RESUMEN
The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.
Asunto(s)
Sistemas CRISPR-Cas , Gripe Humana , Estabilidad del ARN , ARN Mensajero , ARN Viral , Animales , ARN Viral/genética , ARN Viral/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Gripe Humana/virología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/virología , Antivirales/farmacología , Perros , Cricetinae , Proteínas Virales/genética , Proteínas Virales/metabolismo , Mesocricetus , Células de Riñón Canino Madin DarbyRESUMEN
Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.
Asunto(s)
COVID-19 , Animales , Ratones , ARN Mensajero/genética , SARS-CoV-2/genética , Polímeros/metabolismo , Pulmón , ARN/metabolismoRESUMEN
The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.
Asunto(s)
Proteínas de la Nucleocápside/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Células A549 , Animales , Chlorocebus aethiops , Humanos , Proteínas de la Nucleocápside/genética , ARN Viral/genética , ARN Viral/metabolismo , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Ribonucleoproteínas/genética , Transcripción Genética , Células Vero , Proteínas Virales/genéticaRESUMEN
There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Expresión Génica , Anticuerpos Anti-VIH/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , ARN Mensajero/administración & dosificación , Vagina , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Aerosoles , Animales , Chlorocebus aethiops , Femenino , Técnica del Anticuerpo Fluorescente , Infecciones por VIH/inmunología , VIH-1/inmunología , Ratones , Pruebas de Neutralización , ARN Mensajero/síntesis química , Ovinos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Vagina/metabolismo , Células VeroRESUMEN
The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.
Asunto(s)
Endosomas/metabolismo , Imagen Óptica/métodos , ARN Mensajero/farmacocinética , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodos , Animales , Transporte Biológico , Carbocianinas/química , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Rayos Infrarrojos , Inyecciones Intramusculares , Ratones , Sondas Moleculares/química , Hibridación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, (64)Cu-labeled SIV Gp120-specific antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.
Asunto(s)
Antirretrovirales/uso terapéutico , Tomografía de Emisión de Positrones/métodos , Virus de la Inmunodeficiencia de los Simios , Imagen de Cuerpo Entero/métodos , Adenina/análogos & derivados , Adenina/uso terapéutico , Animales , Radioisótopos de Cobre , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Emtricitabina , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Naftiridinas/uso terapéutico , Organofosfonatos/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Tenofovir , Proteínas del Envoltorio Viral/metabolismo , Viremia , Replicación ViralRESUMEN
In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , ARN Mensajero/genética , Vacunas Sintéticas/farmacología , Animales , Formación de Anticuerpos , Inmunidad Celular , Inyecciones Intramusculares , Ratones , Células RAW 264.7 , Transcripción Genética , Vacunas Sintéticas/administración & dosificaciónRESUMEN
Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from â¼80nm to â¼10µm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes, their loading into the virion represents a critical step in the creation of infectious particles. RNA imaging tools represent a powerful approach to gain insight into fundamental viral processes, including virus entry, replication, and virion assembly. Imaging viral processes in live cells is critical due to both the heterogeneity of these processes on a per cell basis, and the inherent dynamics of these processes. There are a number of methods for labeling RNA in live cells; we'll introduce the myriad of methods and then focus on one approach for labeling viral RNA, using multiply-labeled tetravalent RNA imaging probes (MTRIPs), which do not require engineering of the target RNAs. We feel this approach is advantageous given many viral genomes may not tolerate large nucleotide insertions into their sequences.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/química , Imagen Molecular/métodos , ARN Mensajero/química , ARN Viral/química , Virus Sincitiales Respiratorios/química , Coloración y Etiquetado/métodos , Animales , Chlorocebus aethiops , Colorantes Fluorescentes/química , VIH-1/genética , VIH-1/metabolismo , Células Hep G2 , Humanos , Oligonucleótidos/química , Sondas ARN/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/metabolismo , Células Vero , Virión/química , Virión/genética , Virión/metabolismoRESUMEN
The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA-protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with ß-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.
