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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
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  • C12N2830/00—Vector systems having a special element relevant for transcription
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
• • C—CHEMISTRY; METALLURGY
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates

Definitions

• the present invention concerns nucleotide sequences used in expression vectors to ensure and optimally address expression of gene constructs.
• the invention describes different sequences isolated from Atlantic salmon (Salmo salar). They can be used independently or in different combinations in DNA vectors in order to elicit efficient and targeted expression, in particular to protect against infections and to enhance the performance of farmed organisms through the delivery of constructs such as DNA vaccines.
• the invention can be employed in salmon as well as in other organisms.
• naked DNA encoding the antigen is injected into the organism, and the antigen peptide is produced within the targeted cell population of the host with a subsequent development of immune response (Gregoriadis 1998; Tighe et al, 1998; Gerloni et al, 1998; Mor, 1998).
• This is in contrast to common recombinant subunit vaccination, where the antigenic peptide is developed (synthesized) outside the host and subsequently injected for the purpose of developing an immune response towards that specific antigen.
• One clear-cut restriction of the subunit strategy is the difficulty in, or complete failure of, eliciting cell mediated kill of target cells e.g. virus transformed host cell.
• a commonly used promoter is the one derived from the human cytomegalo virus (CMV). This is a very strong promoter, which addresses most vertebrate cells. Especially in DNA vaccination, this is the most commonly used promoter today. There are however certain drawbacks with this promoter. It is derived from a human pathogen virus, and this fact may be a disadvantage both ethically and politically as well as regarding safety. It cannot be ruled out that the CMV promoter carries a potential risk of promoting neoplastic developments in the host, simply because of its nature and origin.
• CMV promoter It may also be of medical and technical disadvantage as its activity has been shown to be down regulated by cytokine interferon gamma (Gribaudo G. et al, 1993, Kerr IM and GR. Stark 1992)
• Another disadvantage of the CMV promoter is that it is active in all kinds of cells, making it difficult to obtain the desired proper and targeted transcription activity.
• cell or tissue specific expression may be desired, and it may be advantageous to mimic the host driven expression pattern of the gene of interest as carefully as possible, with for example fine tuning of regulation such that expression occurs at the right instant, with the correct strength and in the proper target cells, etc. This is very difficult to achieve with the currently used promoters like the CMV, which underlines the need for alternative and improved or optimalised promoters.
• species-specific promoters will be used. In cases where the expression is desired to be as similar as possible to the "natural" expression, species-specific promoters are preferable. Moreover the species specific promoters are expected to be superior over species-non specific ones (xenopromoters) in terms of functions since all transcription factors required for its "normal" regulation will be present. Moreover a political/ethical issue is that species-specific sequences are by far to be preferred, since the prevailing public opinion is that transfer of gene across species is more controversial than transfer within species
• Another advantage of the present promoters is their use as cell specific promoters. In such cases the expression of the gene of interest will be restricted to cells in which the promoter is active. For example expression will occur in MHC (major histocompatibility complex) class II expressing cells only, when using MHC class II promoters.
• MHC major histocompatibility complex
• Cells constitutively expressing MHC class II are mainly antigen presenting cells of the immune system (Glimcher et al, 1992; Mach et al, 1996; Rammensee 1996).
• DNA vaccination it may of particular advantage to use MHC class II promoters since the antigens thus will be expressed in the antigen presenting cells.
• MHC class II the cells which normally ingest antigens by endocytosis, degrading the proteins and presenting the antigen peptides in the context of MHC class II to CD4+ T-cells.
• MHC class II leader sequence will help the expressed peptide to be directed to the same vesicles as the MHC class II molecules, and the peptides will be degraded and subsequently presented by MHC class II very efficiently.
• MHC class II promoters in DNA vaccination implies that the expression will be cell specific and occur only in those cells that "do the job”. No useless and non-desired expression of the antigen will take place. This will also decrease the risk for negative side effects caused by unwanted expression of the antigen.
• the constitutive promoters of the Ran and 102 snRNP specific A 'protein (U2A 1 ) genes will be advantageous.
• the function of the Ran protein is to transport proteins and mRNA between the cytoplasm and nucleus through the nuclear pores (Rush et al. 1996; Dasso and Pu, 1998) and this protein appears to be essential and highly abundant in all cells.
• the U2A' protein is involved in the splicing machinery and is also needed in all cells constantly (Luhrmann 1988; Sillekens et al. 1989).
• the constitutive promoters will also be of importance for other applications like in gene therapy or other methods, where performance enhancement is of interest, e.g. to affect growth rate and desired colouring of aqua cultured organisms.
• Organisms and cells treated this way may not only be used for production purposes, but may as well represent attractive and valuable model systems for experimental/research use. Finally, this may lead to the use of the promoters in human medicine.
• nucleotide sequences i.e. promoters, leaders and an intron sequence, isolated from Salmo salar to be used in expression vectors for a variety of purposes. Sequences from other organisms homologous or corresponding to the salmon sequences described here, can also be used according to the present claims.
• the method implies the use of salmon DNA elements in gene constructs for use both in salmon and other organisms.
• the promoters of the salmon genes MHC class II , M HC class II , Ra ⁇ and 102 A ' are described.
