EP0996715A1 - Expression heterologue de proteines a l'aide d'un vecteur "sauve" comprenant un intron - Google Patents

Expression heterologue de proteines a l'aide d'un vecteur "sauve" comprenant un intron

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Publication number
EP0996715A1
EP0996715A1 EP98935143A EP98935143A EP0996715A1 EP 0996715 A1 EP0996715 A1 EP 0996715A1 EP 98935143 A EP98935143 A EP 98935143A EP 98935143 A EP98935143 A EP 98935143A EP 0996715 A1 EP0996715 A1 EP 0996715A1
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Prior art keywords
gene
intron
expression
construct
sequence
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EP98935143A
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German (de)
English (en)
Inventor
Alan Colman
Ian Garner
Michael Alexander Dalrymple
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PPL Therapeutics Scotland Ltd
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PPL Therapeutics Scotland Ltd
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Publication of EP0996715A1 publication Critical patent/EP0996715A1/fr
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    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to the expression of proteins in heterologous host systems, particularly in, but not limited to, the mammary gland of transgenic animals.
  • mice transgemc for the latter construct alone only one out of eight mice expressed and this at a level of only 3.9 ⁇ g/ml.
  • an expression level of 108 ⁇ g/ml from a wild type human protein C cDNA construct was achieved (WO-A-9211358). This represents approximately 20% the expression level obtained with an equivalent genomic based construct.
  • the cDNA construct alone gave no expression in 11 lines of transgenic mice.
  • the "rescue" .phenomenon has been rationalised as follows. Strongly expressing genes have an innate ability to 'dominate' their chromosomal environment such that they are able to initiate and maintain a high expressing state. Intronless genes are deficient in some, as yet identified, feature which provides them with this capability. However, the dominant effect of the strong gene extends some way 5' and 3' to the gene itself and therefore by linking a 'weak' and 'strong' gene, some of the properties of the high expressing gene are conferred on the intronless gene. Clark and colleagues propose that this probably results in an open chromatin conformation associated with the actively expressing gene which encompasses adjacent intronless genes.
  • the actively expressing gene may thus create a permissive domain allowing access to the intronless genes by the transcriptional machinery of the cell.
  • the intronless construct may be inaccessible, probably residing in condensed chromatin.
  • Other possible explanations for this phenomenon include enhancer-like sequences present in the actively expressing gene but absent from the intronless construct interacting positively with the latter or simply that the actively expressing gene insulates the intronless gene from the negative effects of adjacent chromatin.
  • the two different genes may be present but not at the same locus. Subsequently they may segregate upon breeding. Finally, the physical structure of a BLG/pMAD array is not determined prior to injection and there is no control over it. The relative copy numbers of the two genes may vary especially if the DNA concentrations of the two constructs are not tightly controlled.
  • an expression construct comprising:
  • the intron (b) is not derived from the same gene as that from which either the promoter (a) or the coding sequence (c) is derived, and, in particular wherein the promoter (a) drives expression of the coding sequence (c) at a level which is elevated by virtue of the presence of the intron (b) .
  • Elevated levels of expression include expression where previously none was measurable (or obtained). Elevated levels is optionally defined as a level higher than obtained by the construct without the intron (and optionally the 3' flanking sequence) described above.
  • the expression construct is a DNA expression construct.
  • the coding sequence is a protein-coding sequence although it may code non-protein substances such as ribozymes.
  • the construct is effective for two particular reasons; firstly, the promoter (a) drives expression of the coding sequence (c) at a level which is elevated by virtue of the presence of the intron (b) and/or secondly the coding sequence (c) is more likely to be expressed in a transgenic host, by virtue of the presence of the intron (b).
  • the second effect is particularly important when taking into account the length of time and the efforts required to produce transgemc animals useful as bioreactors for the production of useful proteins, etc. It is also important in laboratory scale trials to determine and obtain transgenic hosts.
  • constructs as described in the claims have shown that an increased number of transgenic hosts express the coding sequence over use of the constructs without specific intron described herein (e.g see number of expressing founders in Table 3).
  • the elevated level of expression of the coding sequence and/or the expression of the coding sequence may be by virtue of the presence of the intron (b) and the 3'- flanking sequence (d).
  • the DNA expression construct may be useful for expression in any suitable host system such as, for example, prokaryotes, (e.g. E.col ⁇ ), fungi, plant and animal (including mammalian) cell lines and transgenic plants and animals (including mammals). However, it is in transgenic animal hosts that the expression constructs of the invention are most useful. In principle, the invention is applicable to all animals, including birds such as domestic fowl, amphibian species and fish species.
  • the protein may be harvested from body fluids (such as milk, blood or urine) or other body products (such as eggs, where appropriate). In practice, it will be to (non-human) mammals, particularly placental mammals, that the greatest commercially useful applicability is presently envisaged.
  • constructs of the invention may also be useful in genetic therapy in humans or other animals.
  • the promoter can be any suitable promoter chosen from a gene different from the source of the intron (b). Within that constraint, it will be chosen having regard to its desired properties in the construct of the expression system to be used and its ability to derive expression of heterologous sequences in cell culture or in a transgenic organism.
  • a promoter is any sequence which drives expression of a coding sequence.
  • the BLG promoter does not express particularly highly in cells which do not respond to prolactin (such as COS cells).