Asunto(s)
Imagen Molecular/métodos , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Línea Celular , Dactinomicina/farmacología , Proteínas ELAV/metabolismo , Humanos , Microscopía Fluorescente , Sondas Moleculares/química , Proteínas de la Nucleocápside/metabolismo , Oligopéptidos , Péptidos/química , ARN Mensajero/metabolismo , ARN Viral/metabolismoRESUMEN
Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl2 or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.
Asunto(s)
ADN/química , Microesferas , Movimiento (Física) , Tampones (Química) , Difusión , Magnesio/química , ViscosidadRESUMEN
The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P(RM). CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P(RM) regulation. We additionally report that different configurations of DNA loops are central to this regulation--one configuration further enhances autoactivation and another is essential for autorepression of P(RM).
Asunto(s)
Bacteriófago lambda/química , ADN Superhelicoidal/química , ADN Viral/química , Proteínas Represoras/química , Proteínas Virales/química , Bacteriófago lambda/metabolismo , ADN Superhelicoidal/metabolismo , ADN Viral/metabolismo , Lisogenia/fisiología , Proteínas Represoras/metabolismo , Proteínas Virales/metabolismoRESUMEN
Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.
Asunto(s)
Antivirales , Virus del Dengue , Dengue , Modelos Animales de Enfermedad , Nanopartículas , ARN Mensajero , Animales , Dengue/tratamiento farmacológico , Ratones , Virus del Dengue/genética , Virus del Dengue/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nanopartículas/química , Antivirales/farmacología , Antivirales/administración & dosificación , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Lípidos/química , Carga Viral/efectos de los fármacos , Femenino , LiposomasRESUMEN
Transport of messenger RNAs (mRNAs) in the cytoplasm is essential for localization to translation sites and for post-transcriptional regulation. Utilizing single-RNA sensitive probes and real-time fluorescence microscopy, we accurately quantified the dynamics of native, non-engineered, ß-actin mRNAs within the cytoplasm of epithelial cells and fibroblasts for the first time. Using single-particle tracking and temporal analysis, we determined that native ß-actin mRNAs, under physiologic conditions, exhibit bursts of intermittent, processive motion on microtubules, interspersed between time periods of diffusive motion, characterized by non-thermal enhanced diffusivity. When transport processes were perturbed via ATP depletion, temperature reduction, dynamitin overexpression and chemical inhibitors, processive motion was diminished or eliminated and diffusivity was reduced. These data support a model whereby processive, motor-driven motion is responsible for long-distance mRNA transport.
Asunto(s)
Actinas/metabolismo , Citoplasma/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Actinas/genética , Adenosina Trifosfato/metabolismo , Complejo Dinactina , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Microscopía Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Transporte de ARN , ARN Mensajero/genética , Temperatura , Células Tumorales CultivadasRESUMEN
Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon ß mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , ARN Helicasas DEAD-box/inmunología , Inmunidad Innata , Cuerpos de Inclusión Intranucleares/inmunología , Nucleoproteínas/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aves , Línea Celular Tumoral , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Genoma Viral/genética , Genoma Viral/inmunología , Humanos , Helicasa Inducida por Interferón IFIH1 , Interferón beta/biosíntesis , Interferón beta/genética , Interferón beta/inmunología , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Cuerpos de Inclusión Intranucleares/patología , Cuerpos de Inclusión Intranucleares/virología , Enfermedad de Newcastle/genética , Enfermedad de Newcastle/inmunología , Enfermedad de Newcastle/metabolismo , Enfermedad de Newcastle/patología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/metabolismo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/inmunología , ARN Viral/metabolismo , Receptores Inmunológicos , Infecciones por Virus Sincitial Respiratorio/genética , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/metabolismo , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Stereochemistry can alter small-molecule pharmacokinetics, safety and efficacy. However, it is unclear whether the stereochemistry of a single compound within a multicomponent colloid such as a lipid nanoparticle (LNP) can influence its activity in vivo. Here we report that LNPs containing stereopure 20α-hydroxycholesterol (20α) delivered mRNA to liver cells up to 3-fold more potently than LNPs containing a mixture of both 20α- and 20ß-hydroxycholesterols (20mix). This effect was not driven by LNP physiochemical traits. Instead, in vivo single-cell RNA sequencing and imaging revealed that 20mix LNPs were sorted into phagocytic pathways more than 20α LNPs, resulting in key differences between LNP biodistribution and subsequent LNP functional delivery. These data are consistent with the fact that nanoparticle biodistribution is necessary, but not sufficient, for mRNA delivery, and that stereochemistry-dependent interactions between LNPs and target cells can improve mRNA delivery.
