WO1990014428A1 - Vecteurs ameliores d'expression du baculovirus - Google Patents

Vecteurs ameliores d'expression du baculovirus Download PDF

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Publication number
WO1990014428A1
WO1990014428A1 PCT/US1990/002814 US9002814W WO9014428A1 WO 1990014428 A1 WO1990014428 A1 WO 1990014428A1 US 9002814 W US9002814 W US 9002814W WO 9014428 A1 WO9014428 A1 WO 9014428A1
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promoter
vector
baculovirus
gene
modified
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PCT/US1990/002814
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Lois K. Miller
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University Of Georgia Research Foundation, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C12N15/68—Stabilisation of the vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to methods and compositions for improving the expression of a gene. More particularly, the present invention relates to classes of novel promoters that improve the expression of a heterologous gene in a baculovirus system.
  • the term "expression" may be characterized in the following manner.
  • a cell is capable of synthesizing many proteins. At any given time, many proteins which the cell is capable of synthesizing are not being synthesized.
  • that gene is said to be expressed.
  • the DNA sequence coding for that particular polypeptide must be properly located with respect to the control region of the gene. The function of the control region is to permit the expression of the gene under its control.
  • vector refers to an extra-chromosomal molecule of duplex DNA comprising an intact replicon that can be replicated in a cell.
  • vectors are derived from viruses or plasmids of bacteria and yeasts.
  • a baculovirus vector comprises a baculovirus replicon.
  • gene refers to those DNA sequences which transmit the information for and direct the synthesis of a single protein chain.
  • infection refers to the invasion by agents
  • transfection refers to a technique for infecting cells with purified nucleic acids of viruses.
  • heterologous gene in reference to the baculovirus vectors hereof, refers to DNA that encodes polypeptides ordinarily not produced by the virus from which the vector is derived, but which is introduced into the cell as recombinant DNA or within viruses carrying recombinant DNA genomes.
  • passenger gene or “passenger DNA” as used herein are equivalent to the term “heterologous gene.”
  • Exogenous as used herein, has the same meaning as heterologous.
  • transplacement plasmid means a bacterial vector which is used as an intermediate in the construction of a virus vector.
  • a transplacement plasmid facilitates the transfer of exogenous genetic information, such as the combination of a novel promoter and a heterologous structural gene under the regulatory control of that promoter, to a specific site within the viral genome by homologous recombination. That homologous recombination occurs via the DNA sequences flanking the chimeric gene.
  • the technology of transferring genes from one species and expressing them in another is made possible because the DNA of all living organisms is chemically similar in that it is composed of long chains containing the same four nucleotides. Nucleotide sequences are arranged in codons (triplets) which code for specific amino acids with the coding relationship between the amino acid and nucleotide sequence being essentially the same for all species of organisms.
  • the DNA is organized into genes which are comprised of control regions which mediate initiation of expression of the gene and coding regions. These control regions are commonly referred to as
  • promoter An enzyme, called RNA polymerase, binds to the promoter region and is either activated or in some way is signalled so that it travels along the coding region and transcribes the encoded information from the DNA into messenger ribonucleic acid (mRNA).
  • mRNA messenger ribonucleic acid
  • the mRNA contains recognition signals: signals for ribosome binding, signals for translational start and stop, and for polyadenylation.
  • Cellular ribosomes then translate the nucleotide codon information of the mRNA into protein with an amino acid sequence specified by the nucleotide codon sequence.
  • the general use of restriction endonucleases and the ability to manipulate DNA sequences has been greatly improved by the availability of chemically synthesized double stranded oligonucleotides containing desired nucleotide sequences including useful restriction site sequences.
  • Virtually, any naturally occurring, cloned, genetically altered or chemically synthesized segment of DNA can be coupled to any other segment by attaching an oligonucleotide containing the appropriate sequences or recognition sites to the ends of the DNA molecule.
  • any DNA sequence can be inserted into a vector molecule to construct an artificial recombinant molecule or composite, sometimes called a chimera or hybrid DNA.
  • the vector utilized is a duplex extra- chromosomal DNA molecule comprising an intact replicon such that the recombinant DNA molecule can be replicated when placed into bacteria or yeast by transformation.
  • Vectors commonly in use are derived from viruses or plasmids associated with bacteria and yeast. Because of the nature of the genetic code, the inserted gene or portions thereof will direct the production of the amino acid sequence for which it codes if the gene or gene portion is attached to a control region (promoter) which is capable of regulating expression in the cell in which the vector replicates.
  • promoter a control region
  • the general techniques for constructing expression vectors with cloned genes located in the proper relationship to promoter regions are described in the literature (e.g., See T. Maniatis, et al. (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory).
  • a number of vector systems utilizing the above-described general scheme and techniques have been developed for use in the commercial or experimental synthesis of proteins by genetically modified organisms.
  • vector systems utilize prokaryotic bacterial hosts for vector replication and heterologous gene expression. Additionally, systems have been utilized which employ eukaryotic cells for vector replication and heterologous gene expression. Such systems are employed for hepatitis B virus surface antigen synthesis and for human tissue plasminogen activator synthesis.
  • Eukaryotic hosts are preferred for the production of some eukaryotic proteins which require modification after synthesis (i.e., glycosylation) to become biologically active. Prokaryotic cells are generally incapable of such modifications.
