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1.
N Engl J Med ; 383(2): 151-158, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640133

RESUMO

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.


Assuntos
Esclerose Amiotrófica Lateral/terapia , MicroRNAs/uso terapêutico , Superóxido Dismutase-1/líquido cefalorraquidiano , Esclerose Amiotrófica Lateral/líquido cefalorraquidiano , Esclerose Amiotrófica Lateral/genética , Dependovirus , Evolução Fatal , Inativação Gênica , Terapia Genética , Vetores Genéticos , Humanos , Injeções Espinhais , Masculino , Meningoencefalite , Pessoa de Meia-Idade , Mutação , Estudo de Prova de Conceito , Medula Espinal/química , Medula Espinal/patologia , Superóxido Dismutase-1/análise , Superóxido Dismutase-1/genética , Capacidade Vital , Adulto Jovem
2.
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
3.
Proc Natl Acad Sci U S A ; 117(18): 10003-10014, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32300008

RESUMO

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.


Assuntos
Cromatina/genética , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Epigenômica/métodos , Fatores de Transcrição/genética , Algoritmos , Animais , Anticorpos/genética , Sítios de Ligação/genética , Cromatina/virologia , Dependovirus/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Integrases/genética , Camundongos , Distribuição Tecidual/genética
4.
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
5.
Sci China Life Sci ; 63(6): 875-885, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32266609

RESUMO

Brain-to-brain interfaces (BtBIs) hold exciting potentials for direct communication between individual brains. However, technical challenges often limit their performance in rapid information transfer. Here, we demonstrate an optical brain-to-brain interface that transmits information regarding locomotor speed from one mouse to another and allows precise, real-time control of locomotion across animals with high information transfer rate. We found that the activity of the genetically identified neuromedin B (NMB) neurons within the nucleus incertus (NI) precisely predicts and critically controls locomotor speed. By optically recording Ca2+ signals from the NI of a "Master" mouse and converting them to patterned optogenetic stimulations of the NI of an "Avatar" mouse, the BtBI directed the Avatar mice to closely mimic the locomotion of their Masters with information transfer rate about two orders of magnitude higher than previous BtBIs. These results thus provide proof-of-concept that optical BtBIs can rapidly transmit neural information and control dynamic behaviors across individuals.


Assuntos
Interfaces Cérebro-Computador , Encéfalo/fisiologia , Locomoção/fisiologia , Imagem Óptica/métodos , Animais , Controle Comportamental , Comportamento Animal/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Simulação por Computador , Dependovirus/metabolismo , Células HEK293 , Humanos , Cinética , Camundongos , Modelos Biológicos , Neurocinina B/análogos & derivados , Neurocinina B/fisiologia , Neurônios/fisiologia , Núcleos da Rafe/fisiologia , Máquina de Vetores de Suporte , Transfecção
6.
EMBO Mol Med ; 12(5): e12059, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32320128

RESUMO

Undoubtedly, vaccination is one of the health interventions showing major impact on humankind. Vaccines remain one of the most effective and safest ways to tackle infections. The current coronavirus pandemic is not an exception, and we all hope that ongoing international efforts will succeed in developing a vaccine soon. In this scenario, the present work published in this edition of EMBO Molecular Medicine by Demminger and colleagues (Demminger et al, 2020) is timeliness to exemplify the steps needed to develop effective vaccines.


Assuntos
Vacinas contra Influenza , Influenza Humana , Dependovirus , Humanos , Pandemias , Receptores de IgG
7.
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
8.
Proc Natl Acad Sci U S A ; 117(11): 6023-6034, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32132200

RESUMO

Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.


Assuntos
Cerebelo/patologia , Clorzoxazona/administração & dosagem , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Mitocôndrias/patologia , Degenerações Espinocerebelares/genética , Adolescente , Animais , Animais Recém-Nascidos , Linhagem Celular , Cerebelo/citologia , Análise Mutacional de DNA , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mutação com Perda de Função , Camundongos , Oócitos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/tratamento farmacológico , Degenerações Espinocerebelares/patologia , Transfecção , Sequenciamento Completo do Exoma , Xenopus
9.
BMC Infect Dis ; 20(1): 185, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111171

