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Supplementary Figure 2: PMTRIPs FISH-PLA is specific, effective, and sensitive without dextran sulfate. A). 5 nM of V5-tagged PMTRIPs targeting polyadenylated mRNA were delivered as FISH probes, with FISH hybridization buffer containing either 0%, 5% or 10% dextran sulfate. PLA (white) was performed between the V5 tag and HuR. Extended focus images are shown. Scale bars indicate 10 µm. B) Quantification of PLA is shown. Statistics were performed with a one-way ANOVA with a Dunn's multiple comparisons test, where n=30 and **** p < 0.0001. 95% confidence intervals are shown in red. C) 5 nM V5 tagged MTRIPs targeting polyadenylated mRNA were assembled using either PNA or 2'OM oligos and were delivered as FISH probes. PLA (white) was performed between the V5 tag and HuR. Extended focus images are shown. Scale bars indicate 10 µm. Quantification of PLA is shown. Statistics were performed with a one-way ANOVA with a Dunn's multiple comparisons test, where n=30 and ** p < 0.002 and **** p < 0.0001. 95% confidence intervals are shown in black. D) Representative extended focus control images for figure 2A are shown. PLA is in white. Scale bars indicate 10 µm. E) Representative extended focus control images for figure 2B are shown. PLA is in white. Scale bars indicate 10 µm. F) Representative extended focus control images for figure 2C are shown. PLA is in white. Scale bars indicate 10 µm. G) Representative extended focus control images for figure 2D are shown. PLA is in white. Scale bars indicate 10 µm.

Supplementary Figure 4: PMTRIP FISH-PLA can quantify COX-2-TIAR interactions in colon tissue. A) 20 nM of V5 tagged PMTRIPs targeting COX-2 mRNA were delivered as FISH probes to colon cancer samples. PLA (green) was performed between the V5 tag and TIAR. Extended focus images are shown, with a 1 mm scale bar. B) Extended focus images of FISH-PLA (green) controls are shown. Scale bar is 1 mm. C) 20x cropped image for (A) are shown, as indicated by the white boxes. Extended focus images are shown, with a 100 μm scale bar. D) Extended focus image of tissue FISH-PLA at 63x is shown, with a 10 μm scale bar. COX-2 FISH shown in red. E) Extended focus cropped images of full tissues in (B) are indicated by white boxes. Extended focus images of tissue FISH-PLA at 63x are also shown. COX-2 FISH shown in red. Scale bars are 100 μm for 20x and 10 μm for 63x images. F) Quantification of PLA signal in (a-b). Statistics were performed with a one-way ANOVA with a Tukey's multiple comparisons test, where n=3 and * p < 0.046. Standard deviations are shown in red. G) Immuohistochemistry for TIAR was performed on colon cancer and healthy colon tissue. Single plane representative images are shown. Scale bars are 50 μm. Quantification of the area of HuR expression normalized by the area of nuceli is shown. Statistics were a Mann-Whitney t-test, where n= 5 and * p < 0.032.

Supplementary Figure 3: PMTRIP FISH-PLA can quantify PolyA-TIAR interactions in colon tissue. A-C) Cells were treated with 5 μ g/ml of ActD 1 h prior to fixation. 5 nM of V5 tagged PMTRIPs targeting polyadenylated mRNA were delivered as FISH probes. PLA was performed between the V5 tag and HuR. PLA signal is quantified by A) the number of PLA puncta, B) volume of PLA signal, or C) the sum of intensities of PLA signal per cell. Statistics were performed with a one-way ANOVA with a Dunn's multiple comparisons test, where n=30, ** p < 0.002, and **** p < 0.0001. 95% confidence intervals are shown in red. D) 5 nM of V5 tagged PMTRIPs targeting polyadenylated mRNA were delivered as FISH probes to colon cancer samples. PLA (green) was performed between the V5 tag and TIAR. Extended focus images are shown, with a 1 mm scale bar for full tissue. E) Extended focus images of FISH-PLA (green) controls are shown. Scale bar is 1 mm. F) Extended focus cropped images of full tissues in (D) are indicated by white boxes. Scale bars are 100 μm. G) Extended focus image of cancer tissue FISH-PLA at 63x is shown. Scale bar is 10 μm. h) Extended focus cropped images of full tissues in (E) are indicated by white boxes. Extended focus images of control tissue FISH-PLA at 63x are also shown. Scale bars are 100 μm for 20x and 10 μm for 63x images. I) Quantification of PLA signal in (D-E). Statistics were performed with a one-way ANOVA with a Tukey's multiple comparisons test, where n=3 and ** p < 0.0075. Standard deviations are shown in red.