  • virus vectors in eukaryotic hosts has been the subject of a considerable amount of recent investigation.
  • Viral vector systems may suffer from significant disadvantages and limitations which diminish their utility.
  • some viral vectors are not able to achieve high enough levels of gene expression for economic protein production in costly eukaryotic cell culture systems.
  • Some eukaryotic viral vectors are either pathogenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with accidental infection.
  • Some virus vectors have severe limitations on the size of the heterologous gene that can be stably inserted into the virus particles.
  • the genetic engineering technology becomes more sophisticated, there will be an increased interest in inserting more than one heterologous gene, i.e., genes coding for more than one protein, into a host cell to achieve coordinated expression and possibly to obtain coordinated activity of the various gene products.
  • An ideal viral vector should be capable of stably carrying a large segment of heterologous DNA, efficiently infecting cells and converting virtually all the protein biosynthesis of the infected cell to the high level expression of the foreign gene.
  • a virus that appears to be well suited as a vector for the propagation and high level expression of many heterologous genes in a higher eukaryotic environment is the baculovirus
  • AcMNPV Autographa californica nuclear polyhedrosis virus
  • the baculovirus group includes the subgroups of nuclear polyhedrosis viruses (NPV) and granulosis viruses (GV); baculoviruses infect only arthropod hosts.
  • the virus particles of NPV and GV are occluded in proteinaceous crystals.
  • the virions envelopeed nucleocapsids
  • This structure referred to as an inclusion or occlusion body, is the form found extraorganismally in nature and is responsible for spreading the infection between organisms.
  • the subgroup NPV produces many virions embedded in a single, large
  • the crystalline protein matrix in either form is primarily composed of a single 25 to 33 kDa polypeptide which is known as polyhedrin or granulin in NPV or GV, respectively. More general information on the subject of baculovirus structure and the process of infection is available in the following reviews: Carstens (1980) "Baculoviruses - Friend of Man, Foe of Insects?,” Trends and Biochemical Science. 52:107- 10; Harrap and Payne (1979) "The Structural Properties and Identification of Insect Viruses” in Advances in Virus Research. Vol. 25, M. A. Lawer et al. (eds.), Academic Press, New York, pp. 273-355; and Miller, L. K. (1981) supra.
  • Baculovirus helper-independent viral vectors are particularly useful for the high-level production of biologically active eukaryotic proteins. Expression levels for some foreign genes have been reported to be 10% to 25% of the total protein of the recombinant infected cell. Appropriate post-translational modifications, including signal peptide cleavage, glycosylation, phosphorylation, oligomerization, complex formation, isolation and proteolysis, have been reported for a variety of different heterologous proteins produced using this expression system.
  • the baculovirus expression vectors described to date use very late promoters, such as the polyhedrin or polypeptide 10
  • baculovirus vectors are needed which can express more than one heterologous gene.
  • the disadvantage to such vectors is that they are often genomically unstable if promoter sequences are duplicated within the vector. To minimize such instability, a different promoter must be employed for each heterologous gene to be expressed. These promoters must be different (nonhomologous) from naturally occurring viral promoters within the vector to more fully avoid genomic instability problems.
  • Vectors should also have the ability to include promoters that express genes at an earlier stage of protein production to improve protein quality and to ensure that the protein has the necessary post- translational modifications to confer biological activity or immunological authenticity. Such post-translational modifications appear to decline during the very late phase of infection when the very late promoters of AcMNPV vectors, such as polyhedrin and p10, are activated.
  • the new promoters should be flexible enough so that either a single heterologous gene or a series of heterologous genes can be inserted into the vector system so that various proteins can be produced at the same or at different times.
  • the polyhedrin gene should be present if the virus is to be administered orally to the appropriate insect host.
  • the present invention provides an improved means of employing baculovirus gene expression vectors for expression of heterologous genes by placing the genes under the control of novel or modified promoters; preferably a novel promoter which promotes high level expression of a gene and allows the proper post-translational modifications of the gene product in insect cells. More specifically, in a preferred embodiment, the present invention comprises providing a modified baculovirus promoter to which a heterologous gene is attached. The promoter is inserted into a virus via a transplacement plasmid or by direct insertion to produce a recombinant virus vector which can be used to infect suitable host cells. The infected host cells are then used to produce the heterologous gene product.
  • the present invention includes a novel promoter which contains a modification of a natural baculovirus promoter or a synthetic modified promoter functional in a baculovirus expression system.
  • the new promoter may be a combination promoter such as a promoter with a double start site (i.e., two ATAAG's) or a combination of two different promoters such as an early/very late promoter or a late/very late promoter. It is contemplated that the present invention also includes foreign gene placement in a novel location or genomic orientation.
  • a modified baculovirus promoter of the present invention has one or more features selected from the group consisting of an ATAAG sequence flanked by a very A+T rich region, an upstream activator sequence, similar to those of linker scan promoters LSXVI, LSXIV and LSXVII, of about 10bp of GC-rich sequence, placed from about 10 to about 30bp upstream of the transcription start site, a polyadenylation site (A 2 UA 3 site) in the orientation opposite to desired transcription to block antisense transcripts which may originate from fortuitous TAAG sequences in the foreign gene, and selected components of one or more of three very strong late promoters (vp39, p10, and p6.9), and/or of the very strongly expressed very late genes (polyhedrin and p10).