  • a 'cell' promoter according to the invention is the HCMV (human cytomegalovirus) IE gene promoter.
  • promoters of the invention include, endothelial promoters such as vascular cell adhesion molecule (VCAM), platelet endothelial cell adhesion molecule- 1 (PEC AM), inter-cellular adhesion molecule-2 (ICAM) and smooth muscle promoters, such as Desrnin E and Desmin P.
  • VCAM vascular cell adhesion molecule
  • PEC AM platelet endothelial cell adhesion molecule- 1
  • IAM inter-cellular adhesion molecule-2
  • smooth muscle promoters such as Desrnin E and Desmin P.
  • a preferred promoter is one which drives expression of the protein coding sequence in mammalian cells.
  • the preferred expression system involves expression in the mammary gland of transgenic placental mammals.
  • milk protein promoters will generally be used, preferably but not necessarily derived from the species chosen as an expression host.
  • the promoter may be a casein promoter (such as an ⁇ -, ⁇ - or ⁇ -casein promoter), but it is preferred that it be a non-casein promoter, such as the human Bile Salt Stimulated Lipase (BSSL) promoter, more preferably a whey protein promoter, such as that of whey acidic protein (WAP), ⁇ -lactalbumin or, most preferred of all, ⁇ - lactoglobulin.
  • Figure 3 is a schematic representation of the cloning of pCASLAC and obtaining transgene constructs therefrom.
  • pCASLAC corresponds to pCASMAD ⁇ (see Fig. 2, 4 and 7) only using the more tightly regulated ⁇ -lac promoter.
  • the present invention covers promoters, in the constructs described, which have not yet been isolated or characterized.
  • One general way for isolating specific promoters (such as mammalian promoters) for use in the present invention is to isolate specific cDNAs by differential display or from subtractive cDNA libraries. These, in turn, are used to screen genomic libraries for the cognate promoters.
  • the present invention encompasses the use of a modified low expressing naturally occurring promoter in vitro to an increased level of expression (eg. by addition of an enhancer) or to use a promoter with a higher level of expression in a crossed species (eg. the human ⁇ -lactalbumin promoter expresses better in mice than the endogenous mouse promoter).
  • a modified low expressing naturally occurring promoter in vitro to an increased level of expression (eg. by addition of an enhancer) or to use a promoter with a higher level of expression in a crossed species (eg. the human ⁇ -lactalbumin promoter expresses better in mice than the endogenous mouse promoter).
  • a promoter according to the invention may also be a viral or modified cellular promoter or a completely artificial promoter having the properties of high level expression (preferably mammalian species). Details of suitable promoters can be found in Houdibine, J-M., J. Biotech., 34: 269-287 (1994); Garner, I. & Dalrymple, M., in "Encyclopedia of Molecular Biology: Fundamentals and Applications ", Robert A. Myers (Ed.), Weinheim, NY.
  • Element (b) of a construct of the invention is an intron whose natural position is within the 5 '-untranslated region (5'-UTR) of its natural gene (i.e the gene with which it is naturally associated).
  • the whole intron is not necessarily required. Fragments or portions may be sufficient.
  • the requirement for the present invention is that the level of protein expression, from any construct according to the invention, is elevated by virtue of the presence of the intron, or parts thereof. It has been shown that the first and third portions of an intron (which has been divided into three fairly equal parts), recombined, are often effective. Generally speaking, the intron for inclusion in a construct according to the invention will be the first intron of such a gene.
  • genes with such known introns include; human and rat aldolase A, human type II IL-1 receptor, human UDP-N- acetylglucosaminyl transferase, mouse involucrin and mouse adenosine deaminase.
  • Some genes have more than one intron whose natural positions are all within the 5' untranslated region of its natural gene.
  • the present invention recognises this and covers, within element (b), one or more of such introns (for example in a gene with two introns naturally positioned in the 5' UTR, they may separately, together or parts of each cojoined be included in a part of a construct according to the invention).
  • introns whose natural position is within the 5' untranslated region of its natural gene may be used according to the present invention. Also included are: the introns of several gene families including; the actin family (two skeletal muscle actins-alpha cardiac and alpha skeletal, two smooth muscle actins-alpha smooth and gamma smooth, and two non- muscle actins-beta and gamma cytoplasmic actin), the troponin family (cardiac, skeletal and foetal troponins) and the casein family ( ⁇ SI, ⁇ S2, ⁇ and K).
  • the intron is the first intron of the family.
  • the most preferred gene family from which the intron may come is the casein family or the actin family.
  • the intron may preferably be from the same source of organism as the promoter and/or the expression system which it is in (e.g. mammalian, bovine, ovine, etc.).
  • DNA expression constructs of the present invention are different from that of Barash et al. (Nucl. Acids Res. 24(4) 602-610 (1996)), in that Barash et ⁇ /. 's constructs include ⁇ -lactoglobulin intragenic sequences which are not within the 5'- untranslated region. Barash et al. do not refer to the possibility of using an intron whose natural position is within the 5 '-untranslated region of its natural gene.
  • Caseins whose genes represent a preferred source of the 5' -UTR introns useful in the present invention, are the major mammalian milk proteins and are encoded by a small gene family, which in cows and sheep consists of four members, ⁇ sl , ⁇ , ⁇ s2 and K, and in mice and rats five, ⁇ , ⁇ , ⁇ , ⁇ and K (Yu-Lee & Rosen, J. Bio I Chem., 258 10794-10804 (1983); Jones et al, J. Biol. Chem., 206 7042- 7048(1985); Thompson et al, DNA, 4 263-271 (1985)/ reviewed by Mercier & Vilotte, J.