Supplementary Figure 5: PMTRIP FISH-PLA can quantify PolyA-TIAR interactions in lung tissue. A) 5 nM of V5 tagged PMTRIPs targeting polyadenylated mRNA were delivered as FISH probes to colon cancer samples. PLA (green) was performed between the V5 tag and TIAR. Extended focus images are shown, with a 1 mm scale bar for full tissue. B) Extended focus images of FISH-PLA (green) controls are shown. Scale bar is 1 mm. C) Extended focus cropped image of full tissues in (A) are indicated by white boxes. Scale bars are 100 μm. D) Extended focus image of tissue FISH-PLA at 63x is shown. Scale bar is 10 μm. E) Extended focus cropped images of full tissues in (B) are indicated by white boxes. Extended focus images of tissue FISH-PLA at 63x are also shown. Scale bars are 100 μm for 20x images and are 10 μm for 63x images. F) Quantification of PLA signal in (A, B). Statistics were performed with a one-way ANOVA with a Tukey's multiple comparisons test, where n=3 and ** p < 0.0087. Standard deviations are shown in red.

Supplementary Figure 6: PMTRIP FISH-PLA can quantify COX-2-TIAR interactions in lung tissue. A) 20 nM of V5 tagged PMTRIPs targeting COX-2 mRNA were delivered as FISH probes to colon cancer samples. PLA (green) was performed between the V5 tag and TIAR. 20x cropped image is indicated by the white box. Extended focus images are shown, with a 1 mm scale bar for full tissue. B) Extended focus images of FISH-PLA (green) controls are shown. Scale bar is 1 mm. C) Extended focus cropped images of full tissues in (A) are indicated by white boxes. Scale bars are 100 μm. D) Extended focus image of cancer tissue FISH-PLA at 63x is also shown. Scale bars for (D) are 10 μm. E) Extended focus cropped images of full tissues in (B) are indicated by white boxes. Extended focus images of tissue FISH-PLA at 63x are also shown. COX-2 FISH shown in red. Scale bars are 100 μm for 20x and 10 μm for 63x images. F) Quantification of PLA signal in (A,B). Statistics were performed with a one-way ANOVA with a Tukey's multiple comparisons test, where n=3 and * p < 0.015 and ** p < 0.0077. Standard deviations are shown in red.

Supplementary Figure 1: PNA increases mRNA-protein PLA specificity upon live delivery and reduces the need for formamide and dextran sulfate for FISH. A) 5 nM of of V5-tagged MTRIPs with either PNA or 2OM oligonucleotides targeting polyadenylated mRNA were delivered to live cells via SLO. PLA (white) was performed between V5 and HuR. Extended focus images are shown. Scale bars indicate 10 μm. Quantification of PLA is shown. Statistics were performed with a two-way ANOVA with a Tukey's multiple comparisons test, where n=30 and **** p < 0.0001. 95% confidence intervals are shown in black. B) Cells were infected with RSV, they were exposed to FISH hybridization buffer with either 0% or 10% formamide overnight, and stained for F (red) and M (green). Extended focus images are shown. Scale bars indicate 10 μm. C) 10 μg of Dylight 650 pre-labeled luciferase mRNA (red), either naked or complexed with Viromer Red, was delivered to mouse muscle. FISH (green) was performed either with cy3B-PMTRIPs or Quazar 570 DNA oliognucleotides. D) 200 ng of cy3B pre-labeled luciferase mRNA (red) was delivered to cells via L2K transfection. FISH (green) was performed either with PMTRIPs or DNA oliognucleotides. E) Representative images of sham controls for (C) and (D). F) PMTRIP FISH was performed in A549s for polyadenylated mRNA or for a scrambled sequence. G) PMTRIP FISH was performed in HT-29s for COX-2, Actin-β or for a scrambled sequence. H) PMTRIP FISH was performed in DLD-1s for COX-2. In all images scale bars represent 10 μm.

PNA Oligonucleotide Sequences

Abnormal post-transcriptional regulation induced by alterations of mRNA–protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA–protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA–protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA–protein interactions are of interest.Significance:This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.

