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1.
Science ; 368(6494): 993-1001, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467389

RESUMO

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.


Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Estimulação Elétrica/instrumentação , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Tecnologia sem Fio/instrumentação , Animais , Biônica , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Engenharia Celular , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Próteses e Implantes , Transcrição Genética , Transgenes
2.
Anticancer Res ; 40(5): 2687-2694, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366413

RESUMO

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays dual suppressive and oncogenic roles in mammary carcinogenesis. MATERIALS AND METHODS: To analyze whether TGF-ß exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-ß receptor (TßRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. RESULTS: Lobular proliferative lesions, showing high ß-casein expression, developed in the mammary glands of TßRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TßRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. CONCLUSION: The functional decline of TGF-ß signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-ß signaling may act as a tumor suppressor during mammary tumor development.


Assuntos
Carcinogênese/patologia , Genes Dominantes , Neoplasias Mamárias Animais/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Neoplasias Pulmonares/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/induzido quimicamente , Neoplasias Mamárias Animais/patologia , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Transgenes , Uretana
3.
PLoS One ; 15(3): e0222619, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150577

RESUMO

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-ß1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-ß1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.


Assuntos
Eczema/genética , Epiderme/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ceratose/genética , Pele/metabolismo , Transgenes , Acetamidas/farmacologia , Animais , Citocinas/metabolismo , Doxiciclina/farmacologia , Eczema/tratamento farmacológico , Feminino , Homeostase/genética , Hiperplasia/tratamento farmacológico , Hiperplasia/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Queratinócitos/metabolismo , Ceratose/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transativadores/metabolismo , Compostos de Tritil/farmacologia
4.
Mol Biotechnol ; 62(4): 260-272, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32144553

RESUMO

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-ßGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Cirrose Hepática/terapia , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Tetracloreto de Carbono/administração & dosagem , Ciclosporina/uso terapêutico , Terapia Genética , Imunização , Imunossupressores/uso terapêutico , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Ratos , Distribuição Tecidual , Transgenes , Ativador de Plasminogênio Tipo Uroquinase/farmacocinética
5.
Mol Biotechnol ; 62(4): 252-259, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146690

RESUMO

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.


Assuntos
Linhagem Celular , Túbulos Renais Proximais/efeitos dos fármacos , Testes de Toxicidade , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fluorescência , Engenharia Genética , Humanos , Concentração de Íons de Hidrogênio , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Transgenes
6.
PLoS One ; 15(3): e0230362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176712

RESUMO

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.


Assuntos
Ascomicetos/genética , Resistência à Doença/genética , Farmacorresistência Fúngica/genética , Inativação Gênica , Genes Fúngicos , Engenharia Genética , Perileno/análogos & derivados , Tabaco/microbiologia , Ascomicetos/efeitos dos fármacos , Resistência à Doença/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Perileno/farmacologia , Plantas Geneticamente Modificadas , Transformação Genética , Transgenes
7.
Nat Commun ; 11(1): 608, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001704

RESUMO

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in ß-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFß and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.


Assuntos
Reprogramação Celular/genética , Genoma , Transdução de Sinais/genética , Proteína 9 Associada à CRISPR/metabolismo , Sinalização do Cálcio , Engenharia Genética , Células HEK293 , Humanos , Imunomodulação , Inflamação/genética , Inflamação/patologia , Insulina/metabolismo , Interleucina-12/metabolismo , Células Jurkat , Ativação Linfocitária/imunologia , Potenciais da Membrana , Linfócitos T/imunologia , Transgenes
8.
Invest Ophthalmol Vis Sci ; 61(2): 14, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049344