  • the first intron of the calcium sensitive casein genes is naturally positioned in the
  • the intron may be obtained by PCR amplification from genomic DNA. The resulting DNA fragment may be cloned into a suitable site of an appropriate vector, such as the pMAD6 vector described above.
  • Constructs of the invention also contain a coding sequence (c), whose expression is driven by the promoter (a) under the beneficial influence of the intron (b).
  • the protein-coding sequence may code for any (natural or modified) protein of interest, particularly those which may be advantageously produced in the preferred mammary gland expression systems.
  • proteins involved in haemostasis including factors V, VII, VIII, IX, X, XIII, PAI-1, PAI-2, TFPI
  • protein C protein C
  • protein S protein S
  • alpha 1-antitrypsin (AAT) details of which can be found in general from Perlino et al EMBO Journal, 6, 2767-2771, 1987 and WO90/05188
  • tPA alpha 1-antitrypsin
  • fibrinogen details for which may be found in WO95/23868 and the references cited therein
  • other protease inhibitors such as serpins, Kazal/Kunitz inhibitors, kinninogens, stefms, cy statins or tissue inhibitors of metalloproteinases
  • growth factors protein hormones
  • structural proteins such as collagens (details of which may be found in
  • BLG promoter + bovine ⁇ -casein intron 1 + BLG 3' sequence (particularly the 3' sequence beginning immediately 3' to the natural ⁇ - lactoglobulin stop codon and continuing to at least about 30 bases 3' of the poly-A site), optionally including ovine beta- lactoglobulin intron 6 (preferred positioned 5' to the flanking sequence and 3' to any coding sequence); in particular the construct pCASMAD ⁇ as described in Fig. 2, 4 or 7;
  • BLG promoter + muscle cardiac actin intron 1 + BLG 3' sequence (particularly the 3' sequence beginning immediately 3' to the natural ⁇ - lactoglobulin stop codon and continuing to at least about 30 bases 3' of the poly-A site), optionally including ovine beta-lactogloulin intron 6 (preferred position 5' to the flanking sequence and 3' to any coding sequence); in particular the construct pACTMAD ⁇ as described in Fig. 2, 5 or 7;
  • BLG promoter + ovine ⁇ -casein intron 1 -I- ovine ⁇ -casein 3' flanking sequence in particular the construct pBOB as described in Fig 2, 6 or 7.
  • 3 '-sequences as may be necessary or appropriate. In the invention at its broadest, it is not thought that the nature of such 3 '-sequences is particularly limited.
  • the 3'- flanking sequence may or may not include its natural intron.
  • Suitable 3' flanking sequences preferably comprise functional elements which are able to direct the correct transcription, termination and 3' end processing. These can be determined, without undue burden, by the person skilled in the art.
  • 3 '-flanking sequences have been found to be particularly useful. These include, but are not restricted, to: (i) a poly-A site (poly A addition site), (ii) a ⁇ -lactoglobulin gene 3 '-sequence beginning immediately 3' to the natural ⁇ -lactoglobulin stop codon and continuing to at least about 30 bases 3' of the poly-A site (as found in pMAD6 and pCASMAD ⁇ and PACTMAD6), or (iii) ⁇ -casein 3' sequences including poly A signal. These sequences (as used in pBOB) consist of 6.5Kbp of DNA incorporating ovine ⁇ -casein exons 7 to 9, introns 7 and 8, and approximately 4.8Kbp of 3 ' sequence .
  • the ⁇ -casein 3' sequences may be cloned from the ⁇ -casein gene cr amplified by PCR and cloned from genomic DNA, which may be of ovine origin.
  • Appropriate signal and/or secretory sequences, operably linked to the construct may be present if necessary or desirable.
  • the invention is directed to:
  • the process comprises linking together selected nucleotide bases and/or nucleotide sequences;
  • the vector may be plasmid, phage, cosmid or other vector type, for example derived from yeast.
  • the vector may be an expression vector;
  • a process for the preparation of a host comprising introducing a DNA expression construct (or a vector), as described above, into a suitable organism; the process, in particular provides a host which expresses elevated levels of the coding sequence in the construct;
  • a host organism preferably an expression host organism
  • the host may be a eukaryotic or prokaryotic cell/organism, such as bacteria, insect or yeast cells, as well as animal tissues (cells in culture) and animals themselves.
  • Such animals are transgenic and preferred transgenic animals include mammals, in particular non-human placental mammals such as pigs, sheep, cattle and goats.
  • the host e.g.
  • transgemc animal according to the invention has the construct (of the first aspect of the invention) integrated into its genome. It is particularly preferred that the transgenic animal transmits the construct to its progeny, thereby enabling the production of at least one subsequent generation of producer animals.
