mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA.

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.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

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.

Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection.

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.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

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.

Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

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.

Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques.

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.

In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity.

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.

Imaging viral RNA using multiply labeled tetravalent RNA imaging probes in live cells.

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 ∼80nm to ∼10µm 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.

Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation.

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.

Enhanced tethered-particle motion analysis reveals viscous effects.

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.