651. Making the Optimal Messenger RNA for Gene Therapy Applications: Evaluation of Novel Nucleotide Modifications for Improved Activity
Authors: Anton P. McCaffrey, Alexandre Lebedev, Dongwon Shin, Krist Azizian, Julie R. Escamilla-Powers, Brea Midthune, Hiroko Yokoe, Joel Jessee, Richard I. Hogrefe
Journal: Molecular Therapy (2015)

Recently, there has been significant interest in the use of messenger RNA (mRNA) based expression systems for gene therapy applications. Several groups have shown that mRNA is an attractive vehicle for therapeutic gene expression in mammals (Kormann et al. Nat. Biotechnol (2011) 29, 154; Kariko et al. Molecular Therapy (2012) 20, 948). mRNAs are expressed in the cytoplasm of cells which may improve expression in non-dividing cells, which are difficult to transfect. Additionally, Warren et al. demonstrated highly efficient induced pluripotent stem cell (iPS cells) generation by transfection of mRNAs encoding reprogramming factors (Warren et al. Cell Stem Cell (2010) 7, 618). The authors suggested that iPS cells generated in this manner should be safer than iPS cells derived by plasmid transfection or viral transduction since mRNA poses no risk of insertional mutagenesis and subsequent oncogenesis. In addition, transient expression from mRNA is desirable for applications such as genome editing using zinc-finger nucleases, TALENs and Cas9/CRISPR. Lastly, there is considerable interest in using mRNA for vaccines (Geall et al. Expert Rev Vaccines (2015) 14, 151). A key insight for the development of mRNA expression systems was the recognition that mRNA induces innate immune responses in transfected cells. Kariko et al. showed that substitution of uridine and cytidine residues with pseudouridine and 5-methylcytidine dramatically reduced innate immune recognition of mRNA (Kariko et al. Molecular Therapy (2008) 16, 1833). They also showed that pseudouridine modified RNA was translated more efficiently. These studies highlight the importance of the development of stable, non immunogenic mRNA. Activity and immunogenicity of mRNAs likely depends on the chemical modification pattern, the route of delivery and cell type or tissue transfected. To date, however, there have been few studies to assess novel chemically modifications of mRNAs. Here we synthesized a panel of numerous novel nucleotide triphosphates (NTPs). The panel consisted primarily of 5-position-modified pyrimidines but also included other pyrimidine and purine NTPs. A large number of combinations of the modifications were used to synthesize GFP and luciferase mRNAs in order to evaluate the ability of different combinations of modifications to support transcription by T7 RNA polymerase. The translation potential of the mRNAs was evaluated in rabbit reticulocyte lysates. The activity and toxicity profile of these mRNA was further evaluated in panels of different primary and immortalized cell lines. Comparison of in vitro translation with cell culture expression allows us uncouple translation from cytotoxicity, RNA stability and innate immune stimulation in cells. While trends were seen, cell type specific differences in expression were also observed. These studies greatly expand our knowledge of the optimal chemical modification of mRNA required to achieve maximal expression in different cell types.