RESUMO

BACKGROUND: Staphylococcus aureus (S. aureus) arthritis is one of the most detrimental joint diseases known and leads to severe joint destruction within days. We hypothesized that the provision of auxiliary immunoregulation via an expanded compartment of T regulatory cells (Tregs) could dampen detrimental aspects of the host immune response whilst preserving its protective nature. Administration of low-dose interleukin 2 (IL2) preferentially expands Tregs, and is being studied as a treatment choice in several autoimmune conditions. We aimed to evaluate the role of IL2 and Tregs in septic arthritis using a well-established mouse model of haematogenously spred S. aureus arthritis. METHODS: C57BL/6 or NMRI mice we intravenously (iv) injected with a defined dose of S. aureus LS-1 or Newman and the role of IL2 and Tregs were assessed by the following approaches: IL2 was endogenously delivered by intraperitoneal injection of a recombinant adeno-associated virus vector (rAAV) before iv S. aureus inoculation; Tregs were depleted before and during S. aureus arthritis using antiCD25 antibodies; Tregs were adoptively transferred before induction of S. aureus arthritis and finally, recombinant IL2 was used as a treatment starting day 3 after S. aureus injection. Studied outcomes included survival, weight change, bacterial clearance, and joint damage. RESULTS: Expansion of Tregs induced by IL2 gene therapy prior to disease onset does not compromise host resistance to S. aureus infection, as the increased proportions of Tregs reduced the arthritis severity as well as the systemic inflammatory response, while simultaneously preserving the host's ability to clear the infection. CONCLUSIONS: Pre-treatment with IL2 gene therapy dampens detrimental immune responses but preserves appropriate host defense, which alleviates S. aureus septic arthritis in a mouse model.


Assuntos
Artrite Infecciosa/prevenção & controle , Terapia Genética , Interleucina-2/genética , Staphylococcus aureus/patogenicidade , Animais , Anticorpos Monoclonais/uso terapêutico , Artrite Infecciosa/etiologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/metabolismo , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
10.
Curr Opin Ophthalmol ; 31(3): 147-154, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32175942

RESUMO

PURPOSE OF REVIEW: Gene therapy offers, for the first time, the possibility to cure diseases such as retinitis pigmentosa. The positive outcomes that led to the U.S. Food and Drug Administration (FDA) approval of Luxturna to treat Leber congenital amaurosis caused by RPE65 mutations created an optimistic atmosphere in the research, clinical and patient community. Despite this first success, we must understand that this is not a 'one treatment for all'. This review aims to explain the basic concepts of gene therapy and how they translate in different approaches that are utilized in ongoing clinical trials here reviewed. RECENT FINDINGS: In 2017, the FDA approved the first gene therapy treatment. In parallel, other approaches have gained attention. Different delivery methods (adeno-associated virus, lentivirus), injection sites (subretinal, intravitreal, suprachoroidal) and methodologies (gene replacement, silencing, editing) are currently being tested. SUMMARY: Gene therapy is an evolving field in medicine and ophthalmology. Its success and application depends on several factors that are specific to the disease to treat. For now, we know it's a relatively safe approach and we look forward to the continued advancements of current ongoing clinical trials.


Assuntos
Edição de Genes , Inativação Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Degeneração Retiniana/terapia , Dependovirus/genética , Humanos , Lentivirus/genética , Degeneração Retiniana/genética
11.
Am J Physiol Heart Circ Physiol ; 318(4): H840-H852, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142359