  • a host organism in particular expresses elevated levels of the coding sequence in the construct;
  • the protein may be a fusion protein
  • nucleic acid expression construct comprising a promoter, an intron whose natural position is within the 5 '-untranslated region of a gene from which it is derived, a coding sequence and a 3' flanking sequence to obtain a transgenic host, preferably with elevated levels of the expressed coding sequence;
  • nucleic acid construct comprising a promoter, an intron whose natural position is within the 5' untranslated region of a gene from which it is derived, a coding sequence and a 3' flanking sequence to increase the likelihood of expression of the coding sequence from a transgenic host which incorporates the nucleic acid construct; • a process for improving whether an individual or a number of transgenic hosts express a transgene coding sequence, the process comprising introducing into a host, a nucleic acid construct comprising a promoter, an intron whose natural position is within the 5' untranslated region of a gene from which it is derived, a coding sequence and a 3' flanking sequence.
  • an empty 'cassette' including all feamres in claim 1, without the coding sequence.
  • Such a "cassette” provides an easy means by which to provide a high expressing vector for other parties to use by simply introducing coding sequences of interest by restriction endonuclease cutting of the empty cassette and religation (according to standard techniques).
  • the empty "cassette” is for use with an incorporated coding sequence of interest.
  • Preferred feamres for each aspect of the invention are as for each other aspect mutatis mutandis.
  • the present invention also provides, as a separate aspect, the novel expression of collagen cDNA (natural procollagen chains or modified collagen).
  • collagen cDNA naturally procollagen chains or modified collagen
  • the collagen cDNA is expressed via a construct according to the first aspect of the invention.
  • Preferred feamres of and all different aspects of the invention described herein above in relation to the construct also apply to the expression of collagen cDNA.
  • Particular preferred details in relation to collagen are described above under a discussion of the protein-coding sequences, including references thereto.
  • expression hosts may co-express prolyl 4-hydroxylase, which is a post- trans lational enzyme important in the natural biosynthesis of procollagen.
  • FIGURE 1 shows the ⁇ -lactoglobulin (BLG) sequences and the plasmids pMAD and pMAD ⁇ .
  • FIGURE 2 shows the origin of sequences present in plasmids pMAD, pMAD6, pCASMAD ⁇ and pACTMAD ⁇ .
  • FIGURE 3 shows the construction of pCASLAC
  • FIGURE 4 shows the construction of pCASMAD ⁇ .
  • FIGURE 5 shows the construction of pACTMAD ⁇ .
  • FIGURE 6 shows the construction of pBOB.
  • FIGURE 7 shows details of pMAD, pMAD ⁇ , pCASMAD ⁇ , pACTMAD ⁇ and pBOB.
  • Preferred embodiments of the invention are based on the use of the BLG promoter, and are designed to express cDNAs from the BLG gene.
  • the structure of pMAD ⁇ is indicated in Figures 1, 2 and 7. This vector contains the same 5' and 3' flanking sequences present in the ovine BLG gene which itself always gives rise to high level expression in transgenic mice.
  • the resulting DNA fragment of approximately 2 Kbp, was cloned and subsequently subcloned into the EcoRV site of pMAD ⁇ in such a way that the original EcoRV site was destroyed and reformed on the 3' side of the intron.
  • a cDNA encoding human protein C was inserted into the unique EcoRV site of pCASMAD ⁇ and the new construct called pCORI69.
  • the cDNA utilised encodes a mutant form of the natural protein C (PC962): the mutation was designed to allow more efficient processing of the mature protein (Foster et al, Biochemistry, 29:347-354 (1990)).
  • This mutant form of the human protein C cDNA has been incorporated into a construct pCORP9, exactly analogous to pCORP2 (see WO-A-9211358).
  • pCORP9 expressed particularly poorly, the highest expressing line being 3 ⁇ g/ml compared to 108 ⁇ g/ml for the wild type cDNA.
  • All references to the DNA sequence of the ⁇ -lactoglobulin gene utilise the numbering of the sequence allocated ⁇ MBL Accession No. X 12817 (Harris et al , NAR 16: 10379-80 91988).
  • the multiple cloning site of the vector pUC18 (Yanisch-Perron et al. , (1985) Gene 33: 103-119) was removed and replaced with a synthetic, double stranded, oligonucleotide containing the new restriction sites: PvuVMluVSall/EcoRY/Xbal/ Pvull MM, and flanked by 5 '-overhangs compatible with the restriction sites EcoRI and HmdIII.
  • pUC18 DNA was cleaved with both EcoRI and HmdIII and the new linker DNA was ligated into pUC18. The DNA sequence across the new multiple cloning site was confirmed. This new vector was called pUCPM.
  • Plasmid pUCXS The ⁇ -lactoglobulin gene sequences from plasmid pSSltgXS (see WO-A-9201358) were excised on a SaWXbal fragment and recloned into the vector pUCPM, cut with Sail and Xbal, to give plasmid pUCXS.
  • Plasmid pUCXS/RV Plasmid pUCXS/RV
  • the plasmid pSSltgSE contains: ⁇ -lactoglobulin gene sequences from the Sphl site at position 754 to the EcoRI site at 2050, a region spanning a unique NotI site at position 1148.
  • This insert contains a single Pvull site (832) which lies in the 5 '-untranslated region of the ⁇ -lactoglobulin mR ⁇ A.
  • the D ⁇ A sequences bounded by Sphl and NotI were then excised and used to replace the equivalent fragment in the plasmid pUCXS, thus effectively introducing a unique EcoRV site into the ⁇ -lactoglobulin gene placed in such a way as to allow the insertion of any additional D ⁇ A sequences under the control of the ⁇ - lactoglobulin gene promoter and 3' to the initiation of transcription.