Supplementary Figure 7: PMTRIP FISH-PLA can quantify PolyA-HuR interactions in frozen colon tissue. A) 5 nM of V5 tagged PMTRIPs targeting polyadenylated mRNA were delivered as FISH probes to tissue samples. PLA (green) was performed between the V5 tag and HuR. Extended focus images are shown, with a 1 mm scale bar. B) Extended focus cropped images of full tissues in (A) are indicated by white boxes. Extended focus images of tissue FISH-PLA at 63x are also shown. Scale bars are 100 μm for 20x and 10 μm for 63x images. C) Quantification of PLA signal in (A) is shown. Standard deviations are shown in red.

Lipid nanoparticles (LNPs) for the efficient delivery of drugs need to be designed for the particular administration route and type of drug. Here we report the design of LNPs for the efficient delivery of therapeutic RNAs to the lung via nebulization. We optimized the composition, molar ratios and structure of LNPs made of lipids, neutral or cationic helper lipids and poly(ethylene glycol) (PEG) by evaluating the performance of LNPs belonging to six clusters occupying extremes in chemical space, and then pooling the lead clusters and expanding their diversity. We found that a low (high) molar ratio of PEG improves the performance of LNPs with neutral (cationic) helper lipids, an identified and optimal LNP for low-dose messenger RNA delivery. Nebulized delivery of an mRNA encoding a broadly neutralizing antibody targeting haemagglutinin via the optimized LNP protected mice from a lethal challenge of the H1N1 subtype of influenza A virus, and delivered mRNA more efficiently than LNPs previously optimized for systemic delivery. A cluster approach to LNP design may facilitate the optimization of LNPs for other administration routes and therapeutics. Lipid nanoparticles can be optimized for the efficient delivery of therapeutic mRNAs to the lung via nebulization, as shown for the delivery of a therapeutic antibody in mice challenged with a lethal dose of the H1N1 influenza A virus.

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.

CAR T cell immunotherapy relies on CAR targeting of tumor-associated antigens, yet heterogenous antigen expression, interpatient variation, and off-tumor expression by healthy cells remain barriers. Here, we develop synthetic antigens to sensitize solid tumors for recognition and elimination by CAR T cells. Unlike tumor-associated antigens, we design synthetic antigens that are orthogonal to endogenous proteins to eliminate off-tumor targeting and that have a small genetic footprint to facilitate efficient tumor delivery to tumors by viral vectors. Using the RSV-F camelid single-domain antibody (VHH) as a synthetic antigen, we show that adoptive transfer of αVHH CAR T cells to mice bearing VHH expressing tumors reduced tumor burden in multiple syngeneic mouse models of cancer, improved survival, induced epitope spread, and protected against tumor rechallenge. Our work supports in situ delivery of synthetic antigens to treat antigen low or negative tumors with CAR T cells.

BackgroundAppropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, “M2-like” macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4).MethodsTo enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing.ResultsWe observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain.ConclusionThe results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.

Abnormal post-transcriptional regulation induced by alterations of mRNA–protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA–protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA–protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA–protein interactions are of interest. Significance: This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.

CAR T cell immunotherapy relies on CAR targeting of tumor-associated antigens, yet heterogenous antigen expression, interpatient variation, and off-tumor expression by healthy cells remain barriers. Here, we develop synthetic antigens to sensitize solid tumors for recognition and elimination by CAR T cells. Unlike tumor-associated antigens, we design synthetic antigens that are orthogonal to endogenous proteins to eliminate off-tumor targeting and that have a small genetic footprint to facilitate efficient tumor delivery to tumors by viral vectors. Using the RSV-F camelid single-domain antibody (VHH) as a synthetic antigen, we show that adoptive transfer of αVHH CAR T cells to mice bearing VHH expressing tumors reduced tumor burden in multiple syngeneic mouse models of cancer, improved survival, induced epitope spread, and protected against tumor rechallenge. Our work supportsin situdelivery of synthetic antigens to treat antigen low or negative tumors with CAR T cells.