  • vp39, p10, and p6.9 very strong late promoters
  • polyhedrin and p10 very strongly expressed very late genes
  • An upstream activator sequence of a modified promoter is a sequence which acts to increase the level of transcription at a transcription start site located downstream therefrom.
  • the upstream activating sequence is a GC- rich sequence, with a nucleotide composition of at least about 60% G + C.
  • the modified promoter of the present invention shares only limited sequence homology or continuity with any other AcMNPV promoter.
  • a major advantage of the novel promoter is that it can be stably incorporated at any region in the AcMNPV genome without concern for recombination with other regions of the AcMNPV genome.
  • the modified promoter may also be designed to be stronger than the polyhedrin promoter.
  • the modified promoter of the present invention has been incorporated into several different transplacement plasmids by methods well known to those of ordinary skill in the art.
  • plasmid pSynVI contains the synthetic promoter in place of the EcoRV to Kpnl fragment within AcMNPV sequences between 3.1 to 6.16 map units.
  • the orientation of the promoter is such that the direction of expression will be opposite that of normal polyhedrin gene expression, thus taking advantage of the potential increase in gene expression when foreign genes are placed in this orientation.
  • Foreign genes can be placed within the multicloning site of this plasmid and recombinant viruses can be identified on the basis of their occlusion negative phenotype.
  • a second transplacement plasmid contains the synthetic promoter (including a multicloning site for passenger gene insertion) as well as an intact polyhedrin gene under the control of the wild-type polyhedrin promoter.
  • this transplacement plasmid allows construction (or formation) of a recombinant virus that directs the production of both polyhedrin and the foreign gene product.
  • the benefit is two fold: the recombinant virus has an easily visible and rapidly selectable (occlusion positive) phenotype, and the recombinant virus can infect insects orally. Thus be easily employed for mass production in insect larvae.
  • the transplacement plasmids can be further manipulated in any number of additional ways which may increase expression in different situations. For example, it may be useful to include a polyadenylation signal within the multicloning site and downstream from the foreign gene insert so that efficient polyadenylation occurs if an effective polyadenylation site is not included in the foreign gene insert. Portions of the multicloning site which are not used in transplacement plasmid construction may be deleted to improve expression since an A+T rich leader and an AACAAT sequence near the ATG seem to be preferred by highly expressed baculovirus genes (i.e., vp39, p10 and p6.9).
  • a modified promoter is provided that will allow multiple heterologous genes to be expressed with one vector.
  • An object of the present invention is to provide baculovirus vectors with modified promoters that will allow more than one heterologous genes to be expressed within one vector.
  • any baculovirus capable of replication in cultured host cells is useful for conversion into a vector of this invention.
  • the baculovirus used is a nuclear polyhedrous virus, and more preferably is Autographica californica.
  • the vectors of this invention may be prepared by genetic engineering technologies known to the art, preferably insertion of chimeric genes comprising the modified promoters of this invention in combination with heterologous genes placed under regulatory control of said promoters into the genome of a baculovirus by homologous recombination in an area of said genome able to tolerate said insertion without interference with the replication functions of said baculovirus, all as will be readily apparent to those skilled in the art.
  • the host cells useful for expression of the heterologous genes under control of the modified promoters of this invention are insect cells in which the baculovirus vectors of this invention are capable of replication and expression, all as known to the art, and include cultured insect cells in vitro and insect larvae.
  • a preferred upstream activating sequence of this invention is a G + C-rich sequence of about 10 bp, more preferably a Hindlll site linker as exemplified by the sequence CCAAGCTTGG.
  • Any transplacement plasmid known to the art which has sequences flanking the chimeric gene(s) of the present invention which are homologous to the baculovirus which is to serve as the expression vector can be used in the practice of the present invention.
  • transplacement plasmids exemplified by the present invention are pEV55 and derivatives thereof.
  • Fig. 1 is a schematic representation of the plasmid designated phcLSXIV.
  • Fig. 2 is a schematic representation of the plasmid designated pSynVI-.
  • Fig. 3 is a schematic representation of the plasmid designated pSynVI+wtp.
  • Fig. 4 is a schematic representation of the plasmid designated pSynwtVI-.
  • Fig. 5 is a schematic representation of the plasmid designated pSPLSXIWI+CAT.
  • Fig. 6 is a schematic representation of the plasmid designated pLSXIWI+CAT.
  • Fig. 7 is a schematic representation of the plasmid phcwt.
  • Fig. 8 is a schematic representation of the plasmid pEVmod.
  • Fig. 9 is a schematic representation of the plasmid pEVmodXIV.
  • Fig. 10 is a schematic representation of the plasmid pSynVI-CAT.
  • Fig. 11 is a schematic representation of the plasmid pSynVI+wtpCATl.
  • Fig. 12 is a schematic representation of the plasmid pSynwtVI-CATl.
  • Fig. 13 is a schematic representation of the plasmid PLSXIV3VI+.
  • Fig. 14 is a schematic representation of the plasmid PLSXIV2.
  • Fig. 15 is a schematic representation of the plasmid phc39.
  • Fig. 16 is a schematic representation of the plasmid pEWp39/LSXIV CAT.
  • Fig. 17 is the legend for interpreting the plasmid schematics.
  • the present invention provides a plasmid containing a novel promoter that is used to drive foreign gene expression and improve the quality of the proteins produced during infection.
