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1.
Drugs ; 79(11): 1255-1262, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31270752

RESUMO

Onasemnogene abeparvovec (onasemnogene abeparvovec-xioi; formerly AVXS-101; ZOLGENSMA®) is an adeno-associated viral vector-based gene therapy designed to deliver a functional copy of the human survival motor neuron (SMN) gene to the motor neuron cells of patients with spinal muscular atrophy (SMA). It has been developed by AveXis, a Novartis company, and was approved in May 2019 in the USA for the treatment of paediatric patients aged < 2 years with SMA and bi-allelic mutations in the SMN1 gene (the primary gene encoding survival motor neuron protein). Onasemnogene abeparvovec is the first gene therapy to be approved for SMA in the USA. The recommended dose is 1.1 × 1014 vector genomes per kg of bodyweight, administered as a single intravenous infusion over 60 min. Regulatory assessments for this formulation of onasemnogene abeparvovec are underway in the EU and Japan; an intrathecal formulation is currently undergoing clinical development in the USA. This article summarizes the milestones in the development of onasemnogene abeparvovec leading to this first approval for the treatment of paediatric patients aged < 2 years with SMA and bi-allelic mutations in SMN1.


Assuntos
Atrofia Muscular Espinal/terapia , Proteínas do Complexo SMN/metabolismo , Dependovirus/genética , Aprovação de Drogas , Terapia Genética , Vetores Genéticos , Humanos , Lactente , Recém-Nascido , Infusões Intravenosas , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Mutação , Proteínas do Complexo SMN/genética , Estados Unidos , United States Food and Drug Administration
2.
Int J Mol Sci ; 20(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064116

RESUMO

Hypercholesterolemia may be causally related to heart failure with preserved ejection fraction (HFpEF). We aimed to establish a HFpEF model associated with hypercholesterolemia and type 2 diabetes mellitus by feeding a high-sucrose/high-fat (HSHF) diet to C57BL/6J low-density lipoprotein receptor (LDLr)-/- mice. Secondly, we evaluated whether cholesterol-lowering adeno-associated viral serotype 8 (AAV8)-mediated LDLr gene transfer prevents HFpEF. AAV8-LDLr gene transfer strongly (p < 0.001) decreased plasma cholesterol in standard chow (SC) mice (66.8 ± 2.5 mg/dl versus 213 ± 12 mg/dl) and in HSHF mice (84.6 ± 4.4 mg/dl versus 464 ± 25 mg/dl). The HSHF diet induced cardiac hypertrophy and pathological remodeling, which were potently counteracted by AAV8-LDLr gene transfer. Wet lung weight was 19.0% (p < 0.001) higher in AAV8-null HSHF mice than in AAV8-null SC mice, whereas lung weight was normal in AAV8-LDLr HSHF mice. Pressure-volume loop analysis was consistent with HFpEF in AAV8-null HSHF mice and showed a completely normal cardiac function in AAV8-LDLr HSHF mice. Treadmill exercise testing demonstrated reduced exercise capacity in AAV8-null HSHF mice but a normal capacity in AAV8-LDLr HSHF mice. Reduced oxidative stress and decreased levels of tumor necrosis factor-α may mediate the beneficial effects of cholesterol lowering. In conclusion, AAV8-LDLr gene therapy prevents HFpEF.


Assuntos
Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/prevenção & controle , Terapia Genética/métodos , Insuficiência Cardíaca/prevenção & controle , Hipercolesterolemia/terapia , Receptores de LDL/genética , Animais , Colesterol/sangue , Dependovirus/genética , Diabetes Mellitus Tipo 2/etiologia , Cardiomiopatias Diabéticas/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/efeitos adversos , Feminino , Insuficiência Cardíaca/fisiopatologia , Hipercolesterolemia/complicações , Hipercolesterolemia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Receptores de LDL/metabolismo , Volume Sistólico , Fator de Necrose Tumoral alfa/sangue
3.
Nat Commun ; 10(1): 2315, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127098

RESUMO

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Medo/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Condicionamento (Psicologia)/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Modelos Animais , Neurônios/metabolismo , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Técnicas Estereotáxicas
4.
Methodist Debakey Cardiovasc J ; 15(1): 62-69, 2019 Jan-Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31049151