Understanding viral rebound in pediatric HIV-1 infection may inform the development of alternatives to lifelong antiretroviral therapy (ART) to achieve viral remission. We thus investigated viral rebound after analytical treatment interruption (ATI) in 10 infant macaques orally infected with SHIV.C.CH505 and treated with long-term ART. Rebound viremia was detected within 7 to 35 days of ATI in 9 of 10 animals, with posttreatment control of viremia seen in 5 of 5 Mamu-A*01+ macaques. Single-genome sequencing revealed that initial rebound virus was similar to viral DNA present in CD4+ T cells from blood, rectum, and lymph nodes before ATI. We assessed the earliest sites of viral reactivation immediately following ATI using ImmunoPET imaging. The largest increase in signal that preceded detectable viral RNA in plasma was found in the gastrointestinal (GI) tract, a site with relatively high SHIV RNA/DNA ratios in CD4+ T cells before ATI. Thus, the GI tract may be an initial source of rebound virus, but as ATI progresses, viral reactivation in other tissues likely contributes to the composition of plasma virus. Our study provides potentially novel insight into the features of viral rebound in pediatric infection and highlights the application of a noninvasive technique to monitor areas of HIV-1 expression in children.

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-β-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.

Nanoparticles are tested in mice and non-human primates before being selected for clinical trials. Yet the extent to which mRNA delivery, as well as the cellular response to mRNA drug delivery vehicles, is conserved across species in vivo is unknown. Using a species-independent DNA barcoding system, we have compared how 89 lipid nanoparticles deliver mRNA in mice with humanized livers, primatized livers and four controls: mice with 'murinized' livers as well as wild-type BL/6, Balb/C and NZB/BlNJ mice. We assessed whether functional delivery results in murine, non-human primate and human hepatocytes can be used to predict delivery in the other species in vivo. By analysing in vivo hepatocytes by RNA sequencing, we identified species-dependent responses to lipid nanoparticles, including mRNA translation and endocytosis. These data support an evidence-based approach to making small-animal preclinical nanoparticle studies more predictive, thereby accelerating the development of RNA therapies.

Lipid nanoparticles (LNPs) for the efficient delivery of drugs need to be designed for the particular administration route and type of drug. Here we report the design of LNPs for the efficient delivery of therapeutic RNAs to the lung via nebulization. We optimized the composition, molar ratios and structure of LNPs made of lipids, neutral or cationic helper lipids and poly(ethylene glycol) (PEG) by evaluating the performance of LNPs belonging to six clusters occupying extremes in chemical space, and then pooling the lead clusters and expanding their diversity. We found that a low (high) molar ratio of PEG improves the performance of LNPs with neutral (cationic) helper lipids, an identified and optimal LNP for low-dose messenger RNA delivery. Nebulized delivery of an mRNA encoding a broadly neutralizing antibody targeting haemagglutinin via the optimized LNP protected mice from a lethal challenge of the H1N1 subtype of influenza A virus, and delivered mRNA more efficiently than LNPs previously optimized for systemic delivery. A cluster approach to LNP design may facilitate the optimization of LNPs for other administration routes and therapeutics.

Here, Cas13a has been used to target and mitigate influenza virus A (IAV) and SARS-CoV-2 using a synthetic mRNA-based platform. CRISPR RNAs (crRNA) against PB1 and highly conserved regions of PB2 were screened in conjunction with mRNA-encoded Cas13a. Screens were designed such that only guides that decreased influenza RNA levels in a Cas13-mediated fashion, were valid. Cas13a mRNA and validated guides, delivered post-infection, simulating treatment, were tested in combination and across multiplicities of infection. Their function was also characterized over time. Similar screens were performed for guides against SARS-CoV-2, yielding multiple guides that significantly impacted cytopathic effect. Last, the approach was utilized in vivo, demonstrating the ability to degrade influenza RNA in a mouse model of infection, using polymer-formulated, nebulizer-based mRNA delivery. Our findings demonstrate the applicability of Cas13a in mitigating respiratory infections both in vitro and in a mouse model, paving the way for future therapeutic use.

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.

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., Graphical Abstract, Santangelo and colleagues report on the aerosol delivery of synthetic mRNAs, diluted in water, to the cervicovaginal epithelium, which expresses membrane-anchored broadly neutralizing antibodies against HIV. In two large animal models, anchored PGT121 was widely dispersed in the reproductive tract and was protective against a SHIV explant challenge.

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro , depending on the delivery vehicle, resulting in variations in protein expression levels . Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels . This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.

Endothelial cells and macrophages play active roles in disease, and as a result, are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.

