We identified 198 up-regulated and 175 down-regulated lncRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. We also identified 412 up-regulated and 127 down-regulated mRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. GO and KEGG analyses suggested that altered mRNAs were enriched in immune system process, exopeptidase activity, ECM-receptor interaction and protein digestion and absorption. Four validated lncRNAs (ENSMUST00000165968, ENSMUST00000153785, ENSMUST00000134409, and ENSMUST00000154285) and the nearby coding gene pairs were analyzed. A CNC network profile based on those validated altered lncRNAs as well as 410 interacted mRNAs was composed of 509 connections. Moreover, the GO and KEGG analyses demonstrated that these interacted mRNAs mainly enriched in blood vessel development, angiogenesis, cell adhesion molecules and leukocyte transendothelial migration pathways. Conclusion: Our data highlight the utility of altered lncRNA and mRNA profiling in understanding the pathogenesis of ischemia-induced retinal neovascularization and further suggest that therapeutic potential of altered lncRNA for retinal neovascularization.
Assuntos
RNA Longo não Codificante/genética , RNA Mensageiro/genética , Neovascularização Retiniana/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Ontologia Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , RNA Longo não Codificante/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Transdução de Sinais/genéticaRESUMO
Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.
Assuntos
Redes Reguladoras de Genes , RNA Circular/metabolismo , Retina/patologia , Neovascularização Retiniana/genética , Animais , Biologia Computacional , Modelos Animais de Doenças , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/toxicidade , RNA Circular/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sistema Renina-Angiotensina/genética , Retina/efeitos dos fármacos , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/patologia , Regulação para CimaRESUMO
Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.
Assuntos
Inibidores da Angiogênese/biossíntese , Calreticulina , Dependovirus , Retinopatia Diabética , Neovascularização Retiniana , Transdução Genética , Inibidores da Angiogênese/genética , Angiografia , Animais , Calreticulina/biossíntese , Calreticulina/genética , Retinopatia Diabética/diagnóstico por imagem , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Eletrorretinografia , Feminino , Vetores Genéticos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Camundongos , Ratos , Ratos Sprague-Dawley , Neovascularização Retiniana/diagnóstico por imagem , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/fisiopatologia , Tomografia de Coerência Óptica , Proteína Vermelha FluorescenteRESUMO
Corneal neovascularization, the growth of new blood vessels in the cornea, is a leading cause of vision impairment after corneal injury. Neovascularization typically occurs in response to corneal injury such as that caused by infection, physical trauma, chemical burns or in the setting of corneal transplant rejection. The NADPH oxidase enzyme complex is involved in cell signalling for wound-healing angiogenesis, but its role in corneal neovascularization has not been studied. We have now analysed the role of the Nox2 isoform of NADPH oxidase in corneal neovascularization in mice following chemical injury. C57BL/6 mice aged 8-14 weeks were cauterized with an applicator coated with 75% silver nitrate and 25% potassium nitrate for 8 s. Neovascularization extending radially from limbal vessels was observed in corneal whole-mounts from cauterized wild type mice and CD31+ vessels were identified in cauterized corneal sections at day 7. In contrast, in Nox2 knockout (Nox2 KO) mice vascular endothelial growth factor-A (Vegf-A), Flt1 mRNA expression, and the extent of corneal neovascularization were all markedly reduced compared with their wild type controls. The accumulation of Iba-1+ microglia and macrophages in the cornea was significantly less in Nox2 KO than in wild type mice. In conclusion, we have demonstrated that Nox2 is implicated in the inflammatory and neovascular response to corneal chemical injury in mice and clearly VEGF is a mediator of this effect. This work raises the possibility that therapies targeting Nox2 may have potential for suppressing corneal neovascularization and inflammation in humans.
Assuntos
Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/enzimologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Animais , Biomarcadores/metabolismo , Queimaduras/enzimologia , Queimaduras/patologia , Cauterização , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/patologia , Regulação da Expressão Gênica , Imuno-Histoquímica , Inflamação/patologia , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
Assuntos
Dependovirus , Vetores Genéticos , Humanos , Dependovirus/genética , Vetores Genéticos/genética , Terapia GenéticaRESUMO
Retinal neovascularization is one of the leading causes of vision loss and a hallmark of proliferative diabetic retinopathy (PDR). The immune system is observed to be involved in the pathogenesis of diabetic retinopathy (DR). The specific immune cell type that contributes to retinal neovascularization can be identified via a bioinformatics analysis of RNA sequencing (RNA-seq) data, known as deconvolution analysis. Previous study has identified the infiltration of macrophages in the retina of rats with hypoxia-induced retinal neovascularization and patients with PDR through a deconvolution algorithm, known as CIBERSORTx. Here, we describe the protocols of using CIBERSORTx to perform the deconvolution analysis and downstream analysis of RNA-seq data.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Neovascularização Retiniana , Ratos , Animais , Retinopatia Diabética/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Retina/metabolismo , Hipóxia/complicações , Expressão GênicaRESUMO
Translational research is heavily dependent on animal models, and reliable disease models are essential for the development of novel therapies. Here, we outline the methods for culturing mouse and human retinal explants. In addition, we show efficient adeno-associated virus (AAV) transduction of the mouse retinal explants to aid the study and development of AAV-based therapeutics against ocular diseases.