RESUMO

Cardiovascular disease is the leading cause of death worldwide, and elevated lipid levels is a major contributor. Gene delivery, which involves controlled transfer of nucleic acids into cells and tissues, has been widely used in research to study lipid metabolism and physiology. Several technologies have been developed to somatically overexpress, silence, or disrupt genes in animal models and have greatly advanced our knowledge of metabolism. This is particularly true with regard to the liver, which plays a central role in lipoprotein metabolism and is amenable to many delivery approaches. In addition to basic science applications, many of these delivery technologies have potential as gene therapies for both common and rare lipid disorders. This review discusses three major gene delivery technologies used in lipid research-including adeno-associated viral vector overexpression, antisense oligonucleotides and small interfering RNAs, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system-and examines their potential therapeutic applications.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Dislipidemias/terapia , Terapia Genética/métodos , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Animais , Sistemas CRISPR-Cas , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Dependovirus/genética , Dislipidemias/sangue , Dislipidemias/epidemiologia , Dislipidemias/genética , Edição de Genes/métodos , Terapia Genética/efeitos adversos , Vetores Genéticos , Humanos , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Resultado do Tratamento
5.
Cell Physiol Biochem ; 52(6): 1267-1279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31026390

RESUMO

BACKGROUND/AIMS: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. METHODS: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. RESULTS: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del. CONCLUSION: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Dependovirus/genética , Retículo Endoplasmático/genética , Linhagem Celular , Fibrose Cística/terapia , Terapia Genética , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Deleção de Sequência , Transfecção
6.
Nat Commun ; 10(1): 1802, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996254

RESUMO

The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart. Previously, we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation. Here, we report that expression of miR-19a/19b, members of the miR-17-92 cluster, is induced in heart failure patients. We show that intra-cardiac injection of miR-19a/19b mimics enhances cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction (MI) injury. miR-19a/19b protected the adult heart in two distinctive phases: an early phase immediately after MI and long-term protection. Genome-wide transcriptome analysis demonstrates that genes related to the immune response are repressed by miR-19a/19b. Using an adeno-associated virus approach, we validate that miR-19a/19b reduces MI-induced cardiac damage and protects cardiac function. Finally, we confirm the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI. Our study establishes miR-19a/19b as potential therapeutic targets to treat heart failure.


Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/patologia , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Infarto do Miocárdio/terapia , Animais , Proliferação de Células/genética , Dependovirus/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Insuficiência Cardíaca/terapia , Ventrículos do Coração/patologia , Humanos , Injeções Intralesionais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Regeneração/genética
7.
Proc Natl Acad Sci U S A ; 116(12): 5785-5794, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30833387

RESUMO

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1ß were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Transdução Genética/métodos , Animais , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Regiões Promotoras Genéticas/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transgenes , Visão Ocular/genética , Visão Ocular/fisiologia
8.
Neuron ; 101(5): 839-862, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30844402

RESUMO

Gene transfer has long been a compelling yet elusive therapeutic modality. First mainly considered for rare inherited disorders, gene therapy may open treatment opportunities for more challenging and complex diseases such as Alzheimer's or Parkinson's disease. Today, examples of striking clinical proof of concept, the first gene therapy drugs coming onto the market, and the emergence of powerful new molecular tools have broadened the number of avenues to target neurological disorders but have also highlighted safety concerns and technology gaps. The vector of choice for many nervous system targets currently is the adeno-associated viral (AAV) vector due to its desirable safety profile and strong neuronal tropism. In aggregate, the clinical success, the preclinical potential, and the technological innovation have made therapeutic AAV drug development a reality, particularly for nervous system disorders. Here, we discuss the rationale, opportunities, limitations, and progress in clinical AAV gene therapy.


Assuntos
Doença de Alzheimer/terapia , Marcação de Genes/métodos , Terapia Genética/métodos , Doença de Parkinson/terapia , Animais , Dependovirus/genética , Marcação de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Humanos
9.
Curr Protoc Neurosci ; 87(1): e66, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30883041

RESUMO

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.


Assuntos
Cálcio/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Neuroanatomia , Optogenética , Animais , Expressão Gênica/genética , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Optogenética/métodos
10.
Curr Protoc Neurosci ; 87(1): e67, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30901512

RESUMO

Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain. Each viral type offers unique advantages and limitations. To establish strategies for selecting a suitable viral type, this article aims to provide readers with an overview of viral recombinant technology, viral structure, tropism, and differences between serotypes and pseudotypes for three of the most commonly used vectors in neurobiology research: adeno-associated viruses, retro/lentiviruses, and glycoprotein-deleted rabies viruses. © 2019 by John Wiley & Sons, Inc.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Neurociências , Animais , Terapia Genética/métodos , Glicoproteínas/metabolismo , Humanos , Lentivirus/isolamento & purificação
11.
Nat Commun ; 10(1): 1365, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911003