Asunto(s)
Lípidos , Nanopartículas , Lípidos/química , ARN Mensajero/genética , Distribución Tisular , Nanopartículas/químicaRESUMEN
The λ repressor (CI) protein-induced DNA loop maintains stable lysogeny, yet allows efficient switching to lysis. Herein, the kinetics of loop formation and breakdown has been characterized at various concentrations of protein using tethered particle microscopy and a novel, to our knowledge, method of analysis. Our results show that a broad distribution of rate constants and complex kinetics underlie loop formation and breakdown. In addition, comparison of the kinetics of looping in wild-type DNA and DNA with mutated o3 operators showed that these sites may trigger nucleation of nonspecific binding at the closure of the loop. The average activation energy calculated from the rate constant distribution is consistent with a model in which nonspecific binding of CI between the operators shortens their effective separation, thereby lowering the energy barrier for loop formation and broadening the rate constant distribution for looping. Similarly, nonspecific binding affects the kinetics of loop breakdown by increasing the number of loop-securing protein interactions, and broadens the rate constant distribution for this reaction. Therefore, simultaneous increase of the rate constant for loop formation and reduction of that for loop breakdown stabilizes lysogeny. Given these simultaneous changes, the frequency of transitions between the looped and the unlooped state remains nearly constant. Although the loop becomes more stable thermodynamically with increasing CI concentration, it still opens periodically, conferring sensitivity to environmental changes, which may require switching to lytic conditions.
Asunto(s)
ADN Bacteriano/química , Proteínas Represoras/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , ADN Bacteriano/metabolismo , Cinética , Conformación de Ácido NucleicoRESUMEN
Programmable control of gene expression via nuclease-null Cas9 fusion proteins has enabled the engineering of cellular behaviors. Here, both transcriptional and epigenetic gene activation via synthetic mRNA and lipid nanoparticle delivery was demonstrated in vivo. These highly efficient delivery strategies resulted in high levels of activation in multiple tissues. Finally, we demonstrate durable gene activation in vivo via transient delivery of a single dose of a gene activator that combines VP64, p65, and HSF1 with a SWI/SNF chromatin remodeling complex component SS18, representing an important step toward gene-activation-based therapeutics. This induced sustained gene activation could be inhibited via mRNA-encoded AcrIIA4, further improving the safety profile of this approach.
Asunto(s)
Sistemas CRISPR-Cas , Liposomas , Activación Transcripcional , ARN Mensajero/genética , Proteína 9 Asociada a CRISPR/genéticaRESUMEN
Despite the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines, there remains a clear need for new classes of preventatives for respiratory viral infections due to vaccine hesitancy, lack of sterilizing immunity, and for at-risk patient populations, including the immunocompromised. While many neutralizing antibodies have been identified, and several approved, to treat COVID-19, systemic delivery, large doses, and high costs have the potential to limit their widespread use, especially in low- and middle-income countries. To use these antibodies more efficiently, an inhalable formulation is developed that allows for the expression of mRNA-encoded, membrane-anchored neutralizing antibodies in the lung to mitigate SARS-CoV-2 infections. First, the ability of mRNA-encoded, membrane-anchored, anti-SARS-CoV-2 antibodies to prevent infections in vitro is demonstrated. Next, it is demonstrated that nebulizer-based delivery of these mRNA-expressed neutralizing antibodies potently abrogates disease in the hamster model. Overall, these results support the use of nebulizer-based mRNA expression of neutralizing antibodies as a new paradigm for mitigating respiratory virus infections.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , ARN Mensajero/genética , Anticuerpos Neutralizantes/uso terapéuticoRESUMEN
Recently, it was proposed that DNA looping by the lambda repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the lambda repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to -0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.
Asunto(s)
Bacteriófago lambda/genética , ADN Viral/química , Proteínas Represoras/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Sitios de Unión , ADN Viral/metabolismo , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , TermodinámicaRESUMEN
Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.