CA2375423A1 - Promoters from atlantic salmon - Google Patents

Abstract

The invention relates to nucleotide sequences comprising one or more of the sequences of SEQ ID. No. 1-6 or sequences with at least 80 % homology to these, or parts thereof, to be incorporated in nucleotide constructs for the purpose of regulating the expression of such constructs.

Description

PROMOTERS FROM ATLANTIC SALMON
Field of the invention 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 salaf°). 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.
Background of the invention Transfer of gene constructs either through somatic or germ line routes has a wide array of applications with increasing importance not the least in the post genomic area within e.g. molecular research/experimental models, human and veterinary medicine, agriculture, biotechnology processing and production.
These methods share the characteristic that certain genes, or parts thereof, either species specific or species non specific are artificially assembled in a gene construct and artificially transferred to a host organism, where a more or less well addressed expression takes place in the entire organism or in specific cells thereof.
In the case of DNA vaccination, 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. It is well established that a successfully virus vaccine is crucially depending on such an event. The classical virus vaccine overcame this challenge by employing attenuated live virus whereas DNA vaccines mimic the natural virus infection and hence its protection, but avoids the potential pathogenic hazard of natural vaccines.
The results of various methods using such constructed gene expressions are of course dependent on the design of the gene, and in many cases even more dependent on the choice of promoter. A commonly used promoter is the one derived from the human cytomegalovirus (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.
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, Ken 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. In many cases, 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 WO 00/77232 .~ PCT/NO00/00202 alternative and improved or optimalised promoters. In fish, a number of successful experiments on DNA-vaccination has been carned out, most of which has employed the CMV promoter (Andersen et al, 1996; Russell et al, 1998;
Heppell et al, 1998; Lorenzen et al, 1998).
Advantages of the invention 1. One advantage of using the described promoters in salmon is that 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 2. In other cases it may be preferable to use DNA that is non homologous to the host genome. Such cases are those where it is imperative that the risk of genomic insertion of the recombinant DNA due to homologous recombination between the plasmid construct and the chromosomal DNA of the host is eliminated (see guidelines from Federal Drug Administration, UST, docket no 96N-0400). Hence the availability of heterologous DNA, like for example salmon promoters for applications in other organisms than salmon would be useful.

3. 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. 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). In 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. These are 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. In addition to the MHC class II
promoter, the 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. The use of 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.
4. In DNA vaccination against viral infections it may be desired to express the gene of interest constitutively in all cells close to the injection site.
Hence the use of the constitutive promoters of the Ran and U2 snRNP specific A' protein (U2A') 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 (Liihrmann 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.
Closer description of the invention WO 00/77232 ~ PCT/NO00/00202 This invention describes 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. In particular the promoters of the salmon genes MHC class 11 , M HC class II , Ra nand U2A' are described. In addition 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. Especially with emphasis on DNA vaccines against different pathogenic fish virus i.e. like IPN (infectious pancreas necrosis) and ISA
(infectious salmon anaemia) the promoters are expected to have a clear-cut advantage.
The promoter SEQ ID2 may be used to produce specific cytokines as for example interferon y 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. Hence, 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).

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 (3-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 given proteins within these organisms or cell/cell lines of such.
Example 1 Isolation of MHC classII ~3 and a promoters and intron 1 The Salmon MHC ClassII (3 and a promoters and intron 1 were isolated by the use of PromoterFinder kit as follows: 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 (3 is derived from cDNA sequences described by Hordvik et al, 1993, and primers for class II a is derived from cDNA sequences as described by Grimholt et al, 2000). Primers for MHC class II a first reaction (oML 71):

