RESUMEN
Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.
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Dependovirus , Terapia Genética , Vectores Genéticos , Transgenes , Dependovirus/genética , Humanos , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Terapia Genética/métodos , Evasión Inmune , Animales , Ingeniería Genética/métodos , Técnicas de Transferencia de Gen , Tropismo ViralRESUMEN
Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.
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Dependovirus , Vectores Genéticos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Humanos , Vectores Genéticos/genética , Animales , Células HEK293 , Ratones , Terapia Genética/métodosRESUMEN
Lactococcus lactis (L. lactis), the first genetically modified Generally Recognized As Safe (GRAS) category Lactic Acid producing Bacteria (LAB), is best known for its generalized health-promoting benefits and ability to express heterologous proteins. However, achieving the optimal probiotic effects requires a selective approach that would allow us to study in vivo microbial biodistribution, fate, and immunological consequences. Although the chemical conjugation of fluorophores and chromophores represent the standard procedure to tag microbial cells for various downstream applications, it requires a high-throughput synthesis scheme, which is often time-consuming and expensive. On the contrary, the genetic manipulation of LAB vector, either chromosomally or extra-chromosomally, to express bioluminescent or fluorescent reporter proteins has greatly enhanced our ability to monitor bacterial transit through a complex gut environment. However, with faster passage and quick washing out from the gut due to rhythmic contractions of the digestive tract, real-time tracking of LAB vectors, particularly non-commensal ones, remains problematic. To get a deeper insight into the biodistribution of non-commensal probiotic bacteria in vivo, we bioengineered L. lactis to express fluorescence reporter proteins, mCherry (bright red monomeric fluorescent protein) and mEGFP (monomeric enhanced green fluorescent protein), followed by microencapsulation with a mucoadhesive and biodegradable polymer, chitosan. We show that coating of recombinant Lactococcus lactis (rL. lactis) with chitosan polymer, cross-linked with tripolyphosphate (TPP), retains their ability to express the reporter proteins stably without altering the specificity and sensitivity of fluorescence detection in vitro and in vivo. Further, we provide evidence of enhanced intragastric stability by chitosan-TPP (CS) coating of rL. lactis cells, allowing us to study the spatiotemporal distribution for an extended time in the gut of two unrelated hosts, avian and murine. The present scheme involving genetic modification and chitosan encapsulation of non-commensal LAB vector demonstrates great promise as a non-invasive and intensive tool for active live tracking of gut microbes.
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Lactococcus lactis , Proteínas Luminiscentes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Animales , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Vectores Genéticos , Genes Reporteros , Ratones , Probióticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína Fluorescente RojaRESUMEN
Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.
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Células Endoteliales , Edición Génica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Humanos , Edición Génica/métodos , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Sistemas CRISPR-Cas , Fosforilación , ARN Guía de Sistemas CRISPR-Cas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Neovascularización Patológica/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Retina/metabolismo , Vectores Genéticos , AngiogénesisRESUMEN
To date, 3,900 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our March 2023 update, we have entries on 3,900 trials undertaken in 46 countries. We have analyzed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at https://a873679.fmphost.com/fmi/webd/GTCT. We also provide an overview of the progress being made around the world, and discuss key trends since the previous review, namely the unprecedented increase in gene therapy clinical trial activity, including the implementation of genome editing technology with the potential to transform the field moving forward.
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Ensayos Clínicos como Asunto , Terapia Genética , Humanos , Terapia Genética/métodos , Terapia Genética/tendencias , Edición Génica/métodos , Vectores GenéticosRESUMEN
Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19.
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Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Ratones , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Muromegalovirus/inmunología , Muromegalovirus/genética , Femenino , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ratones Endogámicos BALB C , Humanos , Vectores Genéticos , Inmunidad Celular , Inmunidad Humoral , Modelos Animales de EnfermedadRESUMEN
One major limitation of effective vaccine delivery is its dependency on a robust cold chain infrastructure. While Vesicular stomatitis virus (VSV) has been demonstrated to be an effective viral vaccine vector for diseases including Ebola, its -70 °C storage requirement is a significant limitation for accessing disadvantaged locations and populations. Previous work has shown thermal stabilization of viral vaccines with a combination of pullulan and trehalose (PT) dried films. To improve the thermal stability of VSV, we optimized PT formulation concentrations and components, as well as drying methodology with enhanced vacuum drying. When formulated in PT films, VSV can be stored for 32 weeks at 4 °C with less than 2 log PFU loss, at 25 °C with 2.5 log PFU loss, and at 37 °C with 3.1 log PFU loss. These results demonstrate a significant advancement in VSV thermal stabilization, decreasing the cold chain requirements for VSV vectored vaccines.