Assuntos
Dependovirus , Roedores , Humanos , Camundongos , Animais , Dependovirus/genética , Retina , Visão Ocular , Vetores Genéticos/genética , Transdução GenéticaRESUMO
Purpose: Age is the main risk factor for age-related macular degeneration (AMD), a leading cause of blindness in the elderly, with limited therapeutic options. Methods: Here, we analyze the transcriptomic characteristics and cellular landscape of the aging retinas from controls and patients with AMD. Results: We identify the aging genes in the neural retina, which are associated with innate immune response and inflammation. Deconvolution analysis reveals that the estimated proportions of M2 macrophages are significantly increased with both age and AMD severity. Moreover, we find that proportions of Müller glia are significantly increased only with age but not with AMD severity. Several genes associated with both age and AMD severity, particularly C1s and MR1, are strong positively correlated with the proportions of Müller glia. Conclusions: Our studies expand the genetic and cellular landscape of AMD and provide avenues for further studies on the relationship between age and AMD.
Assuntos
Degeneração Macular , Transcriptoma , Humanos , Idoso , Retina , Degeneração Macular/genética , Envelhecimento/genética , NeurogliaRESUMO
Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.
Assuntos
Oftalmopatias , RNA , Humanos , RNA/genética , RNA/uso terapêutico , Edição de Genes , Sistemas CRISPR-Cas/genética , Terapia Genética , Oftalmopatias/genéticaRESUMO
Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line ARPE-19 with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that it retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 caused a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2 in ARPE-19, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.
Assuntos
Degeneração Macular , Superóxidos , Humanos , Superóxidos/metabolismo , Degeneração Macular/genética , Degeneração Macular/patologia , Estresse Oxidativo/genética , Epitélio Pigmentado da Retina/patologia , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Purpose: Previous studies that identify putative genes associated with diabetic retinopathy are only focusing on specific clinical stages, thus resulting genes are not necessarily reflective of disease progression. This study identified genes associated with the severity level of diabetic retinopathy using the likelihood-ratio test (LRT) and ordinal logistic regression (OLR) model, as well as to profile immune and retinal cell landscape in progressive diabetic retinopathy using a machine learning deconvolution approach. Methods: This study used a published transcriptomic dataset (GSE160306) from macular regions of donors with different degrees of diabetic retinopathy (10 healthy controls, 10 cases of diabetes, 9 cases of nonproliferative diabetic retinopathy, and 10 cases of proliferative diabetic retinopathy or combined with diabetic macular edema). LRT and OLR models were applied to identify severity-associated genes. In addition, CIBERSORTx was used to estimate proportional changes of immune and retinal cells in progressive diabetic retinopathy. Results: By controlling for gender and age using LRT and OLR, 50 genes were identified to be significantly increased in expression with the severity of diabetic retinopathy. Functional enrichment analyses suggested these severity-associated genes are related to inflammation and immune responses. CCND1 and FCGR2B are further identified as key regulators to interact with many other severity-associated genes and are crucial to inflammation. Deconvolution analyses demonstrated that the proportions of memory B cells, M2 macrophages, and Müller glia were significantly increased with the progression of diabetic retinopathy. Conclusions: These findings demonstrate that deep analyses of transcriptomic data can advance our understanding of progressive ocular diseases, such as diabetic retinopathy, by applying LRT and OLR models as well as bulk gene expression deconvolution.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Retinopatia Diabética/genética , Progressão da Doença , Seguimentos , Humanos , Inflamação , TranscriptomaRESUMO
PURPOSE: To evaluate anatomic and functional intereye symmetry among individuals with Bietti crystalline dystrophy (BCD) using clinical and multimodal imaging methods, with a focus on the number, area, and distribution of the characteristic retinal crystalline deposits. DESIGN: Observational case series with prospective and retrospective data. METHODS: Setting: Multicenter. STUDY POPULATION: Thirteen Australian and New Zealand participants (26 eyes) with confirmed biallelic CYP4V2 mutations and a characteristic BCD fundus appearance. Procedures and main outcome measures: Crystals visible on color fundus photography were manually counted. Crystals were superimposed on aligned multimodal fundus images. Spearman's correlation coefficients (ρ), intraclass correlation coefficients (ICCs), and Bland-Altman plots were used to quantify symmetry between eyes. MAIN OUTCOME MEASURES: Fundus crystal area and count, and absent-autofluorescence (absent-AF) area. RESULTS: Median participant age was 48 years (interquartile range: 40-60 years). Intereye symmetry was high for fundus crystal area (ρ = 1.00, 95% confidence interval [CI]: 1.00-1.00; ICC = 0.97, 95% CI: 0.88-0.99), fundus crystal count (ρ = 0.98, 95% CI: 0.92-1.00; ICC = 0.97, 95% CI: 0.89-0.99), and absent-AF area (ρ = 0.88, 95% CI: 0.53-0.98; ICC = 0.98, 95% CI: 0.90-0.99). Average foveal volume, foveal crystal count and area, average and central foveal thickness, best corrected visual acuity, and average macular and central foveal sensitivity were not highly correlated between eyes. CONCLUSIONS: This study demonstrated strong intereye symmetry measured by fundus crystal area, fundus crystal number, and absent-AF area. This may influence the choice of outcome measures for future therapeutic trials for BCD and provides valuable clinical information for ophthalmologists involved in the care and counseling of patients with BCD.