5'-AAGTCTGCGTACCACATCTC-3' and nested reaction (oMLlS):
5'-CATGTCCAGTCCATCTGAATC-3' and the primers for MHC class II (3 first reaction (oML70): 5'- TTCCAGGCTTCTGCATTC-3' and nested reaction (oML69):
5'- CTTTGAGGAGTATCGGCAC-3' High molecular DNA from agarose plugs was used for the construction of PromoterFinder libraries. One plug was divided into 5 pieces, each piece containing 2.~ ~g DNA. The DNA was digested in 100 ~l reaction volumes with 80 U of five 6 cutter restriction enzymes, overnight at 37°C. The restriction fragments were isolated from the agarose plugs by gene clean. 10 q1 of the digested DNA was then ligated to the specially designed promoterFinder adapter in a total volume of 20 p1 (adapter primers were constructed, and annealed as described in Clonentech manual). The ligation reaction was terminated by incubation at 70°C for 5 minutes, then diluted 10 fold by adding 180 p1 of 10 mM
tris-HCL, pH 7.5, 1mM EDTA and stored at -20°C. PCR amplifications were performed with 1 ~l DNA in a total volume of 50 p1, 1.5 mM MgOAc2 and the commercially available enzyme mix rTth DNA polymerise, XL (Gene Amp XL
PCR Kit). To walk from the known MHC sequence, 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.
Example 2. Isolation of U2A' and RAN promoters 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.
Example 3. Preparation of the constructs 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) WO 00/77232 PC'T/NO00/00202 of the gene of interest. E.g.: by PCR amplification using primers containing suitable restriction sites (KpnI and XhoI) 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 .
The sequences can be used individually or in various combinations:
PROMOTER I LEADER 1NTRON I "GENE"
Promoter can be SEQ ID 1,2,3,4, or S
Intron 1 can be SEQ ID 6.
Leader can be as published by Hordvik et a1.1993.
"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.
Example 4 DNA "vaccination" of Atlantic salmon against the reporter protein f3-~alactosidase The Salmon MHC class II ~3-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).
The immune response towards (3-galactosidase was measured by Enzyme linked immunosorbent analysis (Elisa), where the plates were coated with ~3-galactosidase protein (commercially available) and serum from the fish was added. The amount of salmon antibodies bound to the plates were detected colorimetrically 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. Four fishes out of eight, which were injected with the MHC
class II promoter containing plasmid, showed an Elisa titer higher than the mean value + 2 times the standard deviation of the eight control fishes used after a period of 4 weeks. In contrast 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. Another experiment also using LacZ as reporter gene showed that the immune response due to DNA vaccination is achieved already after 2 weeks. whereas protein-vaccination against the same antigen including Freunds adjuvans induces the immune response after 6 weeks.
In these experiments no immune response could be detected using DNA-plasmid with LacZ gene but without promoters. Similarly to the shown MHC class II (3-promoter function in DNA vaccination towards (3-Galactosidase, the promoters of MHC class II a, Ran and U2A' were also shown to function.
Example S Optimising DNA vaccination of salmon with emphasis on different immune parameters In order to optimise the DNA vaccination of salmon further experiments are currently ongoing. All constructs will be tested in a larger number of fish to make a more substantial body of data. In addition potential booster effects, duration of the immune response, the amount of DNA injected, injecting methods and the influence of the water temperature will be further tested.
Example 6. DNA vaccination of Atlantic salmon against pathogens.
It is very likely that 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.
It must be further tested in what combination the promoters, leader sequences and introns have their best effect in vaccination against different microorganisms. For instance the MHC class II ~3 promoter (SEQ ID2)+ e.g.
MHC class II (3 leader sequence (Hordvik et al. 1993) + MHC class II (3 intron I
sequence (SEQ ID 6) can be used in a construct in order to drive expression of immunogenic epitopes of ISA, where the nucleotide sequence for the leader peptide + the intron 1 sequence is ligated to the antigen encoding gene in a way that the leader and the antigen-peptide will be in frame and a fusion protein can be made. 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.
Example 7. Morphological studies The use of 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.
Example 8. Expression in other organisms than Salmo salar Experiments will be carried out to test the expression promoting effect of the present salmon promoters in other species. Cell lines were obtained from ATCC. Mainly antigen presenting cell lines, like dendritic cells, macrophages and B-cells from different mammals including human will be transfected using different promoters. As parallel positive control the CMV promoter will be used.
LacZ or GFP will be used as reporter genes. The first results are expected by the end of 2000.

List of claimed sequences Sequence identities are as follows:
SEQ ID 1 (Salmo salar MHC class II a promoter):
SEQ Length: 245 SEQ ID 2 (Salmo salar MHC class II (3 promoter):
SEQ Length: 952 WO 00/77232 PC'f/NO00/00202 SEQ ID 3 (Salmo salar MHC class II (3 promoter):
SEQ Length: 952 WO 00/77232 PC'T/NO00/00202 SEQ ID 4 (Salmo salar RAN promoter):
SEQ Length: 1481 WO 00/77232 PC'T/NO00/00202 SEQ ID 5 (Salmo salar U2A~ promoter):
SEQ Length: 1178 SEQ ID 6 (Salmo salar MHC class II (3 intronl):
SEQ Length: 208 WO 00/77232 PC'T/NO00/00202 REFERENCES
Anderson E, Mourich DV, Gahrenkrug SC, LaPatra S, Shepherd J, Leong JC
(1996) Genetic immunization of rainbow trout (Onchoryncz~s mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol 5:114-122 Dasso, M., Pu. R.T. ( 1998) Nuclear structure '98, Nuclear transport: Run by Ran? Am J Hum Genet 63: 311-316.
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Class-II Transcription. Annu. Rev. Immunol. 10: 13-49.
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Landolfo S. (1993). Interferons inhibit onset of murine cytomegalovirus immediate-early gene transcription. Virology, 197, 303-311.

Heppel J, Lorenzen N. Armstrong NK, Wu T, Lorenzen E. Einer-Jensen K, Schorr J, Davis HL (1998) Development of DNA vaccines for fish: vector design intramuscular injection and antigen expression using viral haemorrhagic septicaemia virus genes as model. Fish & Shellfish Immunol 8: 271-286 Husebye H, Colls P, Alestrom P. (1997) A functional study of the salmon GnRH promoter. Mol Mar Biol Biotechnol. 6(4): 357-363 Kerr IM, Sterk GR. (1992). The antiviral effects of the interferons and their inhibition. Journal of Interferon Research, 12, 237-240.
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Claims (14)

claims

1. Nucleotide sequences which are characterised by comprising one or more of the sequences of SEQ ID. No. 1-6 or sequences with at least 80% homology to these, or parts thereof, to be incorporated in nucleotide constructs for the purpose of regulating the expression of such constructs.

2. Use of the nucleotide sequences of claim 1 in vivo.

3. Use of the nucleotide sequences of in claim 1 in vitro.

4. Use of the nucleotide sequences of claim 2, where the in vivo system is production organisms.

5. Use of the nucleotide sequences of claim 2, where the in vivo system is experimental organisms.

6. Use of the nucleotide sequences of claim 2, where the in vivo system is Homo sapiens.

7. Use of the nucleotide sequences of claim 4, where production organisms are aquatic and marine species.

8. Use according to claim 4 in productivity enhancement.

9. Use according to claim 8 in productivity enhancement achieved through vaccination.

10. Use according to claim 8 in productivity enhancement achieved through somatic routes.

11. Use according to claim 8 in productivity enhancement achieved through germ plasm enhancement e.g. transfer of the construct through germ line routes.

12. Use according to claims 7-11, restricted to bony fish.

13. Use according to claim 12, wherein the bony fish is Salmo spp

14. Use according to claim 12, wherein the bony fish is Oreochromis spp.