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1.
Beijing Da Xue Xue Bao Yi Xue Ban ; 52(5): 845-850, 2020 Oct 18.
Artigo em Chinês | MEDLINE | ID: mdl-33047717

RESUMO

OBJECTIVE: To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa. METHODS: AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 µL purified AAV vectors (1.00×1013 mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope. RESULTS: No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells. CONCLUSION: AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.


Assuntos
Dependovirus , Vetores Genéticos , Animais , Dependovirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Retina , Sorogrupo , Transdução Genética
2.
Nat Commun ; 11(1): 4524, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913280

RESUMO

Traumatic brain injury (TBI) is a leading global cause of death and disability. Here we demonstrate in an experimental mouse model of TBI that mild forms of brain trauma cause severe deficits in meningeal lymphatic drainage that begin within hours and last out to at least one month post-injury. To investigate a mechanism underlying impaired lymphatic function in TBI, we examined how increased intracranial pressure (ICP) influences the meningeal lymphatics. We demonstrate that increased ICP can contribute to meningeal lymphatic dysfunction. Moreover, we show that pre-existing lymphatic dysfunction before TBI leads to increased neuroinflammation and negative cognitive outcomes. Finally, we report that rejuvenation of meningeal lymphatic drainage function in aged mice can ameliorate TBI-induced gliosis. These findings provide insights into both the causes and consequences of meningeal lymphatic dysfunction in TBI and suggest that therapeutics targeting the meningeal lymphatic system may offer strategies to treat TBI.


Assuntos
Lesões Encefálicas/fisiopatologia , Gliose/fisiopatologia , Sistema Glinfático/fisiologia , Meninges/fisiopatologia , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Lesões Encefálicas/terapia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Gliose/etiologia , Gliose/patologia , Gliose/prevenção & controle , Sistema Glinfático/patologia , Humanos , Masculino , Meninges/patologia , Camundongos , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/uso terapêutico
3.
PLoS Biol ; 18(8): e3000851, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822389

RESUMO

High levels of the amyloid-beta (Aß) peptide have been shown to disrupt neuronal function and induce hyperexcitability, but it is unclear what effects Aß-associated hyperexcitability may have on tauopathy pathogenesis or propagation in vivo. Using a novel transgenic mouse line to model the impact of human APP (hAPP)/Aß accumulation on tauopathy in the entorhinal cortex-hippocampal (EC-HIPP) network, we demonstrate that hAPP overexpression aggravates EC-Tau aggregation and accelerates pathological tau spread into the hippocampus. In vivo recordings revealed a strong role for hAPP/Aß, but not tau, in the emergence of EC neuronal hyperactivity and impaired theta rhythmicity. Chronic chemogenetic attenuation of EC neuronal hyperactivity led to reduced hAPP/Aß accumulation and reduced pathological tau spread into downstream hippocampus. These data strongly support the hypothesis that in Alzheimer's disease (AD), Aß-associated hyperactivity accelerates the progression of pathological tau along vulnerable neuronal circuits, and demonstrates the utility of chronic, neuromodulatory approaches in ameliorating AD pathology in vivo.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Córtex Entorrinal/metabolismo , Tauopatias/genética , Proteínas tau/genética , Potenciais de Ação/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Eletrodos Implantados , Córtex Entorrinal/patologia , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Agregados Proteicos , Técnicas Estereotáxicas , Tauopatias/metabolismo , Tauopatias/patologia , Tauopatias/terapia , Ritmo Teta/fisiologia , Transdução Genética , Transgenes , Proteínas tau/metabolismo
4.
J Exp Med ; 217(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-32750141

RESUMO

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Modelos Animais de Doenças , Interferon Tipo I/metabolismo , Camundongos/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Animais , Linhagem Celular Tumoral , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Dependovirus/genética , Feminino , Humanos , Inflamação/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Transdução de Sinais/genética , Replicação Viral/genética
5.
Nat Commun ; 11(1): 4148, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811834

RESUMO

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.


