Persistent and Stable Gene Expression by a Cytoplasmic RNA Replicon Based on a Noncytopathic Variant Sendai Virus

Abstract

Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 ؋ 10 4 copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications.

Development of Defective and Persistent Sendai Virus Vector

“…All of these are indispensable for viral transcription and replication (10). We revealed previously that the L gene of the SeV Cl.151 strain with four missense mutations (V981I, S1088A, C1207S, and V1618L) contributes to long term persistence by providing the mechanism to escape from interferon ␤ (IFN␤) induction (12). Among these mutations, V1618L is most critical; SeV with a mutant L protein (V1618L) is defective in IFN␤ induction as with the Cl.151 strain.…”

“…Replication-competent SeV was reconstituted as described (12). Full-length SeVdp vector genomic cDNA for SeV (Cl.151 strain and Nagoya strain) and for installed genes was constructed on the lambda Dash II vector as described in supplemental Fig.…”

“…This unique variant was originally isolated as a mutant capable of persistent infection at a nonpermissive temperature (38°C) (13). We cloned the entire genome of SeV Cl.151 and determined that more than two genetic elements were responsible independently for the establishment of stable persistent infections (12). We also demonstrated that SeV Cl.151 installed with a single exogenous gene could express it stably without chromosomal insertion (12).…”

“…As SeV can infect various animal cells with an exceptionally broad host range and is not pathogenic to humans, various applications have been explored for SeV as a recombinant viral vector capable of transient but strong gene expression (11). We have demonstrated the potential of SeV as a tool for stable gene expression through an analysis of the Cl.151 strain (12). This unique variant was originally isolated as a mutant capable of persistent infection at a nonpermissive temperature (38°C) (13).…”

“…We cloned the entire genome of SeV Cl.151 and determined that more than two genetic elements were responsible independently for the establishment of stable persistent infections (12). We also demonstrated that SeV Cl.151 installed with a single exogenous gene could express it stably without chromosomal insertion (12). As this characteristic is advantageous for cell reprogramming, we planned to optimize this gene delivery/expression system through a more extensive analysis of SeV-mediated stable gene expression.…”