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1.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33688034

RESUMEN

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.


Asunto(s)
/inmunología , Vectores Genéticos , Virus del Sarampión , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /patología , Cricetinae , Modelos Animales de Enfermedad , Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunización , Inmunogenicidad Vacunal , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Ratones , Ratones Transgénicos , Ratas , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas Sintéticas/genética
2.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33688035

RESUMEN

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.


Asunto(s)
/inmunología , Vectores Genéticos/genética , Vacunas de ADN/inmunología , Virus Vaccinia/genética , /genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , /genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunización , Inmunización Pasiva , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética
3.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33670859

RESUMEN

Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.


Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Cápside/inmunología , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Animales , Proteínas de la Cápside/genética , Humanos , Inmunidad , Inmunogenicidad Vacunal , /inmunología
4.
Methods Mol Biol ; 2244: 115-132, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555585

RESUMEN

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.


Asunto(s)
Citomegalovirus/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Cultivo Primario de Células/métodos , Línea Celular , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Humanos , ARN Interferente Pequeño/genética , Transfección/métodos , Proteínas Virales , Replicación Viral/fisiología
5.
J Vis Exp ; (168)2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33616121

RESUMEN

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.


Asunto(s)
Clonación Molecular/métodos , Genes , Ingeniería Genética , Sistemas CRISPR-Cas/genética , Cartilla de ADN/metabolismo , Replicación del ADN/genética , Escherichia coli/genética , Expresión Génica , Vectores Genéticos/genética , Plásmidos/genética , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Transcripción Genética
6.
Life Sci ; 270: 119142, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33524419

RESUMEN

Adeno-associated virus (AAV) vector, an excellent gene therapy vector, has been widely used in the treatment of various central nervous system (CNS) diseases. Due to the presence of the blood-brain barrier (BBB), early attempts at AAV-based CNS diseases treatment were mainly performed through intracranial injections. Subsequently, systemic injections of AAV9, the first AAV that was shown to have BBB-crossing ability in newborn and adult mice, were assessed in clinical trials for multiple CNS diseases. However, the development of systemic AAV injections to treat CNS diseases is still associated with many challenges, such as the efficiency of AAV in crossing the BBB, the peripheral toxicity caused by the expression of AAV-delivered genes, and the immune barrier against AAV in the blood. In this review, we will introduce the biology of the AAV vector and the advantages of systemic AAV injections to treat CNS diseases. Most importantly, we will introduce the challenges associated with systemic injection of therapeutic AAV in treating CNS diseases and suggest feasible solutions.


Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Dependovirus/genética , Terapia Genética/métodos , Absorción Fisiológica , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Línea Celular , Enfermedades del Sistema Nervioso Central/genética , Dependovirus/metabolismo , Técnicas de Transferencia de Gen/efectos adversos , Terapia Genética/tendencias , Vectores Genéticos/genética , Humanos , Ratones , Transducción Genética/métodos , Transgenes
7.
Viruses ; 13(2)2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33572347

RESUMEN

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.


Asunto(s)
Vectores Genéticos , Lentivirus/crecimiento & desarrollo , Cultivo de Virus , Animales , Reactores Biológicos , Técnicas de Cultivo de Célula , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Humanos , Lentivirus/genética , Lentivirus/aislamiento & purificación , Transducción Genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo
8.
Viruses ; 13(2)2021 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-33572589

RESUMEN

Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives.


Asunto(s)
/métodos , /terapia , Terapia Genética/métodos , Vectores Genéticos/genética , Animales , Antivirales , /administración & dosificación , Humanos , Lentivirus/genética , /aislamiento & purificación
9.
J Korean Med Sci ; 36(6): e54, 2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33559409

RESUMEN

Coronavirus disease 2019 (COVID-19), which started at the end of 2019 and has spread worldwide, has remained unabated in 2021. Since non-pharmaceutical interventions including social distancing are facing limitations in controlling COVID-19, additional absolute means to change the trend are necessary. To this end, coronavirus-specific antiviral drugs and vaccines are urgently needed, but for now, the priority is to promote herd immunity through extensive nationwide vaccination campaign. In addition to the vaccines based on the conventional technology such inactivated or killed virus or protein subunit vaccines, several vaccines on the new technological platforms, for example, nucleic acids-based vaccines delivered by viral carriers, nanoparticles, or plasmids as a medium were introduced in this pandemic. In addition to achieving sufficient herd immunity with vaccination, the development of antiviral treatments that work specifically against COVID-19 will also be necessary to terminate the epidemic completely.


Asunto(s)
/inmunología , /prevención & control , Adenoviridae/genética , /virología , Variación Genética , Vectores Genéticos/genética , Humanos , Inmunidad Colectiva , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , /aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología
10.
Nat Commun ; 12(1): 781, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536416

RESUMEN

After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI.


Asunto(s)
Dependovirus/genética , Miembro Posterior/fisiopatología , Locomoción/fisiología , Neuronas/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Animales , Antipsicóticos/administración & dosificación , Clozapina/administración & dosificación , Clozapina/análogos & derivados , Vectores Genéticos/genética , Miembro Posterior/inervación , Locomoción/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia
11.
Immunity ; 54(3): 542-556.e9, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33631118

RESUMEN

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.


Asunto(s)
/inmunología , Vectores Genéticos/genética , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , /patología , /genética , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Inmunofenotipificación , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Macaca , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunación/métodos , Vacunas de ADN/genética
12.
Methods Mol Biol ; 2238: 95-113, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33471327

RESUMEN

CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.


