841.A Cytoplasmic Gene Therapy with Sendai Virus Vectors: Reduction of Cytopathic and Immunogenic Reaction of New Class Cytoplasmic RNA Vector

Abstract

A new class of CYTOPLASMIC RNA VECTORS is thought to be advanced transgene expression systems that overcome recent problem of genetic disturbance by existing vectors. Our Sendai virus (SeV) vector based on SeV belonging to the family Paramyxoviridae, infects and multiplies its copy 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 (CYTOPLASMIC GENE THERAPY) and vaccination via the expression of therapeutic genes and antigens. In order to establish such SeV system, we have taken the strategy to generate the gene(s)-deleted or modified SeV vectors. Thus, we have succeeded in the recovery in high titers of fusion (F) gene-deleted (SeV/|[Delta]|F), matrix (M) gene-deleted (SeV/|[Delta]|M), hemagglutinin-neuraminidase (HN) gene-deleted (SeV/|[Delta]|HN), both M and F genes-deleted (SeV/|[Delta]|M|[Delta]|F), all of the envelop-related genes-deleted (SeV/|[Delta]|M|[Delta]|F|[Delta]|HN) SeV vectors by using the packaging cell lines which express respective proteins of those deleted gene(s). All the vectors showed efficient infectivity and transgene expression in various types of cell lines and primary cells in vitro. F gene-deletion made SeV vector non-transmissible, M gene-deletion worked well on the vector to become incapable of formation of the particles from infected cells, and HN gene-deletion was expected to reduce the host immune response against SeV vector. From this set of vectors, an appropriate type of SeV vector can be selected depending on the characteristics of applied diseases or protocols. By these vector engineering, a great advance of the SeV vector system has been achieved from the view of cytopathy and immunogenicity originated from SeV. The cytopathic reaction of SeV/|[Delta]|F was reduced from that of transmissible wild type SeV, and the additional gene(s)-deletion(s) from SeV/|[Delta]|F made much further reduction. Because the HN protein that mediates the cell-attachment of SeV is known to be one of the major targets for host immune responsive machineries on SeV infection, the HN gene-deletion from the genome was expected to be one of the important ways to reduce the immune response against SeV vector. As expected, the production of neutralizing antibody was reduced in the case of SeV/|[Delta]|M|[Delta]|F|[Delta]|HN. In addition, no activity of neutralizing antibody was seen when SeV/|[Delta]|M|[Delta]|F|[Delta]|HN was trasduced ex vivo. In this case, anti-HN antibody was not detected in the serum. Therefore, the gene(s)-deletion from SeV genome is very effective to reduce both the immunogenic and cytopathic reaction of SeV vector. Additional introduction of amino acid mutations identified from SeV strains showing persistent infection with slight lowered transcription and replication was also effective for the reduction of cytopathic effect. These modifications of SeV vector would improve the safety and broaden the range of the clinical application of this vector.