  • the resulting plasmid was called pUCXS/RV.
  • pUCXS/RV A derivative of pUCXS/RV, containing only the 4.3 Kbp of the ⁇ -lactoglobulin gene which lie 5' to the transcription initiation site (the promoter), was constructed by subcloning the S ⁇ /I-EcoRV fragment into pUCPM; this plasmid is called pUCSV.
  • Plasmid pBLAClOO A fragment of the 3' flanking sequence of the ⁇ -lactoglobulin gene was subcloned in such a way as to eliminate all introns. Plasmid D ⁇ A of pUCXS/RV was partially digested with Smal by performing an enzyme titration with lower and lower concentrations of enzyme at a fixed D ⁇ A concentration. The Smal protein was removed by phenol-chloroform extraction and ethanol precipitation and the D ⁇ A resuspended in water. This D ⁇ A was subsequently digested to completion with the enzyme Xbal. DNA cut once at the Smal site, position 5286 and then cleaved with Xbal gave a characteristic band of size 2.1 Kbp. This band was purifL ⁇ from an agarose gel slice and ligated into Smal and Xbal cut pBSIISK+ (Stratagene Ltd., Cambridge Science Park, Cambridge, UK) to give the plasmid pBLAClOO.
  • the ⁇ -lactoglobulin cloning vector pMAD was constructed to allow rapid insertion of cDNAs under the control of the ⁇ -lactoglobulin gene promoter and 3 '-flanking sequences. Such constructs contain no introns.
  • the plasmid pBLAClOO was opened by digestion with both EcoRV and Sail, the vector fragment was gel purified. Into this was ligated the 4.3 Kbp promoter fragment from the plasmid pUCSV as a Sall-EcoRV fragment. This construct is termed pSTl and constitutes a ⁇ -lactoglobulin mini-gene encoding the 4.3 Kbp promoter and 2.1 Kbp of 3' flanking sequences.
  • a unique EcoRV site is present to allow blunt-end cloning of any auditional DNA sequences.
  • Mlul the entire mini-gene from pSTl was excised on a Xhol-Notl fragment, the DNA termini made flush with Klenow polymerase, under standard conditions, and blunt-end cloned into the EcoRV site of pUCPM to give pM AD.
  • Two primers complementary to sequences at the 5' and 3' boundaries of the first intron of the ovine BLG gene, were used to amplify a ⁇ 650bp fragment encompassing the entire sequence of intron 1 of the BLG gene from pUCXSRV template.
  • the primers introduce a 5' Smal site and a 3' EcoRV site at the ends of the PCR fragment.
  • This fragment was cloned in Eco RV digested pBluescriptSK to which single 3' dATP overhangs were added, using Taq polymerase.
  • This construct was named pSTIl.
  • the orientation of the insert with respect of the multiple cloning site in pSTIl was determined by restriction digestion.
  • the intron 1 sequence was excised from pSTIl on a 5' Smal -3'Hind l fragment, the recessed 3' terminus generated at the Hindlll end was repaired using Klenow, and the resulting blunt ended fragment was ligated with EcoRV digested pMAD to make pMADl .
  • the correct orientation of the intron fragment with respect to the remainder of the BLG sequences was determined by DNA sequencmg. This step effectively moves the EcoRV site to the 3' end of the BLG intron.
  • Plasmid pCORP2 (see WO-A-9211358)
  • the cDNA was excised as a Kpnl fragment, the 3' overhangs made flush by treatment with T4 DNA polymerase, the fragment gel purified and blunt-end cloned into the EcoRV site of pMAD. Orientation was determined by restriction digest and confirmed by DNA sequencing.
  • This construct is plasmid pCORP2 and contains the human protein C cDNA under the transcriptional control of the ⁇ -lactoglobulin gene 5' and 3' flanking sequences. There are no introns. Plasmid pCORP5
  • the 1450bp protein C cDNA fragment used in the construction of pCORP2 was placed into pMADl ⁇ to make pCORP5.
  • PC962 (Foster et al ., ibid), into pMAD, the plasmid was modified to incorporate EcoRV sites at the extremities of the protein C cDNA insert.
  • a 769 bp Sstll-Pstl fragment encompassing the 3' end of PC962 was cloned between the Sstll and Pstl sites of pBluescript II SK+ (Stratagene, La Jolla, CA). The fragment was excised with Sstll/ EcoRV and purified.
  • the 5' portion of PC962 was modified by PCR.
  • the sense oligonucleotide primer for this reaction covered the 5' ATG region of the cDNA and provided an EcoRV site upstream of this in the product.
  • the antisense oligonucleotide primer covered the Sstll site used to generate the Sstll - EcoRV fragment.
  • the resulting PCR product was digested with EcoRV and Sstll and ligated with the Sstll-EcoRV 3' fragment and EcoRV digested pMAD.
  • the resulting plasmid, designated pCORP9 effectively contained the PC962 cDNA flanked by EcoRV sites in an intronless fusion driven by the ⁇ - lactoglobulin promoter.
  • This genomic construct designated GPClO-1, changed the sequence 16 base pairs upstream of the ATG from the native protein C sequence to the ⁇ -lactoglobulin sequence and introduced mutations in the propeptide cleavage site located in exon 2, and the two-chain cleavage site located in exon 6, as described below.