  • the novel promoter may optionally contain a synthetic promoter, modifications of a natural promoter, and/or combinations of natural, modified or synthetic promoters.
  • the novel promoter may also comprise a combination promoter, including but not limited to, a promoter with a double start site (i.e., two ATAAG's) or a combination of two baculovirus different promoters such as an early/very late promoter or a late/very late promoter. It is contemplated that the present invention also includes promoter-foreign gene placement in a novel location or a genomic orientation.
  • the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) is now widely employed as a gene expression vector (reviewed by Luckow and Summers, Trends in the Development of Baculovirus Expression Vectors, Bio/Technology 6:47-55. 1988; Miller, L. K., Baculoviruses as Gene Expression Vectors, Ann. Review of Microbiology. 42:177- 199, 1988).
  • the helper-independent viral vector systems generally involve the insertion of the foreign gene to be expressed into the AcMNPV genome under the control of a very late viral promoter, either the polyhedrin promoter or the p10 promoter.
  • Transplacement plasmids which include the nucleotides immediately upstream of the +1 (A of the ATG) position of the polyhedrin gene are now recommended for vector use (Luckow and
  • the primary determinant of promoter activity resides within the sequence TAAGTATT at the transcriptional start site located at
  • vp6.9 and vp39 genes are required for both the budded and occluded forms of the virus. Thus, their expression would be expected to continue through very late times postinfection and provide a longer period for foreign gene expression than the polyhedrin or p10 promoters. Specific expression of foreign gene products at very late times has been considered to be preferable for expression of those foreign genes whose products might interfere with budded virus synthesis (Miller, L. K. (1981) supra).
  • the present invention includes the improvement of baculovirus gene expression vectors to provide higher levels of foreign gene expression than currently provided by very late promoters such as natural viral polyhedrin and p10 promoters.
  • the present invention also includes two different approaches: (1) modification of the upstream sequences flanking the transcriptional start point of the polyhedrin promoter and (2) utilization of the late vp39 (capsid) promoter or late/very late promoter combinations to drive foreign gene expression. Reversing the genomic orientation of the promoter and foreign gene seems to have very little impact on the levels of gene expression and has allowed the design of plasmids for the simultaneous transplacement and the expression of two or more foreign genes directed by a single baculovirus expression vector.
  • novel synthetic promoters have been designed which share minimal DNA sequence homology with other natural baculoviral promoters.
  • Such promoters are useful for the expression of more than one foreign gene using a single recombinant virus and eliminate the need for duplicating a natural viral promoter sequence within the vector, thereby minimizing the potential for genomic instability due to homologous recombination between the repeated promoter sequences.
  • transplacement plasmids which mediate the formation of baculovirus expression vectors capable of: (1) elevated levels of gene expression, (2) simultaneous expression of two passenger genes, (3) expression of passenger genes during the late as well as very late phases, and/or (4) simultaneous expression of both a passenger gene and the polyhedrin protein.
  • Transplacement plasmids facilitate the insertion of a foreign gene into the baculovirus genome by providing useful restriction sites for inserting the foreign gene(s) (i.e., a multicloning site at the appropriate site downstream of a promoter), and flanking sequences of viral DNA from the region of the viral genome into which the foreign gene(s) is to be integrated.
  • the flanking viral sequences provide sites for cell-mediated homologous recombination between nonrecombinant viral DNA and recombinant transplacement plasmids to yield the recombinant viral vector.
  • the inventor has found that certain linker scan mutations upstream of the polyhedrin TAAGTATT increase transcription and increase foreign gene expression relative to the wild-type polyhedrin promoter (Rankin et al. (1988) supra).
  • Viruses derived from peEVmodXIV, or derived from phcLSXIV provide higher levels of reporter gene (CAT) expression than other known linker scan modifications.
  • telomeres which contain a wild-type polyhedrin promoter.
  • preferred embodiments of the present invention include several plasmids which contain promoters having various modifications upstream of the TAAGTATT sequences.
  • the plasmid designated phcLSXIV, shown schematically in Fig. 1, is an example of a plasmid containing such modified promoters.
  • plasmids of this nature include phcLSXVI and phcLSXVII (as shown in Table 5). These plasmids contain upstream modified promoters which causes elevated levels of foreign gene expression when incorporated into recombinant viruses.
  • the present invention includes plasmids containing a synthetic promoter. Synthetic promoters provide diversity in the nucleotide sequence of promoters used to drive heterologous gene expression and provide differences in the levels of foreign genes expressed. A sequence for a preferred synthetic promoter is described best by the DNA sequence shown in Table 1. The arrow in the DNA sequence shows the start point and the direction of transcription. It is to be understood that there is much flexibility possible in arranging and ordering the components of the promoter and that the sequence shown in Fig. 2 is only one example of an embodiment of the present invention.
  • transplacement plasmid of the second preferred embodiment which includes the above-referenced synthetic promoter is shown in the general construction in Fig. 2.
  • This plasmid designated pSynVI-, is a transplacement plasmid carrying the synthetic promoter shown in Table 1 and the multi-cloning site (MCS) termed MCS#2 herein.
  • MCS#2 sequence of pSynVI- is shown in Table 2.
  • the orientation of the synthetic promoter is such that foreign gene expression will occur in the genomic orientation opposite that of the polyhedrin gene.