RESUMO

Diabetic cardiomyopathy is a distinct form of heart disease that represents a major cause of death and disability in diabetic patients, particularly, the more prevalent type 2 diabetes patient population. In the current study, we investigated whether administration of recombinant adeno-associated viral vectors carrying a constitutively active phosphoinositide 3-kinase (PI3K)(p110α) construct (rAAV6-caPI3K) at a clinically relevant time point attenuates diabetic cardiomyopathy in a preclinical type 2 diabetes (T2D) model. T2D was induced by a combination of a high-fat diet (42% energy intake from lipid) and low-dose streptozotocin (three consecutive intraperitoneal injections of 55 mg/kg body wt), and confirmed by increased body weight, mild hyperglycemia, and impaired glucose tolerance (all P < 0.05 vs. nondiabetic mice). After 18 wk of untreated diabetes, impaired left ventricular (LV) systolic dysfunction was evident, as confirmed by reduced fractional shortening and velocity of circumferential fiber shortening (Vcfc, all P < 0.01 vs. baseline measurement). A single tail vein injection of rAAV6-caPI3K gene therapy (2×1011vector genomes) was then administered. Mice were followed for an additional 8 wk before end point. A single injection of cardiac targeted rAAV6-caPI3K attenuated diabetes-induced cardiac remodeling by limiting cardiac fibrosis (reduced interstitial and perivascular collagen deposition, P < 0.01 vs. T2D mice) and cardiomyocyte hypertrophy (reduced cardiomyocyte size and Nppa gene expression, P < 0.001 and P < 0.05 vs. T2D mice, respectively). The diabetes-induced LV systolic dysfunction was reversed with rAAV6-caPI3K, as demonstrated by improved fractional shortening and velocity of circumferential fiber shortening (all P < 0.05 vs pre-AAV measurement). This cardioprotection occurred in combination with reduced LV reactive oxygen species (P < 0.05 vs. T2D mice) and an associated decrease in markers of endoplasmic reticulum stress (reduced Grp94 and Chop, all P < 0.05 vs. T2D mice). Together, our findings demonstrate that a cardiac-selective increase in PI3K(p110α), via rAAV6-caPI3K, attenuates T2D-induced diabetic cardiomyopathy, providing proof of concept for potential translation to the clinic.NEW & NOTEWORTHY Diabetes remains a major cause of death and disability worldwide (and its resultant heart failure burden), despite current care. The lack of existing management of heart failure in the context of the poorer prognosis of concomitant diabetes represents an unmet clinical need. In the present study, we now demonstrate that delayed intervention with PI3K gene therapy (rAAV6-caPI3K), administered as a single dose in mice with preexisting type 2 diabetes, attenuates several characteristics of diabetic cardiomyopathy, including diabetes-induced impairments in cardiac remodeling, oxidative stress, and function. Our discovery here contributes to the previous body of work, suggesting the cardioprotective effects of PI3K(p110α) could be a novel therapeutic approach to reduce the progression to heart failure and death in diabetes-affected patients.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/terapia , Terapia Genética/métodos , Animais , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/etiologia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Dieta Hiperlipídica/efeitos adversos , Estresse do Retículo Endoplasmático , Fibrose , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Espécies Reativas de Oxigênio , Remodelação Ventricular
12.
Zhonghua Xue Ye Xue Za Zhi ; 41(1): 34-39, 2020 Jan 14.
Artigo em Chinês | MEDLINE | ID: mdl-32023752

RESUMO

Objective: To evaluate the effects of adeno-associated virus (AAV) carrying hFⅧ by serotype 8 (AAV8/hFⅧ) on hemophilia A (HA) mice by gene therapy strategy. Methods: pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFⅧ, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFⅧ in HEK-293 cells using the optimized package condition. The purified AAV8/hFⅧ were intravenously injected into HA mice and the effects of gene therapy were estimated. Results: The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 µg plasmids, 20 cm-dish to 20 µg, 30 µg and 40 µg plasmids were employed, and the condition of 20 cm-dish to transfect 20 µg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFⅧ was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFⅧ was detected by Western blot. Fractions of AAV8/hFⅧ at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of FⅧ was measured by aPTT assay. Results showed that the activity of FⅧ maintained at the therapeutic level and lasted up to 12 weeks after injection. Conclusion: The purified AAV8/hFⅧ based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFⅧ were observed in vivo.


Assuntos
Dependovirus , Hemofilia A , Animais , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , Camundongos
13.
Nat Rev Genet ; 21(4): 255-272, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042148

RESUMO

Adeno-associated virus (AAV) vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including haemophilia and Duchenne muscular dystrophy. However, current research indicates that the genetic modification of AAV vectors may further facilitate the success of AAV gene therapy. Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Imunidade Adaptativa , Engenharia Genética , Vetores Genéticos/imunologia , Imunidade Inata
14.
Invest Ophthalmol Vis Sci ; 61(2): 11, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049345

RESUMO

Purpose: To study the potential effect of a gene therapy, designed to rescue the expression of dystrophin Dp71 in the retinas of Dp71-null mice, on retinal physiology. Methods: We recorded electroretinograms (ERGs) in Dp71-null and wild-type littermate mice. In dark-adapted eyes, responses to flashes of several strengths were measured. In addition, flash responses on a 25-candela/square meters background were measured. On- and Off-mediated responses to sawtooth stimuli and responses to photopic sine-wave modulation (3-30 Hz) were also recorded. After establishing the ERG phenotype, the ShH10-GFP adeno-associated virus (AAV), which has been previously shown to target specifically Müller glial cells (MGCs), was delivered intravitreously with or without (sham therapy) the Dp71 coding sequence under control of a CBA promoter. ERG recordings were repeated three months after treatment. Real-time quantitative PCR and Western blotting analyses were performed in order to quantify Dp71 expression in the retinas. Results: Dp71-null mice displayed reduced b-waves in dark- and light-adapted flash ERGs and smaller response amplitudes to photopic rapid-on sawtooth modulation and to sine-wave stimuli. Three months after intravitreal injections of the ShH10-GFP-2A-Dp71 AAV vector, ERG responses were completely recovered in treated eyes of Dp71-null mice. The functional rescue was associated with an overexpression of Dp71 in treated retinas. Conclusions: The present results show successful functional recovery accompanying the reexpression of Dp71. In addition, this experimental model sheds light on MGCs influencing ERG components, since previous reports showed that aquaporin 4 and Kir4.1 channels were mislocated in MGCs of Dp71-null mice, while their distribution could be normalized following intravitreal delivery of the same ShH10-GFP-2A-Dp71 vector.


Assuntos
Distrofina/metabolismo , Retina/fisiologia , Doenças Retinianas/fisiopatologia , Animais , Adaptação à Escuridão , Dependovirus/fisiologia , Distrofina/deficiência , Eletrorretinografia , Células Ependimogliais/metabolismo , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Genótipo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/metabolismo , Doenças Retinianas/terapia
15.
Invest Ophthalmol Vis Sci ; 61(2): 45, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32106292

RESUMO

Purpose: Recent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR). Methods: Sprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition. Results: Compared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders. Conclusions: Taken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.


Assuntos
Dependovirus/genética , Técnicas de Silenciamento de Genes/métodos , Terapia Genética/métodos , Neovascularização Retiniana/terapia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley
16.
PLoS One ; 15(2): e0228005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027681

RESUMO

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.


Assuntos
Calcitonina/genética , Carcinoma Neuroendócrino/terapia , Dependovirus/genética , Vetores Genéticos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Neoplasias da Glândula Tireoide/terapia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Masculino , Camundongos SCID , Regiões Promotoras Genéticas , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 11(1): 1105, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107381

RESUMO

Huntington's disease (HD) is caused by Huntingtin (Htt) gene mutation resulting in the loss of striatal GABAergic neurons and motor functional deficits. We report here an in vivo cell conversion technology to reprogram striatal astrocytes into GABAergic neurons in both R6/2 and YAC128 HD mouse models through AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors. We found that the astrocyte-to-neuron (AtN) conversion rate reached 80% in the striatum and >50% of the converted neurons were DARPP32+ medium spiny neurons. The striatal astrocyte-converted neurons showed action potentials and synaptic events, and projected their axons to the targeted globus pallidus and substantia nigra in a time-dependent manner. Behavioral analyses found that NeuroD1 and Dlx2-treated R6/2 mice showed a significant extension of life span and improvement of motor functions. This study demonstrates that in vivo AtN conversion may be a disease-modifying gene therapy to treat HD and other neurodegenerative disorders.


Assuntos
Astrócitos/fisiologia , Técnicas de Reprogramação Celular/métodos , Corpo Estriado/patologia , Neurônios GABAérgicos/fisiologia , Terapia Genética/métodos , Doença de Huntington/terapia , Potenciais de Ação/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Técnicas de Observação do Comportamento , Comportamento Animal , Corpo Estriado/citologia , Dependovirus/genética , Modelos Animais de Doenças , Expressão Ectópica do Gene , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Proteínas de Homeodomínio , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/patologia , Longevidade , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Técnicas Estereotáxicas , Fatores de Transcrição
18.
Nat Biomed Eng ; 4(1): 97-110, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31937940

RESUMO

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann-Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.


Assuntos
Adenina/metabolismo , Citosina/metabolismo , Dependovirus/fisiologia , Edição de Genes/métodos , Animais , Encéfalo/metabolismo , Vetores Genéticos/administração & dosagem , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Retina/metabolismo
20.
Nat Commun ; 11(1): 482, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980606

RESUMO

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.


Assuntos
Dependovirus/genética , Edição de Genes/métodos , Células Fotorreceptoras de Vertebrados/fisiologia , Distrofias Retinianas/genética , Distrofias Retinianas/terapia , Animais , Sistemas CRISPR-Cas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Reparo do DNA por Junção de Extremidades , Proteínas do Olho/genética , Terapia Genética/métodos , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Distrofias Retinianas/fisiopatologia , Reparo Gênico Alvo-Dirigido/métodos , Transducina/deficiência , Transducina/genética , Acuidade Visual/genética , Acuidade Visual/fisiologia
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