  • the construct was assembled using four fragments designated A, B, C and D and encompassed the protein C gene sequence from the ATG to a BamHl site in exon VIII, immediately upstream of the stop codon.
  • the fragments were generated from a human genomic library in Charon 4A phage which was screened with a radiolabeled cDNA probe for human protein C.
  • oligonucleotides ZC6303 (5'-ATT TGC GGC CGC CTG CAG CCA TGT GGC AGC TCA CAA GCC TCC TGC-3') and ZC6337 (5'-CAG GAA GGA GTT GGC GCG CTT GCG CCG TTG CAG CAC CTG CTG GGC-3", a D ⁇ A fragment was generated by polymerase chain reaction (PCR).
  • Oligonucleotide ZC6303 changed the sequence 16 based pairs 5' to the ATG sequence from the native protein C sequence to the equivalent sequence from the ⁇ -lactoglobulin gene and introduced a NotI site.
  • Oligonucleotide ZC6337 changed the propetide cleavage site from Arg-Ile-Arg- Lys-Arg to Gln-Arg-Arg-Lys-Arg.
  • the resulting PCR generated fragment was digested with NotI and BssHlL, and a 1402 base pair fragment was gel purified and designated Al.
  • a second fragment was prepared using a ⁇ gtll clone of PC ⁇ l as a template with oligonucleotides ZC6306 (5' -CTT CTT CCT GAA TTC TGT TTC TTG C-3') and ZC6338 (5' -CGG ATC CGC AAG CGC GCC AAC TCC TTC C-3') in a polymerase chain reaction.
  • the resulting D ⁇ A fragment, designated A3 was digested with 5wHII and EcoRI and gel purified, resulting in a 296 base pair fragment.
  • Fragments Al and A3 were ligated into the Bluescript II KS + phagemid vector (Stratagene. La Jolla, CA).
  • the resulting plasmid, designated GPC 2-2 was digested with NotI and EcoRI, gel purified and the Notl-EcoRI D ⁇ A fragment was designated Fragment A.
  • pCR 2-14 is a subclone which contains an EcoRI to EcoRI DNA fragment of PC ⁇ 8 (Foster et al ., 1985, ibid.).
  • the plasmid was digested with EcoRI and SstI and gel purified. The resulting figment was designated Fragment B.
  • Plasmid pCR 2-14 was used as a template DNA with oligonucleotides ZC6373 (5' -AAA GTA AAA AAA GAT CTA AAA ATT TAA C-3') and ZC6305 (5' - GTG TCT CGT TTT CTT AAG TGA CTG CGC-3'), which introduced za AfTO. site and the RRKR mutation of the native (KR) two-chain cleavage site, in a polymerase chain reaction.
  • the resulting PCR-generated fragment was digested with Bgl ⁇ l and Afl ⁇ and gel purified, resulting in a 1441 base pair fragment, designated ⁇ l.
  • Fragment ⁇ I was used in a ligation reaction with oligonucleotides ZC6302 (5' -TTA AGA AGA AAA CGA GAC ACA GAA GAC CAA GAA GAC CAA GTA GAT CCG C-3') and ZC6304 (5' -GGA
  • a fourth fragment was generated by digestion of a genomic subclone (pHCB7-l) of PC ⁇ 8.
  • pHCB7-l contained a Bglll to Bglll fragment that encompassed exons VI through VIII.
  • pHCB7-l was digested with Sstll and Bam ⁇ l and a 2702 base pair fragment was gel purified. The fragment was designated Fragment D.
  • a five-part ligation reaction was prepared using NotI and Bam ⁇ l digested and linearized Bluescript II KS+ phagemid vector (Stratagene) with Fragment A (5' NotI to 3' EcoRI) that contained exons I and II, Fragment B (5' EcoRI to 3 'SstI) that contained exons III, IV and V, Fragment C (5' SstI to 3' Sstll) that contained the 5' portion of exon VI and Fragment D (5' Sstll to BamHl) that contained the remaining 3' portion of exon VI and exons VII and VIII.
  • Fragment A 5' NotI to 3' EcoRI
  • Fragment B (5' EcoRI to 3 'SstI) that contained exons III, IV and V
  • Fragment C (5' SstI to 3' Sstll) that contained the 5' portion of exon VI
  • Fragment D (5' Sstll to BamHl) that contained the remaining 3
  • the resulting D ⁇ A was 8950 base pairs and designated GPC 10-1.
  • GPClO-1 was originally generated with BLG sequences and a NotI site upstream of the ATG initiator codon and modifications to both cleavage sites.
  • pPC12/BS was generated using PCR amplification of a 1 kb ⁇ Otl- Sc ⁇ l fragment that covered the 5' region of the protein C gene and contained the wild-type ATG codon environment. This introduced an EcoRV site immediately s downstream of the NotI site, adjacent to the ATG codon, and a BamRl site was incorporated 3' of the Seal site to facilitate cloning.
  • the PCR product was cloned into Notl/BamUl digested Bluescript II KS+ phagemid vector (Strategene).
  • the Notl-EcoRV-Scal fragment present in pPC12/BS was excised, purified and ligated to GPClO-1, which had been o linearized with NotI and partially digested with Seal (the pUC amplillicin gene has an internal Seal site).
  • the resulting clone was designated GPC 10-2 and possesses an EcoRV site immedately upstream of the ATG initiator codon.