Assuntos
Desoxirribonucleases/genética , Dependovirus/genética , Infecções Oculares/terapia , Edição de Genes/métodos , Herpes Simples/terapia , Herpesvirus Humano 1/genética , Latência Viral/genética , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Chlorocebus aethiops , Infecções Oculares/genética , Infecções Oculares/virologia , Feminino , Células HEK293 , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/virologia , RNA-Seq , Análise de Célula Única , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/virologia , Células Vero
6.
Anticancer Res ; 40(8): 4425-4444, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727773

RESUMO

BACKGROUND/AIM: Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. MATERIALS AND METHODS: Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. RESULTS: The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. CONCLUSION: Melanoma gene therapy based on rAAV vectors is a possible treatment option.


Assuntos
Dependovirus/genética , Proteínas de Fluorescência Verde/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Melanoma Experimental/genética , Administração Intranasal , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Neoplasias Pulmonares/terapia , Masculino , Melanoma Experimental/terapia , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Transdução Genética , Resultado do Tratamento
7.
Nat Commun ; 11(1): 3279, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606306

RESUMO

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Sistema Nervoso Central/virologia , Microscopia Crioeletrônica , DNA Viral/análise , DNA Viral/genética , Dependovirus/classificação , Dependovirus/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Sorogrupo , Transdução Genética , Montagem de Vírus/genética
8.
Nat Commun ; 11(1): 3708, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709899

RESUMO

The Cre-loxP recombination system is a powerful tool for genetic manipulation. However, there are widely recognized limitations with chemically inducible Cre-loxP systems, and the UV and blue-light induced systems have phototoxicity and minimal capacity for deep tissue penetration. Here, we develop a far-red light-induced split Cre-loxP system (FISC system) based on a bacteriophytochrome optogenetic system and split-Cre recombinase, enabling optogenetical regulation of genome engineering in vivo solely by utilizing a far-red light (FRL). The FISC system exhibits low background and no detectable photocytotoxicity, while offering efficient FRL-induced DNA recombination. Our in vivo studies showcase the strong organ-penetration capacity of FISC system, markedly outperforming two blue-light-based Cre systems for recombination induction in the liver. Demonstrating its strong clinical relevance, we successfully deploy a FISC system using adeno-associated virus (AAV) delivery. Thus, the FISC system expands the optogenetic toolbox for DNA recombination to achieve spatiotemporally controlled, non-invasive genome engineering in living systems.


Assuntos
Engenharia Genética , Integrases/metabolismo , Integrases/efeitos da radiação , Luz , Recombinação Genética , Animais , Linhagem Celular , Sobrevivência Celular , Dependovirus/genética , Vetores Genéticos , Genoma , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Optogenética , Receptor EphB3
9.
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
10.
Nat Commun ; 11(1): 3253, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591505

RESUMO

Optogenetics has become an indispensable tool for investigating brain functions. Although non-human primates are particularly useful models for understanding the functions and dysfunctions of the human brain, application of optogenetics to non-human primates is still limited. In the present study, we generate an effective adeno-associated viral vector serotype DJ to express channelrhodopsin-2 (ChR2) under the control of a strong ubiquitous CAG promoter and inject into the somatotopically identified forelimb region of the primary motor cortex in macaque monkeys. ChR2 is strongly expressed around the injection sites, and optogenetic intracortical microstimulation (oICMS) through a homemade optrode induces prominent cortical activity: Even single-pulse, short-duration oICMS evokes long-lasting repetitive firings of cortical neurons. In addition, oICMS elicits distinct forelimb movements and muscle activity, which are comparable to those elicited by conventional electrical ICMS. The present study removes obstacles to optogenetic manipulation of neuronal activity and behaviors in non-human primates.


Assuntos
Membro Anterior/fisiologia , Córtex Motor/fisiologia , Movimento/fisiologia , Optogenética , Animais , Channelrhodopsins/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Macaca , Neurônios/fisiologia , Estimulação Física
11.
Nat Med ; 26(7): 1096-1101, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483358

RESUMO

Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.


