Design and Production of mRNA-based Gene Vectors for Therapeutic Reprogramming of Cell Fate

Abstract

Recent advances in therapeutically important cell fate reprogramming fuelled a renaissance in the use of mRNAbased gene vectors. Thus, mRNA vectors were successfully employed to induce lasting epigenetic changes in various target cells making them short-listed vector candidates for the manufacture of therapeutic engraftment materials for autologous transplantation, artificial human tissues for drug discovery via high-throughput screening projects and also for therapeutic cell trans-differentiation directly in the human body. De-differentiation of cells into 'induced pluripotent stem cells', transgene-directed differentiation and trans-differentiation require the simultaneous delivery of a number of regulatory factors, and, favourably, potent reprogramming vector cocktails can be straightforwardly assembled from a selection of mRNA species. In addition, several proteins can be conveniently expressed from a single mRNA using internal ribosome entry sites (IRESes) or, alternatively, fusion proteins supplemented with 'polypeptide-cleaving' ribosome skipping sequences. This review is focused on the design and production of cell-fate changing mRNAs.

Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse

“… 3 , 4 To evaluate the transfection ability, biodistribution, and pharmacokinetics of these delivery vectors in vivo , mRNAs coding for reporter genes (e.g., bioluminescent luciferases, fluorescent proteins, β-galactosidase, and others) are often used to optimize the system before therapeutic mRNA delivery is attempted. 5 Additionally, for most vectored mRNA delivery applications, it is useful to identify not only the targeted tissue but also the targeted cell populations within that tissue. For example, cancer immunotherapies would ideally express mRNA encoding antigen in dendritic cells of the lymphatic system; 6 , 7 conversely, protein replacement therapy for cystic fibrosis would target expression of mRNA encoding CFTR in lung epithelial cells.…”

Methods of Transfection with Messenger RNA Gene Vectors

“…One way to achieve this is to transfer an immediate information source for protein biosynthesis, that is, messenger RNA (mRNA), into target cells. Presently, mRNA-based vectors are established multipurpose gene vectors applicable to a diverse range of tasks in gene therapy, gene immunisation and transgene-mediated cell-fate reprogramming [1][2][3][4].…”

“…In general, mRNA-based vectors can be delivered to cells in tissue culture (in vitro) and intracorporeally (in vivo). In vitro gene delivery is a necessary step in ex vivo strategies of gene immunisation [8,9], gene therapy [10] and therapeutic cell-fate reprogramming [4]. As a rule, barriers outside tissues (e.g.…”