  • This promoter construct drives heterologous gene expression at approximately 10%-20% of the level of the wild-type polyhedrin promoter.
  • pSynVI+wtp Another example of the second preferred embodiment of the present invention is the plasmid designated pSynVI+wtp.
  • the schematic representation of this promoter is shown in Fig. 3.
  • This construct is a transplacement vector containing the synthetic promoter described above and MCS#2 as in the foregoing pSynVI- but also expressing the polyhedrin gene under wild-type polyhedrin promoter control. It is an example of a plasmid which is able to express two genes simultaneously.
  • This plasmid provides the selectable occlusion positive phenotype and, because recombinant viruses are occlusion positive, they can be used to orally infect insects.
  • This plasmid construction drives expression of the heterologous gene at approximately 10%-20% of the natural polyhedrin promoter.
  • pSynwtVI- Another plasmid that is included as an example of the second preferred embodiment of the present invention is designated pSynwtVI- and is shown in Fig. 4. This is a transplacement plasmid containing both the polyhedrin promoter
  • This transplacement plasmid is therefore an example of a two-gene expression vector.
  • the plasmids contain a promoter or promoters in an orientation opposite to that of the naturally occurring promoter.
  • the plasmids pSPLSXIWI+CAT and pLSXIWI+CAT are plasmids with the pLSXIV promoter in the opposite orientation (with respect to the original polyhedrin direction). These plasmids are used to test CAT expression from the LSXIV promoter in opposite orientations.
  • the pLSXIWI+CAT plasmid has a tandem double promoter. CAT gene expression from the LSXIV promoter in the opposite orientation has been found by the inventor to be similar to the levels achieved in the original orientation.
  • the promoters can be positioned in different genomic positions/orientations to give elevated levels of expression.
  • the plasmids pSPLSXIWI+CAT (Fig. 5) and pLSXIV3VI+ (Fig. 13) are two examples of high level expression promoters in opposite orientation.
  • a fourth preferred embodiment of the present invention includes plasmids containing the vp39 (capsid) promoter sequence.
  • the DNA sequence of this promoter is shown in Table 3.
  • the transplacement plasmids which can be used for testing reporter CAT expression include phc39 and pEVmod39.
  • the vp39 promoter provides approximately 5 to 120 fold higher levels of expression during the period of time from 12 to 24 hrs post- infection than the naturally occurring polyhedrin promoter. It is contemplated that the present invention also includes a combination of late/very late promoters such as the polyhedrin and vp39 promoters which would provide earlier gene expression and higher final levels (total levels) of heterologous proteins.
  • the inventor has discovered that the major determinant for polyhedrin gene expression appears to be located within the sequence ATAAGTATT. Transcription of this abundantly transcribed late gene also initiates within an ATAAG sequence flanked by an A+T rich region (Wilson et al., 1987, supra).
  • a novel baculovirus promoter has been designed which lacks minimal extended sequence homology to other viral promoters by combining short sequence motifs common to other abundantly expressed late and very late genes.
  • Two additional features incorporated in the design of the novel promoter include: an LSXIV linker, in the context of a G+C-rich region upstream of the ATAAG and a TTTATT sequence within the untranslated region near the ATG.
  • the TTTATT serves as a polyadenylation signal for any transcripts initiating in the opposite orientation from ATAAG sequences which occur by chance in the foreign gene insert.
  • the inventor has found that late and very late promoters contain an ATAAG at the most abundant transcriptional start points. This sequence is important for polyhedrin expression and promoter activity.
  • linker scan mutational analysis of the polyhedrin promoter shows that nucleotides throughout the untranslated leader region appear to contribute to optimum gene expression.
  • the sequences specifying the untranslated leader regions of four promoters of the most abundantly expressed genes of AcMNPV, namely the late gene encoding the major capsid protein (p39), the late gene encoding the basic core protein (p6.9), the very late gene encoding p10 and the polyhedrin gene have been aligned. All contain ATAAGs in the context of an A+T-rich region.
  • TAAATTACA nucleotide sequence ACAAT has been found near the ATG of 3 of 4 promoters (but not polyhedrin)
  • TACTGT was found in both the polyhedrin and p10 promoters
  • TTTGTA has been found in both the p10 and polyhedrin promoters
  • TTTGTA has been found in both the p10 and polyhedrin promoters
  • TCAANTCA has been found in the p10 and the p39 promoters.
  • all the promoters contain stretches of at least 3 T's, usually flanked by A's, and stretches of at least 3 A's, usually preceded by a pyrimidine. All these components have been designed into the model synthetic promoter. The components have been placed to minimize length; for example, the T stretches and A stretches have been placed just downstream of the ATAAG to also serve as an A+T rich region downstream of the ATTAG. However, the ACAAT sequence has been placed near the 3' restriction site
  • the transplacement plasmid pSynVI- (Fig. 2) has the Syn promoter driving expression in the opposite direction as the polyhedrin promoter. It also has a multicloning site (MCS#2) with numerous useful restriction sites. It lacks 627 nucleotides encoding the N-terminus of polyhedrin, and thus the use of this transplacement plasmid results in recombinants with occlusion negative phenotypes.
  • MCS#2 multicloning site
  • MCS#2 can also be moved to other plasmids for transplacement into other AcMNPV genomic locations using flanking restriction endonucleases (e.g., SacI downstream of MCS#2 and Kpnl or Smal upstream of the promoter).
