At the fundamental level, messenger RNA (mRNA)-based therapeutics involves the delivery of in vitro-transcribed (IVT) mRNA into the cytoplasm of a target cell, where it is translated into the desired protein. IVT mRNA presents various advantages compared to DNA and recombinant protein-based approaches that make it ideal for a broad range of therapeutic applications. IVT mRNA, which is translated in the cytoplasm after transfection into cells, can encode virtually any target protein. Notably, it does not enter the nucleus, which avoids its integration into the genome and the risk of insertional mutagenesis. The large-scale production of IVT mRNA is less complex than production of recombinant proteins, and Good Manufacturing Practice-compliant mRNA production is easily scalable, ideally poising mRNA for not only off-the-shelf, but more personalized treatment approaches. IVT mRNA's safety profile, pharmacokinetics, and pharmacodynamics, including its inherent immunostimulatory capacity, can be optimized for different therapeutic applications by harnessing a wide array of optimized sequence elements, chemical modifications, purification techniques, and delivery methods. The value of IVT mRNA was recently proved during the COVID-19 pandemic when mRNA-based vaccines outperformed the efficacy of established technologies, and millions of doses were rapidly deployed. In this review, we will discuss chemical modifications of IVT mRNA and highlight numerous preclinical and clinical applications including vaccines for cancer and infectious diseases, cancer immunotherapy, protein replacement, gene editing, and cell reprogramming.