Assuntos
Anticorpos Neutralizantes/imunologia , Dependovirus/genética , Terapia Genética , Vetores Genéticos/efeitos adversos , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Dependovirus/imunologia , Endopeptidases/imunologia , Vetores Genéticos/uso terapêutico , Humanos , Imunoglobulina G/farmacologia , Fígado/imunologia , Fígado/metabolismo , Camundongos
12.
Virology ; 546: 127-132, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452411

RESUMO

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Regulação Viral da Expressão Gênica , Motivos de Aminoácidos , Proteínas do Capsídeo/genética , Dependovirus/química , Dependovirus/fisiologia , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Montagem de Vírus , Replicação Viral
13.
Arterioscler Thromb Vasc Biol ; 40(7): 1722-1737, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32404007

RESUMO

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Terapia Genética , Linfangiogênese , Vasos Linfáticos/metabolismo , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Interferon gama/metabolismo , Vasos Linfáticos/imunologia , Vasos Linfáticos/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Miocárdio/imunologia , Miocárdio/patologia , Ratos Wistar , Recuperação de Função Fisiológica , Transdução de Sinais , Fatores de Tempo , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Função Ventricular Esquerda
14.
Invest Ophthalmol Vis Sci ; 61(5): 30, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32428231

RESUMO

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.


Assuntos
Dependovirus/genética , Proteínas do Olho/genética , Terapia Genética , Amaurose Congênita de Leber/terapia , Proteínas Associadas aos Microtúbulos/genética , Retina/fisiopatologia , Adulto , Animais , Criança , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Terapia Genética/métodos , Vetores Genéticos , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Fenótipo , Pupila/fisiologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Testes de Campo Visual , Campos Visuais/fisiologia , Adulto Jovem
15.
PLoS Biol ; 18(3): e3000686, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226015

RESUMO

Compact CRISPR/Cas9 systems that can be packaged into an adeno-associated virus (AAV) hold great promise for gene therapy. Unfortunately, currently available small Cas9 nucleases either display low activity or require a long protospacer adjacent motif (PAM) sequence, limiting their extensive applications. Here, we screened a panel of Cas9 nucleases and identified a small Cas9 ortholog from Staphylococcus auricularis (SauriCas9), which recognizes a simple NNGG PAM, displays high activity for genome editing, and is compact enough to be packaged into an AAV for genome editing. Moreover, the conversion of adenine and cytosine bases can be achieved by fusing SauriCas9 to the cytidine and adenine deaminase. Therefore, SauriCas9 holds great potential for both basic research and clinical applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , DNA/metabolismo , Edição de Genes/métodos , Staphylococcus/enzimologia , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , DNA/química , DNA/genética , Dependovirus/genética , Células HEK293 , Humanos , Motivos de Nucleotídeos , Engenharia de Proteínas , Staphylococcus/genética , Especificidade por Substrato
16.
PLoS Biol ; 18(4): e3000665, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275651

RESUMO

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.


Assuntos
Sistemas CRISPR-Cas , Epitopos/genética , Edição de Genes/métodos , Neurônios/imunologia , Proteínas/genética , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Vetores Genéticos , Genoma , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos Transgênicos , Microscopia de Fluorescência , Imagem Molecular/métodos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Proteínas/imunologia , Proteínas/metabolismo , Ratos Wistar , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Análise Espaço-Temporal
17.
J Neurosci ; 40(20): 3896-3914, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32300046

RESUMO

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.


Assuntos
Regulação da Expressão Gênica/genética , Edição de RNA/genética , Células Ganglionares da Retina/metabolismo , gama-Sinucleína/genética , Animais , Sistemas CRISPR-Cas , Dependovirus/genética , Feminino , Deleção de Genes , Terapia Genética , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico/patologia , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/terapia , Células Ganglionares da Retina/patologia , Transgenes/genética
18.
J Vet Sci ; 21(2): e26, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233134

RESUMO

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.


Assuntos
Sistemas CRISPR-Cas , Campylobacter jejuni/genética , Marcação de Genes , Neoplasias Pancreáticas/microbiologia , Streptococcus pyogenes/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos
19.
Nat Methods ; 17(5): 541-550, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313222

RESUMO

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood-brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications.


Assuntos
Encéfalo/virologia , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/genética , Integrases/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Proteínas do Capsídeo/genética , Feminino , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Tropismo Viral
20.
Cell ; 181(1): 136-150, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32243786

RESUMO

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.


Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Edição de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Animais , Humanos
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