Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Scleroproteins – e.g. – fibroin – elastin – silk – etc.
Reexamination Certificate
1999-08-17
2003-09-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Scleroproteins, e.g., fibroin, elastin, silk, etc.
Reexamination Certificate
active
06617431
ABSTRACT:
This application is a 371 of PCT/FR97/02331, filed Dec. 17, 1977, and claims priority to French application 96/16224, Dec. 17, 1996.
The present invention relates to the production by plants of recombinant collagens, in particular Type 1 homocatenary collagen [&agr;I (I)
3
] and other polypeptide derivatives, and their uses.
Patent WO 9603051 is known to the prior art which concerns the production of collagen in the milk of transgenic animals.
Collagen is an extracellular fibrous animal protein, widely found in animal tissues (recently detected in some mushrooms also). Some organs contain high quantities thereof: skin (at the dermis), tendons, bones. It is in fact a polymer whose remarkable properties are due both to the triple coil characteristics of some domains of its molecule and to the regularity of its supramolecular assemblies. It is involved in the organisation of the extracellular matrix grouping together twenty or so different molecules, called “types” that are identified by Roman figures (currently from I to XIX). The characteristic triple coil domain is formed by the coiling of three peptides, or &agr; chains, arranged in a left wound coil and derived from a single conformation, the &agr; coil of collagen. This specific conformation results from the repetition of a triplet of amino acids, Gly-X-Y in which X is frequently represented by proline and Y by hydroxyproline. These amino acids give stability to this type of coil. In a collagen molecule, the three &agr; chains (identified by an index in Arabic figures) arranged in a right wound supercoil may be identical (&agr;1 (or alpha1)), of two types (&agr;1 and a2) or all different (&agr;1, &agr;2, &agr;3). Collagen molecules therefore comprise helical domains (or collagen domains) and non-helical domains. They associate to form homo or heterotype polymers. Thus the collagen fibrils which form the essential part of the dermis are mostly made up of type I collagen [&agr;1 (I)
2
&agr;2 (I)] associated with collagens of type III [&agr;1 (III)
3
), of type V [&agr;1 (V)
2
&agr;2 (V)] and covered by collagens of type XII (&agr;1 (XII)
3
) and/or XIV [&agr;1 (XIV)
3
]. Variants may exist: for example a homocatenary collagen of type 1 [&agr;1 (I)
3
] is found in embryo tissues. During the biosynthesis of collagen some prolyl and lysil residues are hydroxylated, an addition of galactose possibly supplemented with a glucose may be made on some hydroxyl residues and conventional N and O glycosylations may occur on the non-helical domains. The recognition of the three chains forming a molecule and the start of their assembly are under the control of the C-terminal end (C-propeptide). Type I collagen undergoes enzymatic cutting of its non-helical ends subsequent to the previously cleaved peptide signal, the N and C-terminal propeptides are excised during maturation of the collagen leaving short, non-helical terminal extensions (telopeptides). It is these cleaved molecules which group together in arranged polymers (collagen fibrils) and which during the course of time undergo cross-linking via the hydroxylysyl residues of a molecule and the telopeptides of an adjacent molecule. The mechanical and biological properties of collagen have long been put to use; when cross-linked in irreversible manner (tanning process) it gives leather; when denatured by heating it gives rise to gelatin and glues. But it was only in the last decade that collagen truly provided biomaterials for pharmaceutical use (haemostatic compresses, sponges, dressings in particular healing dressings), medical use (prostheses such as cardiac valves, tendons and ligaments, skin substitutes, filling agents), odontological use (gum implants) and cosmetic use (additive, microcontainer for perfumed substances).
Later improved knowledge of this protein and of purification methods have led to the preparation of bovine and human placenta collagens in pre-defined form: gel, sponge, powder, suture and microsphere for example. Engineering of the extracellular matrices is also applied to the production of organoids containing transfected cells for gene therapy applications for example. The collagen mainly used is type I (generally associated with type III) for reasons of abundant availability and low purification costs, and the main sources have been bovine (skins unfit for tanning) ovine (hide and intestine) and human (placenta). This last source was exclusively reserved for pharmaceutical or medical applications.
Although the very useful mechanical and biological properties of collagen are clearly recognised, the use of this protein is questioned owing to the possible risks of contamination by non-conventional infectious agents. While the risks raised by bacterial or viral contamination can be fully controlled, this is not the case for those associated with agents of prion type. These infectious agents which appear to have a protein nature take part in the development of degenerative animal encephalopathy (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeld-Jacob disease, Gerstmann-Straussler syndrome, kuru). The long time of onset for their possible expression means that formal controls are difficult to conduct. These risks have already virtually frozen all marketing of human collagen, and the regulations laid down for the animal collagens concerned complicate purification processes and increase their cost.
Faced with these difficulties and radical deterioration in the image of mammalian collagen, one solution is the production of recombinant collagen which could be easily purified in a system that is not likely to give rise to pathogenic risks for man and whose industrial cost is not prohibitive. The inventors have therefore discovered and developed a production of collagen in plant species. For example we have been able to produce a human collagen of type I. Its molecule comprises a long, unbroken triple coil and is sparingly immunogenic after purification. The inventors have for example caused expression of the &agr;1 (I) chain in order to obtain &agr;1 (I)
3
homocatenary molecules similar to those which exist in some tissues, especially embryo tissues.
Animal cells are, in theory, more adapted to the expression of mammalian genes. Their use however raises problems of protein maturation. The enzymatic equipment which carries out post-translational maturation differs from one tissue, organ or species to another. For example, it has been reported that post-translational maturation of a plasma protein may differ according to whether it is obtained from human blood or produced by a recombinant cell such as Chinese hamster ovary cells or in the milk of a transgenic animal. Moreover, the low levels of expression obtained with mammalian cells involve large volumes of in vitro cultures at high cost. With the production of recombinant proteins in the milk of transgenic animals (mice, ewes and cows) it is possible to reduce production costs and to overcome problems of expression level. However problems remain in respect of ethics and viral and subviral contamination (prions).
For these reasons, the transgenesis of mammalian genes in a plant cell could offer a pathway for the production in great quantities of new recombinant proteins at reduced production cost and with no risk of viral or subviral contamination. In 1983, several laboratories discovered that it is possible to transfer a heterologous gene into the genome of a plant cell, and to regenerate transgenic plants from these genetically modified cells. All the plants cells then have the genetically modified character transmitted to their descent by sexual fertilisation.
Through this work several teams have focused their attention on the production of recombinant mammalian proteins in plant cells or in transgenic plants (Barta et al., 1986; Marx et al., 1982). One of the first truly significant results in this area was the production of antibodies in transgenic tobacco plants. To express a heterologous protein in the grain, w
Bournat Philippe
Comte Jeanne
Exposito Jean-Yves
Garrone Robert
Gruber Veronique
Carlson Karen Cochrane
Feit Irving N.
Hoffmann & Baron , LLP
Meristem Therapeutics S.A.
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