• the leader sequence and intron 1 of MHC class II ca n be used. All the described nucleotide sequences can be incorporated in various constructs for the purpose of performance enhancements such as DNA vaccination of salmon and other fish species against various pathogens like virus, bacteria, parasites and fungi.
• IPN infectious pancreas necrosis
• ISA infectious salmon anaemia
• the promoter SEQ ID2 may be used to produce specific cytokines as for example interferon ⁇ to modulate immune responses subsequent to injection in somatic tissues.
• the promoters may also be combined with MHC alleles which may be of special interest for optimising the expression and presentation of given pathogen antigens.
• injection of a vector containing both the promoter and the desired MHC allele in the salmon may increase the immune response against the antigen of interest.
• the promoters may be used in morphological studies where transfected cells capable of expressing MHC molecules express reporter molecules. These reporter molecules may be any detectable proteins, for example green fluorescent protein (GFP).
• GFP green fluorescent protein
• the promoters may also be used to influence gene expression of given proteins in favourable ways. This can affect certain properties, for example uptake and storage of ⁇ -carotenes, compounds responsible for meat colouring in salmonids.
• the promoters may be used in salmon and other organisms and cell lines thereof where the different effects and applications as mentioned above can be achieved. This will comprise vaccination of a series of organisms and to modify or introduce expression of ven proteins within these organisms or cell/cell lines of such.
• Example 1 Isolation of MHC classll ⁇ and ⁇ promoters and intron 1
• PromoterFinder libraries were constructed for PCR based walking in uncloned genomic DNA, as described by Siebert et al. (1995), with few modifications with an origin in published MHC- sequences.
• Primers for class II ⁇ is derived from cDNA sequences described by Hordvik et al, 1993, and primers for class II ⁇ is derived from cDNA sequences as described by Grimholt et al, 2000).
• the ligation reaction was terminated by incubation at 70°C for 5 minutes, then diluted 10 fold by adding 180 ⁇ l of 10 mM tris-HCL, pH 7.5, lmM EDTA and stored at -20°C.
• PCR amplifications were performed with 1 ⁇ l DNA in a total volume of 50 ⁇ l, 1.5 mM MgOAc2 and the commercially available enzyme mix rTth DNA polymerase, XL (Gene Amp XL PCR Kit).
• the promoterFinder libraries were used as templates in nested PCR reactions, sense primers were adapter specific primers annealing to the adapter and antisense primers were MHC specific primers.
• the PCR products were cloned into the TA PCR cloning kit (Invitrogen) and the inserts sequenced with standard sequencing primers. See sequence list for details.
• the promoters of Atlantic salmon U2A' and Ran were isolated from two differnt cosmid clones as follows: An Atlantic salmon cosmid library was constructed as described by Lundin et al (1998). The cosmid clones cSSML032 and cSSML033 were randomly selected (Lundin et al. 1999) and sequenced directly by use of walking primers and by subcloning into the pBluescriptKS + vector (Stratagene, La Jolla, CA, USA). See sequence list for details.
• promoter sequences SEQID 1-6 The sequences are listed in the sequence list and numbered 1-6. The corresponding fragments were by standard molecular biological methods assembled in constructs to promote expression (promoter sequences SEQID 1-6) of the gene of interest. E.g.: by PCR amplification using primers containing suitable restriction sites (Kpnl and Xhol) the promoter sequences were properly inserted at the 5'end of the LacZ in the reporter gene plasmid which also included the SV40 16S /19S donor and acceptor splice signals as well as a SV40 polyadenylation signal as described in detail by Husebye, et al., 1997). This directed the polypeptide product to the ER-Golgi secretory vesicles for the proper degradation of the peptide product and presentation of MHC class II .
• Promoter can be SEQ ID 1,2,3,4, or 5
• Intron 1 can be SEQ ID 6.
• GENE in the scheme means the gene or part of gene, which is selected to be used in the construct in each case, and is not described in this patent application.
• the Salmon MHC class II ⁇ -promoter SEQ ID 2 was inserted in a plasmid vector carrying the LacZ gene right upstream of the gene in order to promote transcription of the Lac Z gene.
• the resulting plasmid was prepared by use of Quiagen Mega prep, dissolved in sterile phosphate buffered saline (PBS) and injected intraperitoneally of Atlantic salmon pre-smolts (approximately 30 g).
• PBS sterile phosphate buffered saline
• the immune response towards ⁇ -galactosidase was measured by Enzyme linked immunosorbent analysis (Elisa). where the plates were coated with ⁇ - galactosidase protein (commercially available) and serum from the fish was added.
• the amount of salmon antibodies bound to the plates were detected colorimetricaliy by horse radish peroxidase linked rabbit anti mouse antibodies and a mouse anti-trout IgG secondary antibody, which is commonly used in Elisa of Atlantic salmon.
• only 2 individual of eight injected with the same plasmid containing the CMV promoter showed an immune response comparable to those given MHC class II promoter.
• the described salmon promoters can be us in DNA- constructs for protective DNA vaccination of Atlantic salmon and other fish species by the insertion of a nucleotide sequence encoding antigenic peptides derived from pathogens like bacteria, virus or even parasites and fungi in a way that its transcription is driven by the mentioned promoters.
• the plasmid vector containing the promoter and the antigen encoding gene can be injected in fish as described for DNA vaccination and a protective immune memory will be achieved.
• MHC class II ⁇ promoter SEQ ID2
• MHC class II ⁇ leader sequence Hordvik et al. 1993
• MHC class II ⁇ intron 1 sequence SEQ ID 6
• SEQ ID 6 MHC class II ⁇ intron 1 sequence
• the leader peptide will direct the antigen to secretory vesicles where the antigen will be degraded to short peptides and the peptides presented primarily on the MHC class II molecules. In this way antigens like viruses that normally are presented by MHC class I will be presented by MHC class II instead.
• MHC promoters from salmon or other species in reporter gene constructs where the cell specific expression of the reporter gene construct will be used as cell markers, for example in morphological studies in staining of tissue sections immunohistochemically or staining for reporter gene activity or identification of reporter-gene activity directly by use of for example Green fluorescent protein.
• Gerloni M (1998) Durable immunity and immunologic memory to a parasite antigen induced by somatic transgene immunization. Vaccine 16:293-297

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