  • GPC 10-1 and GPC 10-2 both terminated at the final 5 ⁇ mHI site in exon VIII of the protein C gene.
  • oligonucleotides were synthesized with flanking 5 ⁇ mHI (5') and Bglll (3') restriction sites. Following annealing of the oligonucleotides, the product was cloned into 5 ⁇ mHI digested pBST + to generate plasmid pPC3 ' .
  • pBST+ is a derivative of pBS (Stratagene) with a new polylinker. The addition of the polylinker added Bglll, Xhol, Narl and CZ ⁇ l restriction sites from the o vector polylinker downstream of the destroyed Bglll site of the oligonucleotide construct.
  • Notl-BamHl fragment of GPC 10-1 was subcloned into NotI/ BamRl digested pPC3' to add 3' coding sequences of protein C, the TAG termination codon followed by Bglll-Xhol-Narl-Clal.
  • the 3' region of the protein C gene beginning with the EcoRV site in intron V was excised from this plasmid on an EcoRV-C/ ⁇ l fragment.
  • a further genomic construct was generated from pCORP13 which contained only the modified two-chain cleavage site. This was achived using PCR amplification to modify two fragments which result in restoration of the coding capabilitiy of exon 2 from the mutant Gln-Arg-Arg-Lys-Arg to the wild-type Arg-Ile-Arg-Lys- Arg. pCORP13 was used as template for these reaction. The first fragment was
  • the sense primer was designed to add a HmdIII site 5' to the EcoRV site proximal to the ATG initiation codon.
  • the antisense primer was designed to restore the wild-type sequences in exon 2, which included a restored 5 ⁇ m ⁇ I site.
  • a 7.5kb Xhol fragment from pCORP13 was ligated to Xhol digested pGEMPC1.5 to generate a complete protein C genomic sequence covering exons 1-8 with a wild-type propeptide cleavage site and a modified two-chain cleavage site.
  • the plasmid was designated pGEMPC14.
  • the sequence was excised from pGEMPC14 as a Hindl ⁇ l/Sall fragment. The DNA termini was repaired using a Klenow reaction and the fragment was blunt-end ligated into EcoRV digested pMAD ⁇ to generate pCORP14.
  • the modified protein C cDNA (PC962) was excited from the plasmid pCORP9 (see above) as an EcoRV fragment and ligated with EcoRV pMAD ⁇ .
  • the resulting construct has been named pCORPl ⁇ .
  • Bovine ⁇ -Casein intron 1 (BBCI 1; BOVCASl (5'-AGG CCT ATT CAG CTC CTC CTT CAC TTC TT-3') and BOVCAS2 (5'-GAT ATC GGC TCT CAA TTC CTG GGA ATG GG-3') approximately 2 Kbp) was PCR amplified from dairy cow DNA.
  • the 5' primer incorporates a Stul site and the 3' primer incorporates an EcoRV site.
  • the purified 2 Kb fragment was cloned into the pG ⁇ M-T vector (Promega) to give construct p ⁇ ' 10.
  • Plasmid pCASMAD ⁇ pMAD ⁇ was modified by inserting a linker, containing Spe l/Not I/Sac II sites, into the EcoRV site. Both orientations of the linker were obtained and thus two new cloning vectors were obtained. These were called pMAD ⁇ /STOPS (5'
  • the modified protein C cDNA (PC962) was excised from the plasmid pCORP9 (see above) as an EcoRV fragment and ligated with EcoRV digested pCASMAD ⁇ . This places the AUG translation start downstream with respect to the ⁇ -casein intron sequence.
  • the resulting construct was named pCOR169.
  • Two primers (Sequences ACTPl 5'-AGG CCT AGT GCC TGC CAC CAG CGC CAG CC-3' ACTP2 5' -GAT ATC CCT GGC AC A GCT TTG TGT GGT TC-3') complementary to the opposing strands of the 3' end of the first exon and the 5' end of the second exon of the murine cardiac actin gene respectively, were used in a PCR reaction to amplify a 0.8 Kb fragment encompassing the intervening sequences from a template of mouse genomic DNA.
  • the two primers introduced a 5' SnaBl and a 3' EcoRV restriction site at the ends of the PCR product. This DNA fragment was cloned in pG ⁇ M-T to give a construct which was named pG ⁇ M-AI. DNA sequence analysis confirmed that the sequence of the amplified product beyond the primers matched that published for the murine beta actin gene.
  • actin intron 1 sequence was excised from pG ⁇ M-AI on a 5' Sn ⁇ BI- 3 'EcoRV fragment which was then ligated with ⁇ coRV digested pMAD ⁇ to give vector pACTMAD ⁇ .
  • This cloning step effectively moves the EcoRV site from the 3' end of the BLG promoter downstream to the 3 ' end of the actin gene intron segment.
  • the modified protein C cDNA (PC962) was excised from the plasmid pCORP9 as an EcoRV fragment and ligated with EcoRV digested pACTMAD ⁇ . This places the AUG translation start downstream with respect to the actin intron sequence.
  • the resulting construct was named pCOR170.
  • PCR primers were designed to amplify the region of the ovine ⁇ -casein gene from exon 1 to exon 2 (BOB1: 5'-CGG GAT CCG TCG ACC ATT CAG CTT CTC CTT CAC TTC TTC TC-3'; BOB2: 5'-CGG GAT CCG GGT CCC TAC GTA GGC TCT CGA TTC CTG TGA ATG GGA-3').