  • flanking restriction endonucleases e.g., SacI downstream of MCS#2 and Kpnl or Smal upstream of the promoter.
  • viruses are derived originally from AcMNPV L-1 (Lee and Miller (1978) "Isolation of genotypic variants of A. californica nuclear polyhedrosis virus," J. Virol.22 ⁇ 754-767); and are plaque-purified and propagated in the Spodoptera frugiperda IPLB-SF-21 cell line (Vaughn et al. (1977) "Establishment of Two Insect Cell Lines From the Insect Spodoptera frugiperda (Lepidoptera: Noctuidae)" In Vitro 13: 213-217 using methods described previously (Lee and Miller (1978); Miller et al.
  • the CAT gene for all CAT-containing plasmids used in these studies was derived from pCMlCAT (LKB Biotechnology, Piscataway, N.J.) in which the CAT gene is inserted as a Sall cassette.
  • pEVmodXIV transplacement plasmid (Fig. 9) is described.
  • the previously described transplacement plasmid pEV55 (Miller et al. (1986) supra), which serves as the basis for constructing phcwt (Fig 7), also serves as the starting plasmid for pEVmodXIV construction.
  • the pEV55 plasmid contains AcMNPV DNA from 3.18 to 7.3 map units (mu).
  • a multicloning site replaces polyhedrin coding sequences from +1 to +635.
  • the viral sequences between 6.16 mu and 7.3 mu in pEVdel are deleted by digestion with BamHI (6.16 mu) and NotI (7.3 mu) at the right vector junction.
  • pEVmod Blunt end ligation at the deleted junction resulted in pEVmod (Fig. 8).
  • the polyhedrin promoter (from the EcoRV at -92 to the Bglll at +1) of pEVmod is replaced with the small EcoRV-BglII fragment of phcLSXIV (Fig. 1) resulting in the improved and convenient transplacement plasmid pEVmodXIV.
  • Virus vectors derived from pEVmodXIV-based transplacement plasmids provide approximately 50% higher levels of expression than virus vectors derived from pEV55 or any other known plasmids.
  • Example III construction of the phc39 (Fig. 15) and pEVmod39 (Fig. 9) transplacement plasmids is described.
  • a plasmid containing the vp39 promoter is first constructed as follows. The Narl to EcoRV fragment containing the vp39 promoter between 57.6 to 57.9 map units on the AcMNPV genome is cloned into the AccI and EcoRV sites of the plasmid vector Bluescript KS- (Stratagene, San Diego, CA). The resulting plasmid, pSTVNM, is cut at the Xhol and Apal sites.
  • the N-terminal sequences of the vp39 coding sequence are deleted with Exonuclease III and mung bean nuclease digestion.
  • a Bglll linker oligonucleotide is inserted into the gap using DNA ligase.
  • One plasmid is selected which contains the Bglll site at -2 relative to the ATG (+1, +2, +3) of vp39.
  • the 458 bp capsid promoter region is excised from the previously selected plasmid using Kpnl and EcoRV and is ligated into the plasmid Bluescript KS+ (Stratagene, San Diego, CA) which is also cut with Kpnl and EcoRV.
  • transplacement plasmid phc39 To construct the transplacement plasmid phc39, the 458 bp EcoRV to Bglll fragment of p39pro containing the vp39 promoter is excised and inserted into EcoRV and Bglll digested phcwt, thus replacing the polyhedrin promoter of phcwt with the vp39 promoter region.
  • the resulting transplacement plasmid, phc39 (Fig. 15) is used to construct viral vector vhc39.
  • CAT gene expression using this vector is 5 to 10 times higher between 12 and 24 hours post infection than CAT gene expression from virus vectors carrying the CAT gene under polyhedrin promoter control. The earlier expression of the foreign gene provides for more efficient post-translational modification of the protein product.
  • transplacement plasmids such as pEVmod39 (not shown), can be derived easily from phc39 by replacing the CAT gene with a MCS containing Bglll and Kpnl termini (e.g., MCS#1).
  • Example IV To construct the plasmid pSynVI- (Fig. 2), which contains the Syn promoter and an adjoining multicloning site (MCS#2) in place of the polyhedrin promoter, the two Syn promoter oligonucleotides shown in Table 1 are annealed, the duplex is purified by polyacrylamide electrophoresis, and is inserted into the Kpnl- and EcoRV-digested Bluescript plasmid vector pBSKS- (Stratagene, San Diego, CA), which lacks the Smal restriction site in the multicloning site. This allows the forced cloning of the oligonucleotide into the multicloning site of the Bluescript plasmid and the regeneration of both the EcoRV and Kpnl sites.
  • the portion of the multicloning site of this plasmid from the EcoRV to the SacI site contains, in order, EcoRV, EcoRI, Pstl. BamHI, Spel. Xbal, NotI, SacII, and SacI sites.
  • the synthetic promoter and this multicloning site are removed from the Bluescript vector by ordered digestion with SacI. mung bean nuclease, and Kpnl. The small fragment is gel purified and inserted into the large, gel-purified fragment of Kpnl- and EcoRV digested pEVmod (Fig. 8) .
  • the sequence of the promoter from the Smal site through the flanking multicloning site referred to as MCS#2, is determined by sequencing. The sequence is shown in Table 2.
