Transient pacing in pigs with complete heart block via myocardial injection of mRNA coding for the T-box transcription factor 18.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo.

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.

Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection.

The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.

RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane.

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.