Paramyxovirus vectors used for transfer of foreign genes

Details Paramyxovirus vectors used for transfer of foreign genes Paramyxovirus vectors used for transfer of foreign genes

2000-10-31

2004-06-08

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S320100, C514S04400A, C536S023720

Reexamination Certificate

active

06746860

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of Japanese patent application JP 2000/152726 filed May 18, 2000.
FIELD OF THE INVENTION
The present invention relates to a paramyxovirus vector capable of transferring foreign genes and having a replication capacity.
BACKGROUND OF THE INVENTION
Until now, most of the approaches of clinical studies pertaining to gene therapy have utilized viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses. These gene therapy vectors have limitations in gene transfer efficiency and continuous expression. Furthermore, the vectors themselves may have cytotoxicity, immunogenicity, and such problems are crucial when it comes to the medical application of these vectors (Lamb, R. A. & Kolakofsky, D., Paramyxoviridae: the viruses and their replication. in Fields Virology, 3rd edn, (Edited by B. N. Fields, D. M. Knipe & P. P. Howley) pp.1177-1204 (Philadelphia, Lippincott-Raven. (1996)). As a solution, novel vectors based on lentivirus and HSV have been proposed, and research is also being vigorously carried out to modify existing vectors. However, all these vectors exist in the form of DNA within the nucleus, throughout their life cycles. Therefore, it is difficult to overcome concerns of safety regarding random interactions with the chromosomes of the patient.
Rapid progress of reverse genetics technology is beginning to enable the development of vectors based on RNA viruses, which had long been delayed. Recombinant RNA vectors have a high gene transfer efficiency and expression capacity, and therefore, are highly potential vectors for gene therapy (Roberts, A. & Rose, J., K., Virology 247,1-6 (1998); Rose, J., Proc. Natl. Acad. Sci. USA 94, 14998-15000 (1996); Palese, P. et. al., Proc. Natl. Acad. Sci. USA 93, 11354-11358 (1996)). The paramyxovirus vectors that comprise negative strand RNA in the genome have several characteristics that significantly differ from retroviruses, DNA viruses, or plus strand RNA viruses. This genome or antigenome does not directly function as mRNA, and cannot initiate protein synthesis and genome replication of the virus. The RNA genome and antigenome of the virus consistently exists in the form of a ribonucleoprotein complex (RNP), and therefore problems of antisenses observed in plus strand RNA viruses, such as the inhibition of the assembly of genome towards RNP due to the hybridization of mRNAs to complementary naked genomic RNA, rarely occur. These viruses have their own RNA polymerase, and the transcription of viral mRNA or viral genome replication is carried out using the RNP complex as template. Worthy of special notice is the fact that negative strand RNA (nsRNA) viruses proliferate only within the cytoplasm of host cells, and since they do not have a DNA phase, they are not integrated into chromosomes. Furthermore, homologous recombination between RNAs has also not been observed. These features contribute largely to the stability and safety of negative strand RNA viruses as gene expression vectors.
Among the negative strand RNA viruses, the inventors have been focusing their attention on Sendai virus (SeV) that is not pathogenic towards humans, particularly the Z strain that is especially avirulent. SeV is a non-segmented negative strand RNA virus belonging to Paramyxoviruses, and is a type of murine parainfluenza virus. This virus attaches to the host-cell membrane via hemagglutinin-neuraminidase (HN) and fusion protein (F), which are two envelope glycoproteins, initiates membrane fusion, releases its own RNA polymerase and the RNA genome that exists in the form of ribonucleoprotein (RNP) complex into the cytoplasm, and carries out mRNA transcription and genome replication of the virus there (Bitzer, M. et al., J. Virol. 71(7): 5481-5486, 1997). The viral envelope protein F is synthesized as a non-active pre-protein (F
0
), is cleaved into F1 and F2 through proteolytic cleavage by trypsin (Kido, H. et al., Biopolymers (Peptide Science) 51(1): 79-86, 1999), and turns into an active protein causing membrane fusion. This virus is said to be non-pathogenic towards humans. Moreover, a laboratory-attenuated strain (Z strain) has also been isolated, which only induces a slight pneumonia in rodents, its natural hosts. This strain is widely used as a research model for molecular-level studies in the transcription/replication mechanisms of paramyxoviruses, and has also been used in the preparation of hybridomas. Apart from a high safety, this virus shows a high production titer of 10
9
to 10
11
pfu/ml in cell lines and hen-eggs. In one recent successful recovery system from negative strand RNA virus cDNA, Sendai viruses showed an especially high reconstitution efficiency rate. In recombinant wild-type viruses transfected with foreign genes, the capacity to express the foreign gene efficiently as well stably, is gaining wide attention.
Even though Sendai viruses having a foreign gene upstream of the NP gene have been known, it was not known how the viral reconstitution and foreign gene expression will be affected when the foreign gene was inserted into a site other than the above.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a paramyxovirus vector capable of transferring foreign genes and having a replicating capacity.
The inventors constructed viral vector DNA, in which a foreign gene was inserted to a site other than the upstream of the NP gene of the Sendai virus, and examined viral reconstitution and the expression level of the foreign gene. Namely, a new restriction enzyme site for inserting the foreign gene was introduced between the start signal and ATG translation start signal of each gene encoding viral proteins of Sendaivirus (SeV) full-length cDNA. A foreign gene (human secreted alkaline phosphatase (SEAP) gene) was inserted into this restriction enzyme site, and when reconstitution of the Sendai virus was conducted using LLC-MK2 cells, it was seen that only Sendai viruses having the capacity to proliferate were reconstituted. These viruses were amplified within hen-eggs to prepare a viral stock solution. These virus titers were combined and infected into LLC-MK2 cells, and the expression level of the foreign gene was determined to find that foreign gene expression is seen in all cases examined where the foreign gene was inserted into different sites. The foreign gene expression was relatively high when it was inserted between NP gene and P gene, or between P gene and M gene, and it was revealed that the foreign gene expression dropped as the site of insertion neared the downstream (5′ side of the negative strand) of the genome.
These results suggest that it is possible to obtain a relatively high foreign gene expression by placing the foreign gene downstream of the NP gene or the P gene, and that it is possible to decrease the expression level by placing the foreign gene towards the downstream of the genome. Based on these findings, it is possible to regulate the expression level of the foreign gene within the vector, by inserting the foreign gene into the upstream of the genome, namely, in the 3′ side of the negative strand genome, to obtain a high expression of the foreign gene. Conversely, when a high expression is not preferable, such as when the gene is cytotoxic, the foreign gene can be inserted downstream of the genome, namely, in the 5′ side of the negative strand genome. The paramyxovirus vector of the invention is useful in the expression of foreign genes in vivo and in vitro, and would especially be applied in gene therapy taking advantage of the outstanding features of paramyxoviruses.
Though problems regarding genomic stability may be pointed out in RNA viruses, results of heterologous gene expressions using SeV vector showed that there were hardly any nucleotide mutations even when the virus was serially passaged over several generations, and that it is possible to stably express the inserted heterologous genes over a long period of time (Yu, D. et al. Genes cells 2, 457-466 (1997)). Vectors based

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