Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-04-17
2003-05-20
Shukla, Ram R. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S455000, C424S199100
Reexamination Certificate
active
06566093
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is related to polynucleotide molecules and to their use for production of desired products after introduction into human or animal cells. In addition, the present invention is concerned with pharmaceutical compositions comprising said polynucleotide molecules and their use in prophylactic or therapeutic treatment methods. The present invention is also related to use of such polynucleotide molecules in animals to achieve expression of desired products, which can be recovered from the animal but do not give rise to any beneficial, e.g. therapeutical, activity in the said animal.
1. Field of the Invention
More specifically, the present invention is directed to alphavirus cDNA vectors comprised of recombinant cDNA consisting of cDNA derived from an alphavirus and heterologous, i.e. foreign, cDNA coding for a desired substance.
2. Description of Related Art
Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins.
The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV), the Ross River virus and Venezuelan, Western and Eastern equine encephalitis viruses, which are all closely related. In particular, the Sindbis and the Semliki Forest viruses have been widely studied and the life cycle, mode of replication, etc, of these viruses are well known and thus, need not to be specifically discussed herein.
Alphaviruses replicate very efficiently in animal cells which makes them valuable as vectors for production of protein and nucleic acids in such cells.
Expression systems based on the Sindbis virus are disclosed in U.S. Pat. Nos. 5,091,309 an 5,217,879. The Sindbis virus vectors of U.S. Pat. No. 5,091,309 comprise RNA derived from Sindbis defective interfering (DI) RNA having heterologous RNA inserted therein.
In U.S. Pat. No. 5,217,879 self-replicating and self-packaging recombinant Sindbis virus RNA molecules are disclosed comprising a heterologous coding sequence and at least one Sindbis virus junction region able to direct Sindbis virus subgenomic messenger RNA synthesis in a host cell. RNA transcripts are synthesized in vitro by transcription of Sindbis virus cDNA which has been inserted in a plasmid under control of a promoter, such as SP6. The SP6 promoter and other promoters disclosed in connection with cDNA transcription are not functional in animal or human cells.
In WO 92/10578 (Garoff and Liljeström) an expression system based on alphaviruses is disclosed. An illustrative example of such viruses is the Semliki Forest virus (SFV). Earlier it was reported that a full-sized cDNA copy of the SFV RNA genome was contructed (Journal of Virology, Volume 65, pages 4107-4113, 1991). This was engineered into an SP6 transcription vector from which full-sized SFV genomic RNA molecules can be transcribed in vitro. The RNA can be transfected into animal cells, in which cells the RNA molecules will support normal wild-type virus infection, since the RNA molecules are of positive polarity and can function as messenger RNA molecules in the cells. Upon transfection, the first portion of the genome is translated into a polyprotein which self-cleaves into four non-structural proteins (nsP1-nsP4). These proteins constitute the alphavirus replicase and are responsible for the production of new full-length genomic RNA molecules as well as of a subgenomic RNA species starting from an internal promoter (26S promoter). They are also responsible for the capping of the 5′ end of the new RNA molecules. The pSFV4 cDNA plasmid was further engineered into a general DNA expression plasmid by deleting portions of the coding region for the structural proteins and replacing such deleted portions with a linker region for insertion of foreign coding sequences (Bio/Technology, Volume 9, pages 1356-1361, 1991; Bio/Technology, Volume 11, pages 916-920, 1993). When foreign DNA coding sequences are inserted into these vectors, high amounts of foreign protein are obtained when virus structural proteins are translated from the RNA subgenome made by the alphavirus replicase.
According to WO 92/10578, an RNA molecule is provided, which is derived from an alphavirus RNA genome and is capable of efficient infection of animal cells, which RNA molecule comprises the complete alphavirus genome regions, which are essential for replication of the said alpha-virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof. According to WO 92/10578 such RNA molecules can be transferred into animal cells by any means of transfection or by packaging of said RNA molecules into infectious alphavirus particles for later infection of animal cells. In both cases the transfected or infected RNA molecule will be able to replicate within the target animal cell and to express the exogenus RNA sequences inserted into said RNA molecule. Such molecules and strategies for their expression within the cell can be used as vaccines or strategies to vaccinate in order to prevent or treat infection or cancer.
Since it is difficult to engineer RNA molecules by current genetic engineering technology, manipulations of the Alphavirus genome, such as insertion of heterologous coding sequences, have been conducted on the corresponding cDNA molecule. Subsequently, the engineered cDNA molecule has been transcribed in vitro and the RNA transcripts obtained have been used to transform cells. These constructs comprising the engineered cDNA molecule cannot be transcribed in animal or human cells since the promoters used for transcriptional control is not functional in such cells.
Obviously, it would be to advantage if the cDNA molecule could be used per se to transform cells and achieve expression of a desired substance in these cells.
WO 90/11092 describes the use of naked polynucleotides as a pharmaceutical which operatively codes for a biologically active peptide. Such molecules are proposed to be injected into tissue for the in vivo expression of said peptide. Specifically, it is claimed that the polynucleotide is DNA and that the peptide may function as an antigen, and may thus be used as a vaccine (see also Science, Volume 259, pages 1745-1749; DNA and Cell Biology, Volume 12, number 9, entire volume, 1993). However, recombinant viral cDNA constructs comprising heterologous coding sequences which can be expressed in animal and human cells are not disclosed, therein, nor is a cDNA construct disclosed, which is transcribed into self-replicating RNA encoding the replicase necessary for its replication. Even though, use of DNA coding for a polypeptide and for a polymerase for transcribing the DNA is disclosed in WO 90/11092, the initial quantity of polymerase is provided by including mRNA coding therefore in the preparation, which mRNA is translated by the cell.
SUMMARY OF THE INVENTION
Thus, it is an object of the present invention to provide a recombinant cDNA molecule complementary to an alphavirus RNA and comprising an exogenous cDNA sequence, which molecule can be introduced into animal or human cells to achieve transcription or expression of said cDNA, desired products such as polynucleotides or proteins being produced in cells harbouring the cDNA-molecule.
In accordance with the present invention, this object is achieved by placing the complete cDNA molecule under transcriptional control of a promoter sequence functional in an animal or human cell. Said promoter sequence will initiate transcription by the DNA-dependent RNA polymerase encoded by the host cell, i.e. the animal or human cell harbouring the said cDNA molecule.
Accordingly, the present invention is concerned with a cDNA molecule complementary to at least part of an alphavirus RNA genome, which CDNA molecule comprises the complement of the complete alphavirus RNA genome regions, which are essent
Garoff Henrik
Liljeström Peter
Bioption AB
Birch & Stewart Kolasch & Birch, LLP
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