  • the SacI site is regenerated unexpectedly and fortuitously during the cloning process; the action of mung bean nuclease downstream of the SacI site accounts for the observed junction.
  • a pSynVI- plasmid containing the reporter CAT gene is constructed to measure the level of gene expression of the pSynVI- plasmid by measuring the CAT gene expression of the constructed CAT-containing plasmid.
  • the CAT gene is excised from pCMlCAT with Sall, blunt-ended with mung bean nuclease, and inserted into pSynVI- cut with EcoRV.
  • Orientation is checked using the EcoRI site in the N-terminal portion of the CAT gene.
  • the corresponding viral vector, vSyn VI-CAT provides CAT expression at levels approximately 10-20% of the levels observed for viral vectors expressing CAT under polyhedrin promoter control. Although not as high as polyhedrin promoter expression, this level of expression from a component synthetic promoter is significant.
  • the Syn promoter and others derived by the combinatorial approach will be useful for balanced expression of two different foreign genes and/or the expression of genes encoding proteins controlling specific post-translational modifications.
  • transplacement plasmid it is useful for a transplacement plasmid to transfer genetic information to form a recombinant virus which allows both foreign gene expression and polyhedrin gene expression.
  • Expression of both genes results in occlusion, which permits efficient oral infection of insect larvae for inexpensive, bulk protein production.
  • production in insect larvae allows expression in differentiated insect cells, including the fat body which constitutes a major secretory tissue of the insect and may provide more efficient post-translational modification than that found in available lepidopteran cell lines such as SF9 cells.
  • pSynVI- (Fig. 2) is modified to produce a transplacement plasmid, pSynVI+wtp (Fig. 3), which allows simultaneous, high level expression of both polyhedrin and the foreign gene.
  • An additional advantage of pSynVI+wtp is the ability to select recombinant viruses as occlusion positive plaques if the DNA of a polyhedrin deletion mutant (or a polyhedrin/ ⁇ -galactosidase fusion mutant) is used in the initial co-transfections of host cells with viral DNA and transplacement plasmid.
  • the pSynVI+wtp transplacement plasmid is constructed by digesting a plasmid carrying the EcoRI-I fragment of the AcMNPV genome, with EcoRV and Kpnl to remove the polyhedrin promoter and N-terminal polyhedrin region.
  • the approximately 0.7kb EcoRV/Kpnl fragment is gel-purified and inserted into pSynVI- which had been digested with Smal and Kpnl. Ligation eliminates the Smal and EcoRV sites while regenerating the Kpnl site found in the polyhedrin coding region.
  • polyhedrin promoter and N-terminal coding sequences from EcoRV (-92) to Kpnl (+635) are inserted into pSynVI- to produce the transplacement plasmid pSynVI+wtp.
  • Polyhedrin mRNA is thus transcribed in its normal orientation under the control of its normal promoter while the foreign gene is inserted at the multicloning site under synthetic promoter control and is transcribed in the opposite direction to polyhedrin.
  • the features of the plasmid are illustrated in Fig. 3.
  • the sequence of the EcoRV/Smal junction, the synthetic promoter, and the multicloning site (MCS#2) are shown in Table 1.
  • the reporter CAT gene is inserted into the MCS of pSynVI+wtp (Fig. 3) at the EcoRV site resulting in the plasmid pSynVI+wtpCATl (Fig. 11).
  • pSynVI+CAT1 the blunt- ended CAT-containing Sall fragment of pCM-lCAT is inserted into EcoRV-digested pSynVI+wtp.
  • transplacement plasmid pSynwt VI- (Fig. 4) is described.
  • This plasmid has two promoters in a back-to- back relationship allowing simultaneous transplacement of two heterologous genes and concerted expression of the two genes by a single vector.
  • a pSynwtVI- plasmid containing the reporter CAT gene is constructed to provide a measure of gene expression of pSynwtVI-.
  • the plasmid is pSynwtVI-CAT1 (Fig. 12).
  • pSynwtVI-CAT1 the blunt ended CAT-containing Sall fragment of pCM-lCAT is inserted into EcoRV-digested
  • pSynwtVI-CAT1 expresses CAT activity at 10-20% of the levels observed for vectors expressing CAT under polyhedrin promoter control.
  • pSPLSXIWI+CAT Construction of the pSPLSXIWI+CAT and pLSXIWI+-CAT transplacement plasmids (Fig. 5 and 6) are described.
  • pSPLSXIWI+CAT To construct pSPLSXIWI+CAT, the CAT gene and attached LSXIV promoter is removed from phcLSXIV (Fig. 1) by Kpnl digestion (and blunt ended with mung bean nuclease) and EcoRV digestion, and then inserted into EcoRV-digested pSynVI+wtp. This places the LSXIV promoter in tandem with the synthetic promoter. The plasmid is then cut with EcoRV and Kpnl and the large vector fragment is gel-purified.
  • the EcoRV/Kpnl fragment of AcMNPV EcoRI-I fragment containing the wild-type polyhedrin promoter and N-terminus is gel-purified and inserted into the vector fragment (EcoRV/Kpnl) of pSPLSXIWI+CAT to yield pLSXIWI+CAT which contains the CAT gene under LSXIV promoter control and the intact polyhedrin gene under polyhedrin promoter control.
  • the viral vectors derived from these plasmids express equivalent or slightly higher levels of CAT than viruses expressing CAT under wild- type polyhedrin promoter control.