RESUMO

Purpose: Experimental access to specific cell subtypes is essential for deciphering the complexity of retinal networks. Here, we characterized the selective labeling, caused by ectopic transgene expression, of two atypical retinal neurons in the ChAT-Channelrhodopsin-2 (ChR2)-EYFP mouse. Methods: Retinal sections and flat-mounts were prepared for double-staining immunohistochemistry with antibodies against EYFP and various neuronal markers. Sagittal/coronal brain slices were made to visualize EYFP signals in central nuclei. Whole-cell recordings were conducted to test the functionality of ChR2. Results: Two populations of EYFP-positive retinal cells were observed. The inner nuclear layer (INL)-located one (type I cell) distributed regularly throughout the entire retina, whereas the ganglion cell layer (GCL)-residing one (type II cell) was restricted ventrally. None of them was cholinergic, as evidenced by the complete absence of ChAT immunoreactivity. Type I cells were immunolabeled by the amacrine marker syntaxin. However, the vast majority of them were neither positive to GABA/GAD65, nor to GlyT1/glycine, suggesting that they were non-GABAergic non-glycinergic amacrine cells (nGnG ACs), which was confirmed by double-labeling with the nGnG AC marker PPP1R17. Type II cells were immunopositive to melanopsin, but not to Brn3a or Brn3b. They possessed dendrites stratifying in the outermost inner plexiform layer (IPL) and axons projecting to the suprachiasmatic nucleus (SCN) rather than the olivary pretectal nucleus (OPN), suggesting that they belonged to a Brn3b-negative subset of M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs). Glutamatergic transmission-independent photocurrents were elicited in EYFP-positive cells, indicating the functional expression of ChR2. Conclusions: The ChAT-ChR2-EYFP retina exhibits ectopic, but functional, transgene expression in nGnG ACs and SCN-innervating M1 ipRGCs, thus providing an ideal tool to achieve efficient labeling and optogenetic manipulation of these cells.


Assuntos
Células Amácrinas/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Transgenes/fisiologia , Animais , Channelrhodopsins/metabolismo , Colina O-Acetiltransferase/metabolismo , Feminino , Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transgenes/genética
9.
Science ; 367(6481)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32029687

RESUMO

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.


Assuntos
Transferência Adotiva , Sistemas CRISPR-Cas , Edição de Genes , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Idoso , Proteína 9 Associada à CRISPR , Engenharia Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Transgenes
10.
PLoS One ; 15(2): e0228203, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027678

RESUMO

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.


Assuntos
Coração/fisiologia , Miocárdio/metabolismo , Transfecção/métodos , Transgenes/genética , Animais , Creatina Quinase Forma MB/sangue , Dimetilpolisiloxanos/química , Ecocardiografia , Expressão Gênica , Camundongos , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/genética , Plasmídeos/metabolismo , Pressão , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transfecção/instrumentação , Troponina T/sangue
11.
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
14.
PLoS One ; 15(1): e0228164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31995598

RESUMO

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken®, a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.


Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Cadeias lambda de Imunoglobulina/genética , Animais , Animais Geneticamente Modificados/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Galinhas/imunologia , Humanos , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/imunologia , Progranulinas/imunologia , Linfócitos T/imunologia , Transgenes/genética
15.
PLoS One ; 15(1): e0227822, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940417

RESUMO

Peptidylarginine deiminase (PAD) modifies peptidylarginine and converts it to peptidylcitrulline in the presence of elevated calcium. Protein modification can lead to severe changes in protein structure and function, and aberrant PAD activity is linked to human pathologies. While PAD homologs have been discovered in vertebrates-as well as in protozoa, fungi, and bacteria-none have been identified in Drosophila melanogaster, a simple and widely used animal model for human diseases. Here, we describe the development of a human PAD overexpression model in Drosophila. We established fly lines harboring human PAD2 or PAD4 transgenes for ectopic expression under control of the GAL4/UAS system. We show that ubiquitous or nervous system expression of PAD2 or PAD4 have minimal impact on fly lifespan, fecundity, and the response to acute heat stress. Although we did not detect citrullinated proteins in fly homogenates, fly-expressed PAD4-but not PAD2-was active in vitro upon Ca2+ supplementation. The transgenic fly lines may be valuable in future efforts to develop animal models of PAD-related disorders and for investigating the biochemistry and regulation of PAD function.


Assuntos
Drosophila melanogaster/genética , Proteína-Arginina Desiminase do Tipo 2/genética , Proteína-Arginina Desiminase do Tipo 4/genética , Transgenes , Animais , Animais Geneticamente Modificados/genética , Drosophila melanogaster/fisiologia , Feminino , Fertilidade , Resposta ao Choque Térmico , Humanos , Longevidade , Masculino , Regulação para Cima
16.
PLoS One ; 15(1): e0227994, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978124

RESUMO

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.


