DNA expression systems based on alphaviruses

Details DNA expression systems based on alphaviruses DNA expression systems based on alphaviruses

2000-10-17

2004-08-03

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Recombinant virus encoding one or more heterologous proteins...

C424S093100, C424S093200, C424S093600, C424S204100, C435S320100, C435S455000, C435S456000

Reexamination Certificate

active

06770283

ABSTRACT:

The present invention is related to DNA expression systems based on alphaviruses, which systems can be used to transform animal cells for use in the production of desired products, such as proteins and vaccines, in high yields.
The rapid development of biotechnology is to a large extent due to the introduction of recombinant DNA technique, which has revolutionized cellbiological and medical research by opening new approaches to elucidate the molecular mechanisms of the cell. With the aid of the techniques of cDNA cloning, large numbers of interesting protein molecules are characterized each year. Therefore, a lot of research activity is today directed to elucidate the relationship between structure and function of these molecules. Eventually this knowledge will increase our possibilities to preserve healthiness and combat diseases in both humans and animals. Indeed, there is today a growing list of new “cloned” protein products that are already used as pharmaceuticals or diagnostics.
In the recombinant DNA approaches to study biological questions, DNA expression systems are crucial elements. Thus, efficient DNA expression systems, which are simple and safe to use, give high yields of the desired product and can be used in a variety of host cells, especially also in mammalian cells, are in great demand.
Many attempts have been made to develop DNA expression systems, which fulfill these requirements. Often, viruses have been used as a source of such systems. However, up to date none of the existing viral expression systems fulfill all these requirements in a satisfying way. For instance, the Baculovirus expression system for cDNA is extremely efficient but can be used only in insect cells (see Reference 1 of the list of cited references; for the sake of convenience, in the following the cited references are only identified by the number they have on said list). As many important molecules will have to be produced and processed in cells of mammalian origin in order for them to become active, this system cannot be used in such cases. Furthermore, the Baculovirus cDNA expression system is not practically convenient for analysis of the relationship between structure and function of a protein because this involves in general the analysis of whole series of mutant variants. Today it takes about 6-8 weeks to construct a single Baculo recombinant virus for phenotype analyses. This latter problem is also true for the rather efficient Vaccinia recombinant virus and other contemporary recombinant virus cDNA expression systems (2,3). The procedure to establish stably transformed cell lines is also a very laborious procedure, and in addition, often combined with very low levels of protein expression.
Hitherto, most attempts to develop viral DNA expression systems have been based on viruses having DNA genomes or retroviruses, the replicative intermediate of the latter being double stranded DNA.
Recently, however, also viruses comprising RNA genomes have been used to develop DNA expression systems.
In EP 0 194 809 RNA transformation vectors derived from (+) strand RNA viruses are disclosed which comprise capped viral RNA that has been modified by insertion of exogenous RNA into a region non-essential for replication of said virus RNA genome. These vectors are used for expression of the function of said exogenous RNA in cells transformed therewith. The RNA can be used in solution or packaged into capsids. Furthermore, this RNA can be used to generate new cells having new functions, i.e. protein expression. The invention of said reference is generally claimed as regards host cells, (+) strand RNA viruses and the like. Nevertheless, it is obvious from the experimental support provided therein that only plant cells have been transformed and in addition only Bromo Mosaic virus, a plant virus, has been used as transformation vector.
Although it is stated in said reference that it is readily apparent to those skilled in the art to convert any RNA virus-cell system to a useful expression system for exogenous DNA using principals described in the reference, this has not been proven to be true in at least the case of animal cell RNA viruses. The reasons for this seem to be several. These include:
1) Inefficiencies in transfecting animal cells with in vitro transcribed RNA;
2) Inefficiency of apparently replication competent RNA transcripts to start RNA replication after commonly used transfection procedures;
3) The inability to produce high titre stocks of recombinant virus that does not contain any helper virus;
4) The inability to establish stable traits of transformed cells expressing the function of the exogenous RNA.
In Proc. Natl. Acad. Sci. USA, Vol 84, 1987, pp 4811-4815 a gene expression system based on a member of the Alphavirus genus, viz. Sindbis virus, is disclosed which is used to express the bacterial CAT (chloramphenicol acetyltransferase) gene in avian cells, such as chicken embryo fibroblasts.
Xiong et al., Science, Vol 243, 1989, 1188-119; also disclose a gene expression system based on Sindbis virus. This system is said to be efficient in a broad range of animal cells. Expression of the bacterial CAT gene in insect, avian and mammalian cells inclusive of human cells is disclosed therein.
Even though it is known from prior art that one member of the Alphavirus genus, the Sindbis virus, can tolerate insertion and direct the expression of at least one foreign gene, the bacterial chloramfenicol acetyl transferase (CAT) gene, it is evident from the results described that both systems described above are both ineffective in terms of exogenous gene expression and also very cumbersome to use. Hence, neither system has found any usage in the field of DNA expression in animal cells today.
In the first example a cDNA copy of a defective interfering (DI) virus variant of Sindbis virus was used to carry the CAT gene. RNA was transcribed in vitro and used to transfect avian cells and some CAT protein production could be demonstrated after infecting cells with wild-type Sindbis virus. The latter virus provided the viral replicase for expression of the CAT construct. The inefficiency of this system depends on 1) low level of initial DI-CAT RNA transfection (0.05-0.5% of cells) and 2) inefficient usage of the DI-CAT RNA for protein translation because of unnatural and suboptimal protein intitation translation signals. This same system also results in packaging of some of the recombinant DI-CAT genomes into virus particles. However, this occurs simultaneously with a very large excess of wild-type Sindbis virus production. Therefore, the usage of this mixed virus stock for CAT expression will be much hampered by the fact that most of the replication and translation activity of the cells infected with such a stock will deal with the wild-type and not with recombinant gene expression.
Much of the same problems are inherent to the other Sindbis expression system described. In this an RNA replication competent Sindbis DNA vector is used to carry the CAT gene. RNA produced in vitro is shown to replicate in animal cells and CAT activity is found. However, as only a very low number of cells are transfected the overall CAT production remains low. Another possible explanation for this is that the Sindbis construct used is not optimal for replication. Wild-type Sindbis virus can be used to rescue the recombinant genome into particles together with an excess of wild-type genomes and this mixed stock can then be used to express a CAT protein via infection. However, this stock has the same problems as described above for the recombinant DI system. The latter paper shows also that if virus is amplified by several passages increased titres of the recombinant virus particles can be obtained. However, one should remember that the titre of the wild-type virus will increase correspondingly and the original problem of mostly wild-type virus production remains. There are also several potential problems when using several passages to produce a mixed virus stock. As there is no selected pressure for preservation o

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