Asunto(s)
Sistemas CRISPR-Cas , Citidina Desaminasa/antagonistas & inhibidores , Edición Génica , Vectores Genéticos/genética , Oryza/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Transformación Genética , Citidina Desaminasa/genética , Técnicas de Transferencia de Gen , Genoma de Planta , Oryza/genética , Plantas Modificadas Genéticamente/genética , Protoplastos/fisiología , Transgenes/fisiología
13.
Methods Mol Biol ; 2238: 115-134, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33471328

RESUMEN

CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.


Asunto(s)
Sistemas CRISPR-Cas , Productos Agrícolas/crecimiento & desarrollo , Edición Génica , Vectores Genéticos/genética , Oryza/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Transformación Genética , Productos Agrícolas/genética , Técnicas de Transferencia de Gen , Genoma de Planta , Oryza/genética , Fitomejoramiento , Plantas Modificadas Genéticamente/genética , Transgenes/fisiología
15.
Nat Commun ; 12(1): 447, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469018

RESUMEN

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl-, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice.


Asunto(s)
Líquido Cefalorraquídeo/metabolismo , Plexo Coroideo/patología , Hidrocefalia/patología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Animales Recién Nacidos , Plexo Coroideo/diagnóstico por imagen , Plexo Coroideo/crecimiento & desarrollo , Plexo Coroideo/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Hidrocefalia/congénito , Hidrocefalia/diagnóstico , Hidrocefalia/fisiopatología , Inyecciones Intraventriculares , Presión Intracraneal/fisiología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Transgénicos , Miembro 2 de la Familia de Transportadores de Soluto 12/genética
16.
Life Sci ; 268: 119014, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33412216

RESUMEN

AIMS: Hepatocyte growth factor (HGF) is a multifunctional cytokine that plays important roles in pancreatic physiology. Approvals of gene therapy drugs have highlighted gene therapy as an innovative new drug modality, but the very recent reports of deaths in clinical trials have provided a warning that high-dose gene therapy can cause dangerous liver toxicity. The present study aimed to develop a safe and low-dose but therapeutically effective adenovirus-mediated HGF gene therapy for streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. MAIN METHODS: A single intravenous injection of a low dose (3 × 108 plaque forming units) of adenoviral vector expressing the HGF gene under the transcriptional control of a strong promoter, i.e., the cytomegalovirus immediate-early enhancer and a modified chicken ß-actin promoter (Ad.CA-HGF), was given to T1D mice. KEY FINDINGS: Low-dose HGF gene therapy significantly attenuated the elevation of blood glucose concentrations at the acute phase of T1D, and this effect persisted for several weeks. Temporal upregulation of plasma insulin at the acute phase was maintained at a normal level in Ad.CA-HGF-treated mice, suggesting that the therapeutic mechanism may involve protection of the remaining ß-cells by HGF. Liver enzymes in plasma were not elevated in any of the mice, including the Ad.CA-HGF-treated animals, all of which looked healthy, suggesting the absence of lethal adverse effects observed in patients receiving high intravenous doses of viral vectors. SIGNIFICANCE: A low dose of intravenous Ad-mediated HGF gene therapy is clinically feasible and safe, and thus represents a new therapeutic strategy for treating T1D.


Asunto(s)
Adenoviridae/genética , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Factor de Crecimiento de Hepatocito/genética , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Factor de Crecimiento de Hepatocito/administración & dosificación , Hiperglucemia/genética , Hiperglucemia/terapia , Inyecciones Intravenosas , Insulina/sangre , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas
17.
Viruses ; 13(1)2021 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-33478128

RESUMEN

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for 'clustered regularly interspaced short palindromic repeats' and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.


Asunto(s)
Ingeniería Genética , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Agricultura/métodos , Sistemas CRISPR-Cas , Productos Agrícolas/virología , Resistencia a la Enfermedad/genética , Edición Génica , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Genoma Viral , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética
18.
Viruses ; 13(1)2021 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-33445753

RESUMEN

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.


Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Replicación Viral , Biomarcadores , Línea Celular , Células Cultivadas , Dimetilsulfóxido/farmacología , Expresión Génica , Vectores Genéticos/genética , Virus de la Hepatitis B/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos
19.
Sheng Wu Gong Cheng Xue Bao ; 37(1): 321-330, 2021 Jan 25.
Artículo en Chino | MEDLINE | ID: mdl-33501812

RESUMEN

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.


Asunto(s)
Escherichia coli , Vectores Genéticos , Clonación Molecular , Escherichia coli/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Operón Lac/genética , Plásmidos/genética , beta-Galactosidasa/genética
20.
Methods Mol Biol ; 2238: 135-143, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33471329

RESUMEN

The fast-moving CRISPR technology has allowed plant scientists to manipulate plant genomes in a targeted manner. So far, most of the applications were focused on gene knocking out by creating indels. However, more precise genome editing tools are demanded to assist the introduction of functional single nucleotide polymorphisms (SNPs) in breeding programs. The CRISPR base editing tools were developed to meet this need. In this chapter, we present a cytidine deaminase base editing method for editing the point mutations that control the grain size and seed coat color in rice.


Asunto(s)
Sistemas CRISPR-Cas , Citidina Desaminasa/antagonistas & inhibidores , Edición Génica , Oryza/crecimiento & desarrollo , Fitomejoramiento , Polimorfismo de Nucleótido Simple , Semillas/crecimiento & desarrollo , Citidina Desaminasa/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Genoma de Planta , Oryza/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Semillas/genética , Transformación Genética , Transgenes/fisiología
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