Assuntos
Algoritmos , Edição de Genes , Genoma de Planta , Plantas/genética , RNA Guia/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Tabaco/genética , Transgenes
17.
Appl Microbiol Biotechnol ; 104(5): 2125-2135, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932895

RESUMO

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3' ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.


Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , RNA Interferente Pequeno/genética , Transgenes/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Metilação de DNA , Regulação da Expressão Gênica de Plantas/genética , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Genética
18.
Nat Commun ; 11(1): 352, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953404

RESUMO

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.


Assuntos
Tecnologia de Impulso Genético/métodos , Genética Populacional/métodos , Alelos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sistemas CRISPR-Cas , Dípteros , Ecossistema , Feminino , Edição de Genes , Genes Ligados ao Cromossomo X , Masculino , Modelos Teóricos , RNA Guia/genética , Transgenes
19.
N Engl J Med ; 382(1): 29-40, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31893514

RESUMO

BACKGROUND: Adeno-associated virus (AAV)-mediated gene therapy is under investigation as a therapeutic option for persons with hemophilia A. Efficacy and safety data include 3 years of follow-up after a single administration of AAV5-hFVIII-SQ. METHODS: We report durable efficacy, long-term safety, and clinical and biologic results in 15 adults with severe hemophilia A (factor VIII level, ≤1 IU per deciliter) who had received a single infusion of AAV5-hFVIII-SQ at various dose levels. We evaluated the factor VIII level, annualized rate of bleeding events, use of factor VIII, safety, expression kinetics, and biologic markers of AAV transduction for up to 3 years. RESULTS: Three years after infusion, two participants (one who had received 6×1012 vector genomes [vg] per kilogram of body weight and one who had received 2×1013 vg per kilogram) had factor VIII expression of less than 1 IU per deciliter, as assessed on chromogenic assay. Seven participants (who had received 6×1013 vg per kilogram) had a median factor VIII expression of 20 IU per deciliter; the median number of annualized treated bleeding events was 0, and the median use of exogenous factor VIII was reduced from 138.5 infusions to 0 infusions per year. Bleeding in all target joints (major joints with ≥3 bleeding events within 6 months) in this cohort resolved (≤2 bleeding events within 12 months). Two years after infusion, six participants (who had received 4×1013 vg per kilogram) had a median factor VIII expression of 13 IU per deciliter; the median annualized rate of bleeding events was 0, and the median use of factor VIII was reduced from 155.5 infusions to 0.5 infusions per year. Bleeding in target joints resolved in five of six participants. The factor VIII pharmacodynamic profiles reflected cellular turnover in the blood and molecular events leading to episomal DNA stabilization for persistent expression, findings that are consistent with previous observations in two model systems. Transgene-derived human factor VIII (hFVIII) protein activity mirrored native hFVIII in hemostatic ability. No inhibitor development, thromboses, deaths, or persistent changes in liver-function tests were observed. CONCLUSIONS: Gene therapy with AAV5-hFVIII-SQ vector in participants with hemophilia A resulted in sustained, clinically relevant benefit, as measured by a substantial reduction in annualized rates of bleeding events and complete cessation of prophylactic factor VIII use in all participants who had received 4×1013 vg per kilogram or 6×1013 vg per kilogram of the gene therapy. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795; EudraCT number, 2014-003880-38.).


Assuntos
Dependovirus , Fator VIII/genética , Terapia Genética , Vetores Genéticos , Hemofilia A/terapia , Adulto , Biomarcadores , Coagulantes/uso terapêutico , Fator VIII/uso terapêutico , Seguimentos , Terapia Genética/efeitos adversos , Hemofilia A/complicações , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Transgenes , Adulto Jovem
20.
Nucleic Acids Res ; 48(5): 2348-2356, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31960057

RESUMO

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Regiões Promotoras Genéticas , Cromatina/química , Cromatina/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Dosagem de Genes , Inativação Gênica , Transferência Genética Horizontal , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Transgenes
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