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Glucanos , Trehalosa , Trehalosa/química , Glucanos/química , Vacio , Vectores Genéticos , Desecación/métodos , Vacunas Virales/química , Vesiculovirus/genética , Animales , TemperaturaRESUMEN
Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.
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Dependovirus , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Terapia Genética/métodos , Humanos , Dependovirus/genética , Vectores Genéticos , Enfermedades Genéticas Congénitas/terapia , Enfermedades Genéticas Congénitas/genética , Enfermedades Raras/terapia , Enfermedades Raras/genética , Lentivirus/genéticaRESUMEN
Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.
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Proteínas de la Cápside , Cápside , Dependovirus , Vectores Genéticos , Hígado , Dependovirus/genética , Animales , Humanos , Ratones , Vectores Genéticos/genética , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Hígado/metabolismo , Transducción Genética , Técnicas de Transferencia de Gen , Aprendizaje Automático , Terapia Genética/métodos , Macaca , Hepatocitos/metabolismo , Células HEK293 , Ingeniería Genética/métodosAsunto(s)
Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Animales , Vectores GenéticosRESUMEN
BACKGROUND: Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is a childhood dementia caused by inherited mutations in the sulfamidase gene. At present, there is no treatment and children with classical disease generally die in their late teens. Intravenous or intra-cerebrospinal fluid (CSF) injection of AAV9-gene replacement is being examined in human clinical trials; evaluation of the impact on brain disease is an intense focus; however, MPS IIIA patients also experience profound, progressive photoreceptor loss, leading to night blindness. AIM: To compare the relative efficacy of the two therapeutic approaches on retinal degeneration in MPS IIIA mice. METHODS: Neonatal mice received i.v. or intra-CSF AAV9-sulfamidase or vehicle and after 20 weeks, biochemical and histological evaluation of neuroretina integrity was carried out. RESULTS: Both treatments improved central retinal thickness; however, in peripheral retina, outer nuclear layer thickness and photoreceptor cell length were only significantly improved by i.v. gene replacement. Further, normalization of endo-lysosomal compartment size and microglial morphology was only observed following intravenous gene delivery. CONCLUSIONS: Confirmatory studies are needed in adult mice; however, these data indicate that i.v. AAV9-sulfamidase infusion leads to superior outcomes in neuroretina, and cerebrospinal fluid-delivered AAV9 may need to be supplemented with another therapeutic approach for optimal patient quality of life.
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Dependovirus , Terapia Genética , Mucopolisacaridosis III , Retina , Animales , Mucopolisacaridosis III/terapia , Mucopolisacaridosis III/genética , Terapia Genética/métodos , Dependovirus/genética , Retina/patología , Ratones , Modelos Animales de Enfermedad , Hidrolasas/genética , Animales Recién Nacidos , Ratones Endogámicos C57BL , Demencia/genética , Demencia/terapia , Vectores Genéticos/administración & dosificación , Inyecciones IntravenosasRESUMEN
Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.
