z and enhanced catalysis to the ribosomal complex [32][33][34][35][36] . Optimization of the poly(A) tail length (100-300 nucleotides) has proven critical in balancing the synthetic capability of a given mRNA 34,36 . Similarly, improved 5′ cap analogs not only increase translational capacity but also enhance capping efficiency, from 70% to 95%, greatly improving the in vitro transcription process 32,37 . The composition of the 3′ and 5′ UTRs can also be customized for the target cell of interest, increasing the efficiency and tissue specificity of translation 35,38,39 .At present, most mRNA products contain a synthetic UTR sequence from α-globin or β-globin [38][39][40] , but UTR optimization can further improve protein expression by a few fold 41,42 . Careful screening and customization to the target of interest could conceivably offer a wide range of improvements in future UTR sequences, allowing each mRNA to be tailored to the targeted cell and disease-induced microenvironment to maximize protein synthesis per mRNA transcript [41][42][43][44] .Perhaps the most critical advances in mRNA vaccines and therapeutics lie in the discovery that the inclusion of chemically modified nucleosides, particularly in uridine moieties, can markedly increase protein expression after in vitro or in vivo transfection. The chemical modifications of most new RNA formulations to date have been central in intellectual property claims 45,46 . Thus far, over 130 different naturally occurring chemical modifications of RNA have been reported 47,48 . The interest in methylpseudouridine and other modified nucleosides centers on their capacity to greatly reduce (up to 100-fold) detection by the Toll-like receptors of the innate immune system, resulting in an increase in protein expression in vivo compared to unmodified mRNA 40,[49][50][51][52][53] . Combinations of different types of chemical modifications, carriers, for the treatment of chronic conditions. The fifth section provides a comprehensive table and summary of current clinical trends in mRNA therapeutics. Finally, the sixth section considers the scope of mRNA therapeutics and guiding principles for near-term and longer-term clinical development of this novel therapeutic modality.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Aims Retinoic acid (RA) signaling is essential for heart development, and dysregulation of the RA signaling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. Methods and Results To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing downregulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signaling in human cardiogenesis. Conclusion Our study highlights a critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations. Translational Perspectives Dysregulation of the RA signaling can cause cardiac OFT defects, however, the detailed mechanisms by which STRA6 mutations lead to cardiac malformations have remained unclear. Our study highlights a critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. These results shed light on novel and human-specific CHD programs, driven by STRA6 mutations. Thus, our study paves the way for further studies of deciphering the origins and the disease mechanisms of a rare genetic disorder Matthew-Wood syndrome, which would help us develop diagnosis, prevention, and novel treatment for the disease.