491. Messenger RNA (mRNA)-Based Gene Therapy for Introducing Anti-Apoptotic Factor

Abstract

In gene therapy, mRNA delivery lacks the risk of random integration to host genome in contrast to DNA delivery. In introduction of anti-apoptotic factors, uncontrollably enhanced and prolonged expression of such factors after genomic integration would cause cancer, and thus DNA delivery cannot be used for introducing these factors in clinical settings. In this study, we tried to treat fulminant hepatitis, a disease associated with excessive apoptosis, by mRNA-based introduction of an anti-apoptotic factor, Bcl-2. For efficient mRNA delivery with minimal inflammatory responses, mRNA was loaded into our original carrier, polyplex nanomicelles (Adv Drug Deliv Rev 2001, 47, 113, PLoS One 2013, 8, e56220), which are composed of polyethyleneglycol-coated surface and mRNA-containing core, for hydrodynamic injection into mouse liver.In a reporter assay using GFP-expressing mRNA, the nanomicelles effectively introduced mRNA into liver cells by preventing degradation of mRNA, resulting in significantly higher level of GFP expression in liver compared to that after naked mRNA injection. This result is quite different from that of naked plasmid DNA (pDNA) delivery, strongly suggesting that mRNA is unstable in mouse body, and a proper carrier is required for mRNA delivery.In tissue distribution analysis of GFP expression, mRNA delivery induced GFP expression uniformly in almost 100% of liver cells, whereas only a small percentage of liver cells became GFP positive after pDNA delivery. After delivery of Cy5-labeled pDNA, nuclear localization of pDNA was undetected in a large percentage of cells, indicating that nuclear entry was a large obstacle in DNA delivery. In contrast, protein expression from mRNA requires only cytoplasmic uptake of mRNA, and this was observed in almost all liver cells, resulting in uniform GFP expression profile.Then, we performed the treatment of fulminant hepatitis using Bcl-2-expressing gene, after preparing a mouse model of the disease by injecting Fas ligand. After mRNA-based introduction of Bcl-2, the number of apoptotic cells in liver was significantly reduced when compared to saline-treated control. In contrast, delivery of Bcl-2-expressing pDNA had almost no therapeutic effect. It is suggested that, in pDNA delivery, a large percentage of cells were unprotected from apoptotic signals because of inhomogeneous protein expression profile. Thus, these results indicate that mRNA delivery is more suitable for introducing intracellular signaling factors than DNA delivery, because such factors require homogeneous expression to obtain therapeutic effect. This is in sharp contrast to the case of secretable factors, where efficient introduction to only a small percentage of cells is sufficient to obtain therapeutic effect.In summary, mRNA delivery has two advantages over pDNA delivery: homogeneous distribution profile of protein expression, and lack of random genomic integration. Thus, mRNA delivery is promising for the treatment of various intractable diseases associated with massive apoptosis.