RESUMO

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial surface receptor genetically linked to the risk for Alzheimer's disease (AD). A proteolytic product, soluble TREM2 (sTREM2), is abundant in the cerebrospinal fluid and its levels positively correlate with neuronal injury markers. To gain insights into the pathological roles of sTREM2, we studied sTREM2 in the brain of 5xFAD mice, a model of AD, by direct stereotaxic injection of recombinant sTREM2 protein or by adeno-associated virus (AAV)-mediated expression. We found that sTREM2 reduces amyloid plaque load and rescues functional deficits of spatial memory and long-term potentiation. Importantly, sTREM2 enhances microglial proliferation, migration, clustering in the vicinity of amyloid plaques and the uptake and degradation of Aß. Depletion of microglia abolishes the neuroprotective effects of sTREM2. Our study demonstrates a protective role of sTREM2 against amyloid pathology and related toxicity and suggests that increasing sTREM2 can be explored for AD therapy.


Assuntos
Doença de Alzheimer/terapia , Potenciação de Longa Duração/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Microglia/efeitos dos fármacos , Placa Amiloide/terapia , Receptores Imunológicos/genética , Memória Espacial/efeitos dos fármacos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções Intraventriculares , Potenciação de Longa Duração/fisiologia , Masculino , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/metabolismo , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fenótipo , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Cultura Primária de Células , Proteólise , Receptores Imunológicos/administração & dosagem , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Memória Espacial/fisiologia , Técnicas Estereotáxicas
12.
Methods Mol Biol ; 1961: 111-126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912043

RESUMO

Genome editing reagents including the recently introduced CRISPR/Cas9 system have become established and widely used molecular tools to answer fundamental biological questions and to target and treat genetic diseases. The CRISPR system, originally derived from bacteria and archaea, can be delivered into cells using different techniques, comprising (1) transfection of mRNA or plasmid DNA, (2) electroporation of plasmid DNA or the Cas9 protein in a complex with a g(uide)RNA, or (3) use of nonviral or viral vectors. Among the latter, Adeno-associated viruses (AAVs) are particularly attractive owing to many favorable traits: (1) their apathogenicity and episomal persistence, (2) the ease of virus production and purification, (3) the safe handling under lowest biosafety level 1 conditions, and (4) the availability of numerous natural serotypes and synthetic capsid variants with distinct cell specificities. Here, we describe a fast and simple protocol for small-scale packaging of CRISPR/Cas9 components into AAV vectors. To showcase its potential, we employ this method for screening of gRNAs targeting the murine miR-122 locus in Hepa1-6 cells (using AAV serotype 6, AAV6) or the 5'LTR of the human immunodeficiency virus (HIV) in HeLaP4-NLtr cells (using a synthetic AAV9 variant). We furthermore provide a detailed protocol for large-scale production of purified AAV/CRISPR vector stocks that permit higher cleavage efficiencies in vitro and are suitable for direct in vivo applications.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , RNA Guia/genética , Animais , Edição de Genes , Vetores Genéticos/genética , Humanos
13.
Methods Mol Biol ; 1961: 293-306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912053

RESUMO

CRISPR, a revolutionizing technology allowing researchers to navigate in and edit the genome, is moving on the fast track toward clinical use for ex vivo correction of disease-causing mutations in stem cells. As we await the first trials utilizing ex vivo CRISPR editing, implementation of CRISPR-based gene editing as an in vivo treatment directly in patients still remains an ultimate challenge. However, quickly accumulating evidence has provided proof-of-concept for efficacious editing in vivo. Attempts to edit genes directly in animals have largely relied on classical vector systems based on virus-based delivery of gene cassettes encoding the Cas9 endonuclease and single guide RNA, the key components of the CRISPR system. However, whereas persistent gene expression has been the primary goal of gene therapy for decades, things may be different in the case of CRISPR delivery. Is short-term presence of the CRISPR components perhaps sufficient for efficacy and ideal for safety?-and are strategies needed for restricting immune recognition of the bacteria-derived editing tool? Here, while answers to these questions still blow in the wind, we review prominent examples of genome editing with focus on targeting of genes with CRISPR in liver, muscles, and eyes of the mouse.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Terapia Genética/métodos , RNA Guia/genética , Animais , Dependovirus/genética , Humanos
14.
Nat Commun ; 10(1): 1221, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874546

RESUMO

Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensitivity and adaptation needed to restore patterned vision.