  • the size of this product is 2.1Kbp and has, engineered into it, the sites BamRl/Sall at the 5' end and BamRl/ Ppuml/ SnaBl at the 3' end.
  • a Xhol linker had been cloned into the EcoRV site of pMAD ⁇ and the modified plasmid named pMADX.
  • the ovine BLG promoter from pMAD6X was cloned into the Sail site of pBOB ⁇ prom as a SaWXhol fragment giving rise to pBOB.
  • the modified protein C cDNA (PC962) was excised from the plasmid pCORP9 as an EcoRV fragment and ligated with EcoRV digested pBOB. This places the AUG translation start downstream with respect to the actin intron sequence.
  • the resulting construct was named pCORB71.
  • Purified human Protein C stored at 50 ⁇ g/ml in Phosphate Buffered Saline (PBS)/1 % bovine Serum Albumin (BSA) at 20 ° C. Dilute to 500ng/ml in blocking buffer for use. Standard curve range of ELISA is 3.9-125ng/ml.
  • Dako Rabbit Anti-human Protein C antibody Peroxidase conjugate Dilute 1/5000 in blocking buffer.
  • Reference human plasma is used as a protective control at 1/40 dilution.
  • Transgenic mice were prepared as in Example 1. Analysis of AAT in the milk of transgenic mice was according to standard procedures, for example as described in Wright, G. , Carver, A., Cottom, D., Reeves, D. , Scott, A. , Simons, J.P., Wilmut, I., Garner, I., and Colman A., 1991. High level expression of active human ⁇ l antitrypsin in the milk of transgemc sheep. Bio/Technology 9: 830-834.
  • Constructs pMAD ⁇ and pCASMAD ⁇ were prepared incorporating DNA encoding an antibody binding fragment to give constructs pMAD6-AB and pCASMAD ⁇ - AB.
  • the constructs were used to obtain transgenic mice according to Example 1. Expression of the antibody fragment was determined by standard protocols.
  • Const r ucts pMAD ⁇ and pCASMAD ⁇ were prepared incorporating DNA encoding IgG to give constructs pMAD6-IgG and pCASMAD6-IgG.
  • the constructs were used to obtain transgenic mice according to Example 1. Expression of IgG in the mice milk was determined by standard ELISA protocol.
  • pMAD ⁇ and pCASMAD ⁇ were prepared incorporating DNA encoding a soluble adhesion molecule (SAM) to give constructs pMAD ⁇ -SAM and pCASMAD ⁇ -SAM.
  • SAM soluble adhesion molecule
  • the expression level range ( ⁇ g/ml) in pCASMAD ⁇ -SAM transgenic mice was up to 500.
  • the maximum level detected in the pMAD ⁇ -SAM transgenic mice was 80.
  • the CASMAD6 vector was used.
  • Collagen cDNA human truncated pro-collagen ⁇ 2(l) homotrimer
  • This was coinjected with two transgenes expressing ⁇ and ⁇ subunits of prolyl 4- hydro yiase, an enzyme for the post-translational modification of procollagen.
  • Transgenic animals were obtained as in Example 1. Determination of collagen expression in mouse milk was as described according to standard protocols and described in WO97/08311.

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Abstract

La présente invention concerne un produit de recombinaison de l'expression d'un acide nucléique comprenant: (a) un agent promoteur, (b) un intron dont la position naturelle est entre la séquence non traduite 5' d'un gène à partir duquel il est obtenu; (c) une séquence codante, et enfin, (d) une séquence flanquante 3' dans laquelle l'intron (b) est obtenu à partir d'un autre gène que celui utilisé pour obtenir soit l'agent promoteur (a), soit la séquence codant les protéines (c), ainsi que des méthodes, des vecteurs, des hôtes et des utilisations impliquant un produit de recombinaison, permettant d'obtenir notamment une augmentation du taux d'expression de la séquence codante.
EP98935143A 1997-07-17 1998-07-17 Expression heterologue de proteines a l'aide d'un vecteur "sauve" comprenant un intron Withdrawn EP0996715A1 (fr)

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US10610606B2 (en) 2018-02-01 2020-04-07 Homology Medicines, Inc. Adeno-associated virus compositions for PAH gene transfer and methods of use thereof
EP3755795A4 (fr) 2018-02-19 2022-07-20 Homology Medicines, Inc. Compositions de virus adéno-associé destinées à restaurer la fonction du gène f8 et leurs procédés d'utilisation
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US5298422A (en) * 1991-11-06 1994-03-29 Baylor College Of Medicine Myogenic vector systems
CA2134675A1 (fr) * 1992-04-30 1993-11-11 Dennis R. Roop Mise au point d'un vecteur pour cibler l'expression genetique dans l'epiderme d'animaux transgeniques
JP3670003B2 (ja) * 1992-06-15 2005-07-13 ファーミング ビー．ブイ． ウシ種による組み換えポリペプチドの製造及びトランスジェニック法
US5639940A (en) * 1994-03-03 1997-06-17 Pharmaceutical Proteins Ltd. Production of fibrinogen in transgenic animals
IL115873A0 (en) * 1995-11-03 1996-01-31 Peri Dev Applic 1985 Ltd Transgenic protein production
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