Life science
Gene delivery, Immunotherapy, Vaccine

Development of a novel LCMV vector capable of controlling
foreign gene expression
Laboratory of Emerging Viral Diseases, Research Institute for Microbial Diseases
Specially Appointed Associate Professor Masaharu Iwasaki
https://researchmap.jp/m_iwasaki

Life science

Abstract
Lymphocytic choriomeningitis virus (LCMV) generates mRNA
with a 5′-capped untranslated region (UTR) and a non-polyadenylated 3′-UTR (vmRNA). We previously reported that the non-polyadenylated 3′-UTR, which is derived from a non-coding intergenic region (IGR) in the LCMV RNA genome, regulates viral protein
levels at the posttranscriptional level. In this study, we found that ashort 3′-UTR sequence located at the 5′side of the vmRNA (proximal region, PR) was a major determinant of translation efficiency.
Furthermore, the reporter protein level was altered in cells infectedwith recombinant LCMV in which the IGR sequence that corresponded to the PR was modified.

the noncytolytic nature and broad tissue tropism, this flexibility to
manage foreign gene expression levels expands the utility of a recombinant LCMV-based gene delivery system to more demanding
applications, such as the generation of induced pluripotent stem
cells, in which the levels of transduced reprogramming factors
must be precisely controlled for effective derivation.

▍ Background & Results
Several mammalian arenaviruses (mammarenavirus) cause hemorrhagic fever in humans and are a major public health concern inendemic regions. Lassa virus is the most significant mammarenavirus, being estimated to infect approximately 900,000 individuals
in West Africa each year, resulting in high numbers of Lassa fevercases. Case fatality rates among patients hospitalized with Lassa
fever are reported to be approximately 15%. The worldwide-distributed prototypic mammarenavirus, LCMV, is genetically closelyrelated to Lassa virus and can be handled in a biosafety level 2facility, offering surrogate infection models for Lassa virus. On thebasis of our previous finding, we developed live-attenuated vaccines against human pathogenic mammarenaviruses by manipulating the IGR sequence. In this study, we further investigated thecontribution of the 3′-UTR sequence of LCMV mRNA (vmRNA) totranslation regulation employing a reporter system with in vitro transcribed mRNA (vlmRNA) that contained the ZsGreen open reading
frame flanked by 5′-capped and 3′-non-polyadenylated UTRs
derived from the vmRNA (Fig. 1). The analysis using vlmRNAs that
contained chimeric 3′-UTR sequences swapped between nucleoprotein (NP, high translation efficiency) and glycoprotein precursor(GPC, low translation efficiency) mRNAs showed that a 10-nucleotide sequence proximal to the nucleoprotein open reading frameand its predicted secondary structure were essential for promoting
translation. Modification of this 10-nucleotide sequence in recombinant LCMV (Fig. 2) also impacted expression of the reporter gene(Fig. 3), suggesting that the expression of a foreign gene was controlled by modifying the PR sequence.

Fig. 1

Fig. 2▍ Significance of the research and Future perspective
Because LCMV can induce robust long-term CD8+ cytotoxic T
lymphocyte responses against virus antigens, attenuated recombinant LCMV was proposed as a tumor immunotherapy platform
to deliver tumor-associated antigens. Our finding provides theopportunities to improve recombinant LCMV-based gene delivery
systems to fine-tune both the expression of a foreign gene and thedegree of attenuation by altering the PR sequence. In addition toFig. 3

P a t e n t
Hashizume, Mei; Takashima, Ayako; Iwasaki, Masaharu. A small stem-loop-forming region within the 3′-UTR of a non-polyadenylated LCMV mRNA
promotes translation. Journal of Biological Chemistry, 2022, 298 (2): 101576, doi: 10.1016/j.jbc.2022.101576
URL https://iwasaki-lab.biken.osaka-u.ac.jp
K e y w o r d virus vector, untranslated region, translation regulation
Tr e a t i s e