  • An additional advantage of virus vectors derived from pLSXIWI+CAT is that they produce polyhedrin at high levels.
  • Plasmid pLSXIWI+CAT is digested with Bglll and EcoRI; the sticky ends removed with mung bean nuclease. The DNA is then cut with SacI and the large fragment is gel-purified and ligated to a multicloning site (MCS#3) having EcoRV and SacI termini to form pLSXIV3VI+ lacking the CAT gene. This plasmid is then digested with EcoRV and Kpnl.
  • the large fragment is isolated and the EcoRV/Kpnl fragment of pEVmodXIV is inserted into the large fragment resulting in pLSXIV2 which has two back-to-back LSXIV promoters, each with its own MCS.
  • the pLSXIV3VI+ plasmid allows superior levels of foreign gene expression using the powerful LSXIV promoter as well as polyhedrin gene expression.
  • the pLSXIV2 plasmid allows superior expression of two foreign genes, each under LSXIV promoter control.
  • Transplacement plasmid phc39 (Fig. 15) is constructed with the CAT reporter gene under the control of the vp39 promoter. Expression of CAT from vhc39 shows approximately 8 fold higher levels of CAT expression at 12 hrs post-infection than vhcwt, the vector carrying CAT under wild-type promoter control.
  • the CAT gene of phc39 can be replaced with a heterologous gene of interest. Earlier expression during the infection process may allow more efficient post-translational modification of the heterologous protein product.
  • Transplacement plasmids are constructed to allow the simultaneous transplacement of two foreign genes into the virus vector and allow the recombinant viruses to simultaneously and abundantly express both foreign genes.
  • the transplacement plasmid pSynwtVI- (Fig. 4) is one example of such a dual gene transplacement plasmid.
  • This plasmid is constructed by digesting pSynVI- with Smal and Kpnl and inserting the pEV55 EcoRV/Kpnl fragment containing the wild-type polyhedrin promoter and MCS#1. This plasmid thus contains two different multicloning sites, each downstream from back-to-back Syn and wild-type polyhedrin promoters.
  • Example XI Another example which allows even higher levels of the second gene is pLSXIV2 (Fig. 14).
  • This plasmid provides back- to-back LSXIV promoters, each with a multicloning site (MCS#1 and #2) containing useful restriction sites for inserting two different foreign genes, each under the control of an LSXIV promoter.
  • MCS#1 and #2 multicloning site
  • the efficacy of back-to-back promoters is tested using vLSXIWI+CAT, a recombinant virus carrying the pLSXIWI+CAT construct in which the LSXIV promoter drives CAT expression back-to-back with the polyhedrin promoter, which in turn drives polyhedrin gene expression.
  • CAT expression by the vLSXIWI+CAT vector was equivalent to or slightly higher than that observed with phcwt alone which expresses only CAT.
  • Hindlll linkers are inserted into the 462 bp promoter of vp39.
  • the DNA sequence of vp39 is shown in Table 3 (see Rankin et al. (1988) supra. for general linker scan approach).
  • the Hindlll linkers serve to allow interface with the Hindlll sites of the linker-scan-modified promoters to generate late/very late combination promoters.
  • Combination promoters are constructed which contain the OCT-A and OCT-B regions of vp39 (e.g., from -65 to approximately -350) fused to the +1 to approximately -60 of the polyhedrin promoter of the LSXIV promoter of Rankin et al. (1988) supra.
  • a plasmid is selected (e.g., pvp39-65) containing a Hindlll linker at an appropriate site (e.g., -65).
  • the Hindlll to Bglll fragment is replaced with the Hindlll to Bglll fragment of phcLSXIV.
  • the entire Bglll to EcoRV fragment containing the combination vp39/LSXIV promoter can then be transferred to pEV55 to construct a transplacement plasmid, pEVvp39/LSXIVCAT (Fig. 16) with the combination promoter controlling CAT expression.
  • Fig. 16 is achieved in accordance with the method described in Example XII above.
  • the sequence of the hybrid vp39/LSXIV promoter is shown in Table 4.
  • the sequence at the junction of the vp39/LSXIV combination promoter is marked by "N1-10 CCAAGCTTG" which indicates a Hindlll site at the junction (CCAAGCTTG) and approximately 1 to 10 nucleotides (Nl-10) of the vp39 at the junction.

Abstract

On a mis au point un procédé et une composition améliorant le niveau d'expression d'un gène ou d'une collection de gènes, consistant à produire un nouveau promoteur de baculovirus auquel on fixe un gène hétérologue. Le nouveau promoteur peut contenir une modification d'un promoteur viral naturel, ou peut être entièrement synthétique. Ledit nouveau promoteur du baculovirus peut également comprendre un promoteur de combinaison tel qu'un promoteur à double site de départ, ou une combinaison de deux promoteurs différents. L'invention concerne également la mise en place d'un gène étranger dans un nouvel emplacement ou dans une nouvelle orientation génomique.
PCT/US1990/002814 1989-05-17 1990-05-17 Vecteurs ameliores d'expression du baculovirus WO1990014428A1 (fr)

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JPH04501065A (ja) 1992-02-27
EP0426840A1 (fr) 1991-05-15
AU631223B2 (en) 1992-11-19
AU5820790A (en) 1990-12-18
CA2033070A1 (fr) 1990-11-18

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