723. Sendai Virus Vectors without All of the Envelop-Related Genes: Successful Recovery and Characterization of the Three Genes (Matrix, Fusion and Hemagglutinin-Neuraminidase)-Deleted Vectors

Abstract

Sendai virus (SeV) vector is a new class of vector bearing a new concept of “cytoplasmic RNA vector”. This vector based on SeV belonging to the genus Respirovirus of the family Paramyxoviridae, infects and replicates in most mammalian cells, and directs high-level transgene expression. Its replication is independent of nuclear functions and does not have a DNA phase during its life cycle so that the transformation of cells by integration of vector materials into the cellular genome is not a concern. These properties make SeV vectors very promising for application to gene therapy and vaccination via the expression of therapeutic genes and antigens. We have previously succeeded in the recovery of fusion (F) gene-deleted SeV vector (SeV/ΔF), matrix (M) gene-deleted SeV vector (SeV/ΔM), both M and F genes-deleted SeV vector (SeV/ΔMΔF) and other types of the vectors in high titers. F gene-deletion made SeV vector non-transmissible, and M gene-deletion worked well on the vector to become incapable of formation of the particles from infected cells. All of the above SeV vectors maintain efficient infectivity in vitro and in vivo as wild type SeV does. The hemagglutinin-neuraminidase (HN) protein, which mediates the attachment of SeV, is known to be one of the major targets for host immune responsive machineries on SeV infection such as natural killer cell (NK), cytotoxic T lymphocyte (CTL) and neutralizing antibodies. The HN gene-deletion from the genome would be one of the important ways to reduce the immune response against SeV vector. Therefore, the HN gene was further deleted from the genome of SeV/ΔMΔF, and thus SeV/ΔMΔFΔHN was generated. The SeV/ΔMΔFΔHN possesses only nucleoprotein (NP), phosphoprotein (P) and large protein (L)-genes on its genome. All of them are essential for efficient expression of SeV vector by consisting ribonucleoprotein (RNP) complex. The new vector was successfully recovered and propagated in a new packaging cell line expressing M, F and HN proteins by using a Cre/loxP inducible system. The titer of SeV/ΔMΔFΔHN carrying the enhanced green fluorescent protein (GFP) was and above 1×107 cell infectious units (CIU)/ml. This vector showed efficient infectivity and gene expression in various types of cell lines and primary cells in vitro. When this vector was administered into the auricle of the ear of mice (5×106 CIU), a visible transduction was confirmed as in the cases of wild type SeV vector in vivo. The important investigation for immune reaction of this vector is in progress. The SeV/ΔMΔFΔHN vector is the most advanced type of cytoplasmic RNA virus vector and the like Sendai virus vector.