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Edición Génica , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Edición Génica/métodos , Linfocitos T/inmunología , Animales , Neoplasias/terapia , Neoplasias/inmunología , Neoplasias/genética , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Ingeniería Genética , Sistemas CRISPR-Cas , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
MPS IIIC is a lysosomal storage disease caused by mutations in heparan-α-glucosaminide N-acetyltransferase (HGSNAT), for which no treatment is available. Because HGSNAT is a trans-lysosomal-membrane protein, gene therapy for MPS IIIC needs to transduce as many cells as possible for maximal benefits. All cells continuously release extracellular vesicles (EVs) and communicate by exchanging biomolecules via EV trafficking. To address the unmet need, we developed a rAAV-hHGSNATEV vector with an EV-mRNA-packaging signal in the 3'UTR to facilitate bystander effects, and tested it in an in vitro MPS IIIC model. In human MPS IIIC cells, rAAV-hHGSNATEV enhanced HGSNAT mRNA and protein expression, EV-hHGSNAT-mRNA packaging, and cleared GAG storage. Importantly, incubation with EVs led to hHGSNAT protein expression and GAG contents clearance in recipient MPS IIIC cells. Further, rAAV-hHGSNATEV transduction led to the reduction of pathological EVs in MPS IIIC cells to normal levels, suggesting broader therapeutic benefits. These data demonstrate that incorporating the EV-mRNA-packaging signal into a rAAV-hHGSNAT vector enhances EV packaging of hHGSNAT-mRNA, which can be transported to non-transduced cells and translated into functional rHGSNAT protein, facilitating cross-correction of disease pathology. This study supports the therapeutic potential of rAAVEV for MPS IIIC, and broad diseases, without having to transduce every cell.
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Efecto Espectador , Dependovirus , Vesículas Extracelulares , Terapia Genética , ARN Mensajero , Humanos , Terapia Genética/métodos , Dependovirus/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Vesículas Extracelulares/metabolismo , Mucopolisacaridosis III/terapia , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/genética , Vectores Genéticos , Acetiltransferasas/metabolismo , Acetiltransferasas/genéticaRESUMEN
Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method.
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Proliferación Celular , Transgenes , Humanos , Células HEK293 , Proliferación Celular/genética , Expresión Génica/genética , Dosificación de Gen , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transfección , Regiones Promotoras Genéticas , Vectores Genéticos/genéticaAsunto(s)
Cardiomiopatía Hipertrófica , Terapia Genética , Humanos , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/terapia , Cardiomiopatía Hipertrófica/fisiopatología , Animales , Resultado del Tratamiento , Vectores Genéticos , Fenotipo , Predisposición Genética a la EnfermedadAsunto(s)
Colágeno Tipo IV , Modelos Animales de Enfermedad , Terapia Genética , Nefritis Hereditaria , Nefritis Hereditaria/terapia , Nefritis Hereditaria/genética , Terapia Genética/métodos , Humanos , Colágeno Tipo IV/genética , Animales , Mutación , Vectores Genéticos , Técnicas de Transferencia de Gen , Ratones , Membrana Basal/metabolismoRESUMEN
Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: â¢The in vivo cloning was performed by replacing the target gene with the landing pad. â¢Fast eradication of non-recombinant plasmids was possible by adapting key vectors. â¢This strategy is not dependent on in vitro assembly reactions and expensive materials.
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Clonación Molecular , Escherichia coli , Plásmidos , Reacción en Cadena de la Polimerasa , Recombinación Genética , Escherichia coli/genética , Clonación Molecular/métodos , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos , Vectores Genéticos/genética , Sistemas CRISPR-CasRESUMEN
Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.
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Elementos Transponibles de ADN , Pigmentos Biológicos , Elementos Transponibles de ADN/genética , Pigmentos Biológicos/metabolismo , Mutagénesis Insercional/métodos , Vectores Genéticos/genética , Bacterias/genética , Bacterias/metabolismo , Bacterias/clasificación , Triptófano/metabolismo , Endófitos/genética , Endófitos/metabolismoRESUMEN
Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.
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ADN Circular , ADN Viral , Virus de la Hepatitis B , Integrasas , Recombinación Genética , Replicación Viral , ADN Circular/genética , Virus de la Hepatitis B/genética , Animales , Integrasas/genética , Integrasas/metabolismo , Ratones , ADN Viral/genética , Humanos , Vectores Genéticos/genética , Ratones Transgénicos , Plásmidos/genética , ADN Recombinante/genéticaRESUMEN
Mice infected with a recombinant adeno-associated virus carrying a replication-competent hepatitis B virus genome (rAAV-HBV) via the intravenous route establish a persistent HBV replication in hepatocytes and develop immune tolerance. They serve as models to evaluate antiviral immunity and to assess potential therapeutic approaches for chronic HBV infection. Combining selected HBV variants and different mouse genotypes allows for addressing a broad spectrum of research questions. This chapter describes the basic principles of the rAAV-HBV mouse model, rAAV-HBV production and purification methods, and finally, the in vivo application.