Assuntos
Cegueira/prevenção & controle , Opsinas dos Cones/genética , Terapia Genética/métodos , Optogenética/métodos , Degeneração Retiniana/terapia , Animais , Cegueira/etiologia , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Intravítreas , Queratinócitos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/complicações , Degeneração Retiniana/patologia , Rodopsina/genética , Resultado do Tratamento
16.
Exp Mol Med ; 51(2): 13, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755583

RESUMO

Vascular regeneration in ischemic hearts has been considered a target for new therapeutic strategies. It has been reported that ETV2 is essential for vascular development, injury-induced neovascularization and direct cell reprogramming of non-endothelial cells into endothelial cells. Thus, the objective of this study was to explore the therapeutic potential of ETV2 in murine models of myocardial infarction in vivo. Direct myocardial delivery of lentiviral ETV2 into rodents undergoing myocardial infarction dramatically upregulated the expression of markers for angiogenesis as well as anti-fibrosis and anti-inflammatory factors in vivo. Consistent with these findings, echocardiography showed significantly improved cardiac function in hearts with induced myocardial infarction upon ETV2 injection compared to that in the control virus-injected group as determined by enhanced ejection fraction and fractional shortening. In addition, ETV2-injected hearts were protected against massive fibrosis with a remarkable increase in capillary density. Interestingly, major fractions of capillaries were stained positive for ETV2. In addition, ECs infected with ETV2 showed enhanced proliferation, suggesting a direct role of ETV2 in vascular regeneration in diseased hearts. Furthermore, culture media from ETV2-overexpressing cardiac fibroblasts promoted endothelial cell migration based on scratch assay. Importantly, intramyocardial injection of the adeno-associated virus form of ETV2 into rat hearts with induced myocardial infarction designed for clinical applicability consistently resulted in significant augmentation of cardiac function. We provide compelling evidence that ETV2 has a robust effect on vascular regeneration and enhanced cardiac repair after myocardial infarction, highlighting a potential therapeutic function of ETV2 as an efficient means to treat failing hearts.


Assuntos
Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica/genética , Fatores de Transcrição/genética , Transdução Genética , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Dependovirus/genética , Modelos Animais de Doenças , Ecocardiografia , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Fibrose , Fluordesoxiglucose F18 , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Tomografia por Emissão de Pósitrons , Ratos , Fatores de Transcrição/metabolismo
17.
Nat Methods ; 16(3): 247-254, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804551

RESUMO

Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.


Assuntos
Dependovirus/genética , Receptores de Antígenos/genética , Linfócitos T/metabolismo , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Sequências Repetitivas de Ácido Nucleico , Linfócitos T/imunologia
18.
Bull Exp Biol Med ; 166(4): 527-534, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30793234

RESUMO

Mesenchymal stromal cells from rat adipose tissue were transduced with adeno-associated viral (AAV) vector encoding stem cell factor SCF that stimulates proliferation of cardiac c-kit+ cells and improved cardiac function and survival of animals after myocardial infarction. Extracellular vesicles isolated from the medium conditioned by mesenchymal stromal cells by ultracentrifugation were characterized by Western blotting, transmission electron microscopy, nanoparticle tracking analysis, immunostaining, and mass spectrometry analysis. Using proteomic analysis, we identified transgenic SCF in extracellular vesicles released by AAV-modified mesenchymal stromal cells and detected some proteins specific of extracellular vesicles secreted by transduced cells. Extracellular vesicles from AAV-transduced mesenchymal stromal cells could be used for delivery of transgenic proteins as they were readily endocytosed by both cardiosphere-derived cells and cardiac-progenitor cells.


Assuntos
Dependovirus/genética , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Células-Tronco/metabolismo , Tecido Adiposo/citologia , Animais , Células Cultivadas , Espectrometria de Massas , Proteômica/métodos , Ratos
19.
Methods Mol Biol ; 1950: 249-262, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30783978

RESUMO

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative "subILM" injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.).


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Retina/metabolismo , Transdução Genética , Animais , Expressão Gênica , Genes Reporter , Injeções , Macaca , Microscopia de Fluorescência , Células Ganglionares da Retina/metabolismo , Transgenes
20.
Methods Mol Biol ; 1950: 123-139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30783971

RESUMO

Degenerative retinal diseases such as retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA) may lead to blindness without effective treatment. With the rapid advancement of the CRISPR/Cas9 genome editing technology, in vivo application of CRISPR/Cas9 holds immense potential for treatment of these diseases. Adeno-associated virus (AAV) vectors are an ideal gene transfer tool for delivery of CRISPR components to the retina. Here, we describe a protocol for utilizing an AAV-based CRISPR/Cas9 system for in vivo genome editing in the retina.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus/genética , Edição de Genes , Vetores Genéticos/genética , Retina/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Dependovirus/isolamento & purificação , Eletrorretinografia , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/isolamento & purificação , Humanos , Imuno-Histoquímica , RNA Guia , Transdução Genética , Transgenes
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