Optimizing Cardiac Delivery of Modified mRNA.

Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5'-Triphosphate nucleotide modification and achieved using 0.013 µg modRNA/mm2/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 µg/mouse heart (1.6 µg modRNA/µL in 60 µL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.

Male with Altered Mental Status.

CASE PRESENTATION: A 62-year-old male presented to the emergency department with altered mental status and fever. Computed tomography of the head showed enlargement of the left lateral ventricle. Magnetic resonance imaging demonstrated debris and purulence in the ventricle along with edema and transependymal flow of cerebrospinal fluid surrounding both ventricles. DISCUSSION: The patient was diagnosed with ventriculitis. Ventriculitis is an uncommon but serious disease. Early recognition and treatment are essential.

Synthesis of Modified mRNA for Myocardial Delivery.

Cardiac gene therapy shows tremendous promise in combating the growing problem of heart disease. Modified mRNA (modRNA) is a novel gene delivery system used in vitro or in vivo to achieve transient expression of therapeutic proteins in a heterogeneous population of cells. Incorporation of specific modified nucleosides enables modRNA to be translated efficiently without triggering antiviral and innate immune responses. ModRNA has been shown to be effective at delivering short-term robust gene expression to the heart and its use in the field of cardiac gene therapy is expanding. Here, we describe a stepwise protocol for the synthesis of modRNA for in vivo myocardial delivery.