The human respiratory syncytial virus G protein plays an important role in the entry and assembly of filamentous virions. Here, we report the use of fluorescently labeled soybean agglutinin to selectively label the respiratory syncytial virus G protein in living cells without disrupting respiratory syncytial virus infectivity or filament formation and allowing for interrogations of respiratory syncytial virus virion assembly. Using this approach, we discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis. This event is then followed by the dynamic formation of filamentous and branched respiratory syncytial virus particles, and assembly with genomic ribonucleoproteins and caveolae-associated vesicles prior to re-insertion into the plasma membrane. We demonstrate that these processes are halted by the disruption of microtubules and inhibition of molecular motors. Collectively, our results show that for respiratory syncytial virus assembly, viral filaments are produced and loaded with genomic RNA prior to insertion into the plasma membrane., Assembly of filamentous RSV particles is incompletely understood due to a lack of techniques suitable for live-cell imaging. Here Vanover et al. use labeled soybean agglutinin to selectively label RSV G protein and show how filamentous RSV assembly, initiated in the cytoplasm, uses G protein recycled from the plasma membrane.

These authors contributed equally : Kevin E. Lindsay, Sushma M. Bhosle and Chiara Zurla.; International audience; Visualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals.

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

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., Author summary Respiratory syncytial virus is a common respiratory disease among children under the age of five. Yearly, approximately three million children infected require hospitalization for treatment. However, no vaccine or effective antiviral treatment for respiratory syncytial virus yet exists. Understanding how respiratory syncytial virus replicates in cells will provide key knowledge for the development of more effective countermeasures. Here, we investigated the interactions between the large protein subunit of the respiratory syncytial virus polymerase, which is responsible for viral mRNA synthesis and replication, and other viral proteins during infection, as well as the localization of the large protein subunit over time in key viral structures. Our study provides new insights into the localization and activity of the respiratory syncytial virus polymerase, and suggests ribonucleocapsid complex rearrangement plays a key role in the function of the respiratory syncytial virus polymerase in replication of the virus, leading to a better understanding of respiratory syncytial virus biology.

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 ~80 nanometers to ~10 microns 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 virion represent 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.

Therapeutic strategies based on in vitro–transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this “proof-of-principle” study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either Ifn-γ mRNA or Il-4 mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.

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.

As the next generation of gene therapy, synthetic mRNA therapy is known for its intrinsic advantages to avoid genomic integration that associated with DNA-based therapy, and its prolonged production of target proteins over traditional recombinant protein therapy. Synthetic mRNA therapy has been studied in various disease models including cancer, infectious disease and cardiovascular disease. However, these studies focus mainly on expressing proteins with direct therapeutic effect or antigens to elicit an immune response. It is not well established that whether synthetic mRNA can be taken advantage in cell based therapies in vivo to recruit cells of interest and modify them locally. Here we reported a synthetic mRNA based immune-modulation method, exampled by using Ccl2 and Ccl3 synthetic mRNA to recruit a certain population of monocyte in a non-inflamed manner. These recruited monocytes exhibit neither bactericidal or tissue-repairing behaviors, but stay in a neutral, non-programmed state. With additional Ifn-g mRNA and Il-4 mRNA, these cells can be polarized to different phenotypes. Furthermore, the monocytes recruited by Ccl3 and Ifn-g mRNA are able to launch the most rapid and strong superoxide burst, which demonstrates their active functionality upon proper signals. In summary, our data established a synthetic mRNA based immune-modulation system, which allows recruitment and modification of specific immune cell populations in a precise and localized manner, and more importantly, proves the concept that synthetic mRNA can be utilized in cell based therapies to replace the time-consuming and labor-demanding process of modifying patients’ own cells ex vivo and transferring back to patients afterwards.

To date, the mRNA delivery field has been heavily dominated by lipid-based systems. Reports on the use of nonlipid carriers for mRNA delivery in contrast are rare in the context of mRNA vaccination. This paper describes the potential of a cell-penetrating peptide containing the amphipathic RALA motif to deliver antigen-encoding mRNA to the immune system. RALA condenses mRNA into nanocomplexes that display acidic pH-dependent membrane disruptive properties. RALA mRNA nanocomplexes enable mRNA escape from endosomes and thereby allow expression of mRNA inside the dendritic cell cytosol. Strikingly, RALA mRNA nanocomplexes containing pseudouridine and 5-methylcytidine modified mRNA elicit potent cytolytic T cell responses against the antigenic mRNA cargo and show superior efficacy in doing so when compared to RALA mRNA nanocomplexes containing unmodified mRNA. RALA's unique sequence and structural organization are vital to act as mRNA vaccine vehicle, as arginine-rich peptide variants that lack the RALA motif show reduced mRNA complexation, impaired cellular uptake and lose the ability to transfect dendritic cells in vitro and to evoke T cell immunity in vivo.

The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses.