TRAINING ANIMALS OUE PRODUCE OLIGOSACARIDOS AND GLUCOCONJUGADOSTechnical Field The present invention relates to the in vivo production of secondary gene products of heterologous glycosyltransferases. These glycosyltransferases are expressed in the non-human mammary tissue that leads to the production of heterologous oligosaccharides, as well as in different glucoco played which these oligosaccharides carry in the milk of the transgenic animal.
Background Art Carbohydrates are an important class of biological compounds. The term "saccharides" encompasses a wide variety of carbohydrate-containing compounds. These include polysaccharides, oligosaccharides, glycoproteins, and glycosides with aglucons that are not carbohydrate. Biological protein macromolecules or lipids containing oligosaccharide moieties are collectively known as glucoconjugates. The carbohydrate moiety provides many biological functions. In cells, carbohydrates function as structural components in which they regulate viscosity, stored energy, or are key components of the cellular surface.The complex oligosaccharide chains of different glycoconjugates (especially glycoproteins and glycolipids) mediate modulate a variety of biological processes For a general review of the bioactivity of carbohydrates, see: 5 (a) Biology of Carbohydrates, Volume 2, Ginsburg and collaborators, Wiley, NY (1984), and (b) PW Macher et al. , Annual Review of Biochemistry, Volume 57, page 785, (1988) Among other things, it is known that: (a) carbohydrate structures are important for stability, activity, localization, and degradation of glycoproteins; ) certain oligosaccharide structures activate the secretion by the plants of antimicrobial substances, (c) - the glucoconjugates are frequently found on different surfaces. it is cells, and they are important, among other things, for cellular interactions with the surroundings, since they function as receptors or regulators when they bind to the cell surfaces of, for example, peptides, hormones, toxins, viruses, bacteria, and during the cell-cell interaction; (d) the carbohydrate structures are anénénico determinants (for example, blood group antigens); (e) carbohydrates function as cell differentiation antigens during normal tissue development;(f) carbohydrates are important in oncogenesis, since it has been discovered that specific oligosaccharides are antigenic determinants associated with cancer; and 5 (g) oligosaccharides are important for sperm / egg interaction and for fertilization. Isolated oligosaccharides are known which inhibit the agglutination of the uropathogenic coliform bacteria with erythrocytes. It has been shown that other oligosaccharides possessIt has a potent antrogenic activity, increasing the levels of plasminogen activator. This same biological activity has been used, through the covalent union of these oligosaccharides with the surface of medical instruments, to produce surfaces that have effects against thecoagulation. These surfaces are useful in the collection, processing, storage, and use of blood. Still other oligosaccharides have found utility as gram-positive antibiotics and disinfectants. In addition, certain free oligosaccharides have been used in diagnosis and inthe identification of specific bacteria. A considerable future market is foreseen for fine chemicals based on biologically active carbohydrates. Universities and Industry are currently working intensively on the development of usesadditional biologically active oligosaccharides.
'These efforts include, but are not limited to: (a) the development of novel diagnostic and blood typing reagents; (b) the development of a novel type of therapy 5 as an alternative to antibiotics, based on the prevention of adhesion of bacteria and viruses to cell surfaces by means of specific oligosaccharides; and (c) the use of oligosaccharides to stimulate j.? growth of plants and provide protection against certain plant pathogens. A large number of oligosaccharide structures have been identified and characterized. The smallest block or unit of construction of an oligosaccharide is amonosaccharide. The main monosaccharides found in mammalian glycoconjugates are: D-glucose (Glc), D-galactose (Gal), D-mannose (Man), L-fucose (Fue), N-acetyl-D-galactosamine (GalNAc ), N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-neuraminic acid (NeuAc). The abbreviations between 0 parentheses are the standard terminology for monosaccharides according to the recommendations of the International Union of Physics, Chemistry, and Biology Council; Journal Biological Chemistry, volume 257, pages 3347-3354, (1982). These abbreviations will be used later in the present. TODespite the relatively small number of fundamental building blocks, the number of possible combinations is very large because both the anomeric configuration (alpha- or β-glucosidic bond) and the position of the O-glycosidic bond can be varied. Accordingly, a large variety of oligosaccharide structures can exist. It is known that the bioactivity of the oligosaccharides is specific in terms of both the conformation of the sugar and its composition. The individual monosaccharides provide an element of n) bioactivity, but also contribute to the overall conformation of the oligosaccharide, thereby providing another level of specificity and bioactivity. It is the diversity of glucoconjugates and oligosaccharides that produces theBiological specificity of certain structures ofoligosaccharides. However, this diversity also causes a particular problem for the practical utility of these compounds. Glucoconjugates are typically potent immunogens, and biospecificity, as noted above, is determined not only by the particular sequence of themonosaccharide, but also by the nature of the glycosidic bond. Consequently, it is often not possible to use the oligosaccharide structures found in one species of animal, in another species. Similar restrictions on use can also be applied, on aindividual. For example, since it is known that certain blood group antigens are formed from specific oligosaccharides, it is necessary to be especially careful when conjugating a blood group oligosaccharide with a protein, and then that glycoprotein is therapeutically used. Careful consideration should be given to concerns about potential immunogenicity. Despite these potential difficulties, it is well accepted that there is a need to produce large quantities of human oligosaccharides and / or glycoconjugates that_ .. < ) carry those oligosaccharides. Numerous methods have been contemplated as adequate elements to achieve this goal. These methods include the synthesis of the oligosaccharides by conventional organic chemistry, or by the use of in vitro enzymes. The immobilized enzymes arecurrently in preferred mode for large-scale production of oligosaccharides in vitro. This is due to a high regio- and stereo-selectivity of the enzyme, as well as a high catalytic efficiency under light reaction conditions. The literature describes a synthesis number of oligosaccharidescatalyzed above. For example, see the scientific review articles by Y. Ichikawa et al. "Enzyme-catalyzed Oligosaccharide Synthesis" in Analytical Biochemistry, volume 202, pages 215-238, (1992), and K.G. I. Nillson, "Enzymatic Synthesis of Oligosaccharides", Trends inBiotechnology, volume 6, pages 256-264 (1988). Both hydrolases and transferases have been used to facilitate the production of oligosaccharides. The glycosidase enzymes, a subclass of the hydrolases, are especially useful in the synthesis of oligosaccharides by an inversion process of the degradation cycle. However, in general, the enzymatic synthesis of oligosaccharides is based on the biosynthetic path. Although the biosynthetic pathway of oligosaccharide synthesis is regulated primarily by the gene encoding the production of each glycosyltransferase, the-_. < } Real oligosaccharide structures are determined by the specificity of the substrate and the acceptor of the individual glucosyltransferases. Oligosaccharides are synthesized by transferring the monosaccharides from sugar nucleotide donors to moleculesacceptors. These acceptor molecules can be other free oligosaccharides, monosaccharides, or oligosaccharides linked with proteins or lipids. The enzymatic synthesis of oligosaccharides has been conducted in general only on a small scale, due tothat enzymes, particularly glycosyltransferases from natural sources, have been difficult to isolate. Also, sugar nucleotide donors are very difficult to obtain from natural sources, and are very expensive when derived from organic chemistry synthesis. Nevertheless,More recently, a recycle and reuse strategy has been developed to synthesize large quantities of oligosaccharides. U.S. Patent No. 5,180,674, incorporated herein by reference, discloses a novel affinity chromatography method, wherein the reaction products are recycled in a repeating manner onto the glycosyltransferases bound to the matrix or the reein. In addition, recent progress in gene cloning techniques has made available several glycosyltransferases in sufficient quality and quantity to make the oligosaccharide enzymatic synthesis more practical. The literature is replete with descriptions of the recombinant or transgenic expression of a heterologous glycosyltransferase. However, before continuing with a discussion of the literature, it is necessary to clarify the meaning of different terms as used herein and in the claims: (a) Host, host cell, or host animal: Estoe> terms are used to refer to the cell or mammal that is responsible for the biosynthesis of the biological material. (b) Homologous: This word means that the entity so characterized is normally present or is produced by the host. (c) Heterologous: This word means that the entity so characterized is not normally present or produced by the host. In other words, the entity so characterized is foreign to the host. (d) Catalytic activity: This term is used to refer to the inherent property of certain biological compounds to facilitate chemical change in othersubstances. (e) Catalytic Entity: This term is used to refer to biological compounds that inherently possess a catalytic activity, which results in the production of new, different, or altered compounds. The a .., examples are enzymes and antibodies. An enzyme is a biochemical catalyst of a specific biochemical reaction. An enzymatic product is formed as a result of the catalytic activity of the enzyme on a substrate material. 15 (f) Genome: This word is used to refer to the complete genetic material found in the host. This material is configured in chromosomes. (g) Gene: This word refers to a functional portion of the genome, which is responsible for the biosynthesis ofa specific biological entity. (h) Insertion: This word is used to refer to the process by which a portion of the heterologous DNA or a heterologous gene is introduced into the genome of a host. The DNA that is inserted is referred to as "insert".(i) Tranegen: This refers to heterologous genetic material that is transferred by inserting from the genome of one species of animal to the genome of another species of animal. In a simpler way, a transgene is a gene that is heterologous to the host. The transgene codes for a specific biological material. (j) Transgenic mammal or transgenic host: These terms are used to refer to a mammal or cell that has had a transgene inserted into its genome. As a result of this insertion, the transgenic host produces a heterologous biological material that would normally not be synthesized. The heterologous entities are present or are produced by a transgenic host as a result of the insertion of foreign genetic material into the genome of the host cell. (k) Primary gene product: This refers to a biological entity that is formed directly as a result of the transcription and translation of a homologous or heterologous gene. Examples include proteins, antibodies, enzymes, and the like. (1) Secondary gene product: This refers to a product that is formed as a result of the biological activity of a primary gene product. An example is an oligoeaccharide which is formed as a result of the catalytic activity of an enzyme. (m) Biological products: This term is used to refer to products produced or synthesized by a transgenic host as a result of the insertion of a transgene into the mammalian genome. More specifically, the term means biological products that are secondary gene products. An example, as described below, is that of human oligosaccharides produced by transgenic cows. The human oligosaccharides are produced as a result of the catalytic activity of human glucosyltransferase. As discovered herein, when the gene encoding human glucosyltransferase is inserted into the murine genome, the tranexgenic mouse produces a heterologous human glycosyltransferase as the primary gene product. Human glycosyltransferase, using homologous substrate materials, produces oligosaccharides and glycosylated proteins. The oligosaccharide, formed as a result of the enzymatic activity of the primary gene product, is also called a secondary gene product. Glucoconjugates are another example of the class of compounds referred to herein and in the claims as "biological products". (n) Product: This word is used to refer to the secondary gene products of the present invention, and is used as an alternative for "biological product". (o) Humanized milk: This refers to milk obtained from a non-human mammal that, through altering the host's genome, is caused to produce milk that closely resembles human milk. An example of humanized milk is cow's milk that contains products found in human milk, but which are not normally found in cow's milk. Human oligosaccharides are produced in cow's milk as a result of the insertion of the gene encoding human glycosyltraneferases in the bovine genome. Humanized milk also contains glycosylated proteins with- or human oligosaccharides. As noted above, there is a considerable body of literature describing the recombinant or transgenic expression of heterologous glycosyltransferases. However, the literature does not describe or suggest anyIn another way, the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. The examples of the literature are: 1) United States Patent No. 5,032,519 to Paulson, describes a method for designinggenetically cells, in such a way that they produce soluble Golgi processing enzymes and secretablee instead of the enzymes bound with membxan that occur naturally. 2) United States Patent Number 5,047,335, by Paulson, describes the alteration bygenetic engineering of the "Chinese" Hamster Ovary Cell (CHO) in such a way that the Chinese Hamster ovary cells produce a sialyltransferase. 3) International Patent Application Number PCT / US91 / 08216 describes a transgene capable of producing proteinsheterologous recombinants in the milk of transgenic bovine species. This published patent application describes a method for obtaining the primary gene product only. This published patent application also describes methods for producing and using the altered milk obtained from a. J these transgenic animals. 4) International Patent Application Number PCT / US91 / 05917 describes methods for producing intracellular DNA segments by homologous recombination of smaller overlapping DNA fragments. ThisThe published patent application describes a method for obtaining the primary gene product only. 5) International Patent Application Number PCT / GB87 / 00458 describes methods for producing a peptide, involving this method incorporating a DNA sequence thatcodes for the peptide in the gene of a mammal encoding a whey protein, such that the DNA sequence ee is expressed in the mammary gland of the adult female mammal. This published patent application describes a method for obtaining only the gene productThe primary protein, the peptide, in the milk of the transgenic mammal, and also describes methods for producing and using the altered milk obtained from these transgenic animals. 6) International Patent Application Number PCT / GB89 / 01343 describes methods for producing materialsproteinaceous in transgenic animals that have genetic constructs integrated in their genomes. The construct comprises a 5 'flanking sequence from a mammalian milk protein gene, and DNA encoding a heterologous protein other than a milk protein. This published application pertains to a method for obtaining only the product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. 7) European Patent Application Number 88301112.4 describes methods for directing specific genestowards the mammary gland, which results in the efficient synthesis and secretion of biologically important molecules in the milk of these transgenic animals. This published patent application also describes methods for producing and using altered milk obtained fromThese transgenic animals, and a method for obtaining only the primary gene product in the milk of the transgenic mammal. 8) International Patent Application Number PCT / DK93 / 00024 describes methods for producing kappa-caseinhuman in the milk of transgenic animals. The genetic construct comprises a 5 'flanking sequence from a mammalian milk protein gene, such as casein or whey acid protein, and DNA encoding human kappa-caffeine. The DNA sequence contains at least one intron. This published patent application describes a method for obtaining only the primary gene product, the heterologous human kappa-casein, in the milk of the transgenic mammal. 9) International Patent Application Number? .u PCT / US87 / 02069 describes a method for producing mammals capable of expressing recombinant proteins in their milk. Each of these publications teaches, in one way or another, a means to obtain the transgene primary gene product, this gene product being the active protein or theenzyme that is encoded by the transgene. This literature describes transgenic elements to obtain glucosyltransferases in non-human milk. However, none of the aforementioned publications describes or suggests the use of tranegenic animals as a means toTo obtain a desired secondary gene product that is the product of the active enzyme. However, in a more particular way, none of the aforementioned publications describes or suggests, nor does it reveal in any other way, the use of transgenic human glycosyltransferases innon-human milk to produce human oligosaccharides or glucoconjugates that carry those oligosaccharides. These oligosaccharides, which are the product of the active glucosyltransferases, are referred to later in the. present as the "secondary gene product". Therefore, the different oligosaccharides found in human milk are formed as a direct result of the genetically regulated expression of certain specific glycosyltransferases. In this regard, oligosaccharides can appropriately be considered as "secondary gene products" since they are synthesized as a result of the biochemical activity of the primary gene product, the heterologous glycosyltransferase enzymes. Human milk contains a variety of oligosaccharides and proteins. Free, soluble oligosaccharides are not normally produced by animal cells and tissues, with the exception of highly differentiated lactation mammary glands. Oligosaccharides make up the bulk of the total carbohydrate content of human and bovine milk. The main constituent of carbohydrates in milk from mammals is the disaccharide lactose. Lactose is typically found at a concentration greater than 10 milligrams / milliliter, and is synthesized by the binding of galactose with glucose. This reaction is catalyzed by the enzyme, β-1,4-galactosyltransferase. The milk of most mammals, including cows, contains only very small amounts of a few additional oligosaccharides, in contrast, human milk contains substantial quantities of a number of additional soluble oligoeaccharides that are larger than lactose. All human oligosaccharides are synthesized by sequential addition of monosaccharides to lactose. The repugnant oligosaccharides found in human milk are shown in Table 1.
TABLE 1 OLIGOSACAIDS PRESENT IN HUMAN MILKStructure Common Name Concentration (mg /1. Gal-ß-l, 4-Glc Lactose 50,000 2. Fuc-al, 2-Gal-ß-l, 4-Glc 2-fucosyl-lactose 200 3. Gal-ß-1, 3 -GlcNac-ß -1, 3-Gal-ß-1, 4-Glc Lacto-N-tetraose 400 4. Gal-ß-l, 4-GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Lacto-N -neotetraose 60 5. Fuc-al, 2-Gal- / -1, -GlcNAc-ß-l, 3-Gallium ß-l, 4-Glc Lacto-N-fucopentaose I 200 6. Gal-ß-1,3 [Fuc-al, 4] GlcNAc-ß-l, 3-Gal-ß-l, 4-Glc Lacto-N-fucopentase II 20 7. Gal-ß-l, 4 [Fuc-al, 3] GlcNAc-ß -l, 3-Gal-ß-l, 4-Glc Lacto-N-fucopentase III 50 15 8. Fuc-al, 2-Gal-ß-l, 3 [Fuc-a-1,4] -GlcNAc- ß -l, 3-Gal-ß-l, 4-Glc Lacto-N-difucopentaose I 25 9. NeuAc-a-2, 6-Gal-al, 4-Glc 6-sialyl-lactose 25. NeuAc-a-2, 3-Gal-ß-l, 4-Glc 3-sialyl-lactose 1011. NeuAc-a-2, 3-Gal-ß-l, 3-R Sialyltetrasaccharide at 1012. Gal-ß-1,3- [NeuAc-a-2,6] GlcNAc-ß-l, 3-R Sialyltetrasaccharide b 3513. NeuAc-a-2,6-Gal-ß-l, 4-GlcNAc-ß-l, 3-R Sialyltetrasaccharide c 50 5 14. NeuAc-a-2, 3-Gal-ß-l, 3 [NeuAc-a -2, 6] - GlcNAc-ß-1,3-Gal-ß-l, 4-Glc Disialyltetrasaccharide 60 15. NeuAc-a-2, 3-Gal-ß-l, 3 [Fuc-a-1,4 ] - GlcNAc-ß-1, 3-Gal-ß-l, 4-Glc Sialyl-Lacto-N-fucopentaose 50-. 10 -a-: denotes an alpha-glucosidic bond. R: Gal-β-1, 4-Glc.
The oligosaccharides in human milk are present as a result of the activity of certain specific glycosyltransferases that are found in human breast tissue. For example, alpha-1,2-linked fucose residues in structures 2, 5, and 8, are produced by a single human fucosyltransferase, and characterize a phenotype known in the field of immunohematology, as "secretors". These individuals are therefore characterized, because they synthesize the substances of the human blood group in their salivary secretions and other mucosae secretions wherein the oligosaccharides are covalently linked to different proteins. The alpha-1, 4-linked fucose residues in structures 6, 8, and 15 are formed as a result of the enzymatic action of a different fucosyltransferase. These oligosaccharides represent a phenotype present in individuals characterized by having a "Lewis-poeitive" blood type. These individuals use ethe fucoeiltraneferasa to synthesize an oligosaccharide structure corresponding to an antigen of the human blood group. This oligosaccharide is also found in saliva, and in other mucous secretions, and is covalently bound to the lipids that are found on the red blood cell membrane of "Lewis-positive" individuals. Structure 5 is related to the H antigen of the ABO blood group; structure 6 is the blood group antigen "Lewis a"; structure 8 is the blood group antigen "Lewis b". At least 15 human milk proteins have been identified. Some of these proteins are generally recognized as glycosylated, that is, they are covalently linked to certain specific oligosaccharides. Particular oligosaccharides that are covalently linked to the protein are the same as, or similar to, the oligosaccharides described above, and their formation is the result of the genetically regulated normal expression of certain specific glycosyltransferase genes. The presence of a heterologous glycosyltraneferase would also affect the modification of proteins after the delivery. The heterologous protein glycosyltransferases are also appropriately known as "secondary gene products". Both homologous and heterologous proteins would be modified by glycosyltransferases in a manner different from that resulting from the activity of homologous glycosyltransferases. It has long been known that these oligosaccharides and glycosylated proteins promote the growth of desirable bacteria in the human intestinal tract. It is also believed that the oligosaccharides in human milk inhibit the binding of harmful microorganisms to the mouth and throat. These human oligosaccharides and specifically glycosylated proteins are absent from, or present in markedly different amounts in, bovine milk. In addition, as noted above, bovine milk contains predominantly lactose only. Human milk contains not only lactose, but also numerous other oligosaccharides. Also, the amino acid composition of the proteins of human milk is significantly different from the amino acid composition of the corresponding cow's milk proteins. As a consequence, infants fed infant formula comprising cow's milk may be more susceptible to intestinal disorders such as diarrhea or its proportions and amino acid levels in blood plasma may differ from breastfed infants. For the same reasons, the elderly, the immunocompromised, and critically ill patients also urgently need the availability of a nutritional product that is biochemically very similar to the composition of human milk. The complicated chemistry of proteins and oligosaccharides in human milk has made it extremely difficult to synthesize on a large scale. Before they can be incorporated into a commercial nutritional product, a practical method must be devised to obtain large quantities of proteins and oligosaccharides from human glycated milk. A potential solution for this problem is the use of transgenic animals, more particularly transgenic cows that express genes or cDNAs that encode enzymes that catalyze the formation of oligosaccharides and / or glycosylated proteins with the same human oligosaccharides. Tranegenic domestic animals that have milk, talee like rabbit, pig, sheep, goats, and cows, are proposed here as a means to produce milk containing human oligosaccharides and proteins glycosylated with human oligosaccharides. More specifically, transgenic cows are highly suitable for the production of oligosaccharides and recombinant proteins because a single cow can produce more than 10,000 liters of milk containing as much as 300 kilograms of protein (mainly casein) per year at a time. minimal cost. Therefore, the transgenic cow appears to be a less expensive production route than other methods of recombinant protein production, since the invention would not be required in fermentation facilities. Also, the mammary glands of the cow are more effective for the cost than the cultivated cells, they have possibilities to produce continuously, and since the milk is collected several times a day, the time between the actual synthesis and the harvest can be as short as a few hours. The genetic stability of the cow is greater than that of microbial or cell-based production systems. Also, cows are relatively easy to reproduce using artificial insemination, embryo transfer, and embryo cloning techniques. In addition, the downstream processing of cow's milk containing human transgenic proteins may require little or no purification. The publications that teach this methods are referred to below. However, none of these publications teaches, deecribes, or otherwise suggests the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. "Molecular Farming: Transgenic Animáis as Bioreactors" by J. Van Brunt, Biotechnology, Volume 6, pages 1149-1154, 1988, describes the alteration of the genome of different large domestic animals that have milk, which produce transgenic animals capable of producing different entities. heterologous This publication suggests methods to obtain the primary gene product only. International Patent Application Number PCT / US91 / 08216 describes a tranegen capable of producing heterologous recombinant proteins in the milk of transgenic bovine species. This published patent application describes a method for obtaining the primary gene product only. This application also describes methods for producing and using the altered milk obtained from these transgenic animals.
International Patent Application Number PCT / GB87 / 00458 describes methods for producing a peptide, this method involving incorporating a DNA sequence encoding the peptide in the gene of a mammal coding for a whey protein, in such a way that the DNA sequence is expressed in the mammary gland of the adult female mammal. This published patent application describes a method for obtaining only the primary gene product, the peptide, in the milk of the transgenic mammal. This application also describes methods for producing and using the altered milk obtained from these transgenic animals. International Patent Application Number PCT / GB89 / 01343, describes methods for producing proteinaceous materials in transgenic animals that have genetic constructs integrated in their genome. The construct comprises a 5 'flanking sequence from a mammalian milk protein gene, and DNA encoding a heterologous protein other than a milk protein. This published patent application describes a method for obtaining only the product of the primary gene, the heterologous protein, in the milk of the transgenic mammal. European Patent Application Number 88301112.4 designates method for targeting specific genes to the mammary glands, which results in the efficient synthesis and secretion of the biologically important molecules in the milk of these transgenic animals. This published application also describes methods for producing and using the altered milk obtained from these transgenic animals, and teaches a method for obtaining only the primary gene product in the milk of the transgenic mammal. International Patent Application Number PCT / US87 / 02069 describes a method for producing mammals capable of expressing recombinant proteins in the milk of lactating animals. This patent application does not disclose or suggest in any other way the production of secondary gene products in the milk of non-human transgenic mammals, as claimed in the present invention. Although transgenic animals can be used for the production of large amounts of human proteins, they have not been used for the production of secondary gene products, such as human oligosaccharides, or proteins and glycosylated lipids with certain specific oligosaccharides, or human milk proteins and glycosylated lipids with certain specific oligosaccharides. None of the aforementioned publications describes or suggests a method for producing human and glucoconjugate oligosaccharides in milk from non-human mammals. The aforementioned publications also do not describe or suggest a method for obtaining glucoconjugates in milk from non-human mammals, where the glycosylation is with the desired oligosaccharides. Achieving this result requires that the genome of non-human mammals having milk be altered to ensure that the breast tissue electively expresses a desired human glycosyltransferase, which would then glycosylate certain proteins with the desired oligosaccharide. This approach requires incorporating the DNA encoding the desired human glycosyltransferases into the genome. The literature also does not disclose or suggest a method for obtaining human glycosylated proteins in milk from non-human mammals, where the glycosylation is with the desired oligosaccharides. The literature also does not disclose or suggest a method for obtaining glycosylated human milk proteins in the milk of a non-human mammal, where the glycosylation is with the desired oligosaccharide fractions. Achieving this result would require that the genome of non-human mammals having milk be altered to ensure that their breast tissue selectively expresses both human glycosyltransferase and the desired human proteins, which are then appropriately glycosylated with the desired oligosaccharides by the glycosyltransferase active human This approach requires not only that the DNA encoding the desired glycosyltransferase be inserted into the genome, but also that the DNA that encodes the desired human proteins in that genome is incorporated. In accordance with the foregoing, it is an aspect of the present invention to provide methods for detecting successful transgenesis of fertilized oocytes prior to implantation, such that the transplanted oocytes contain the genetic constructs required to achieve the desired glycosylation and production of the oligosaccharide. It is also an aspect of the present invention to provide non-human transgenic mammalian species that have milk, which are capable of producing human glycosyltransferases that are secreted extracellularly through the mammary tissue of those mammalian species. Furthermore, it is also an aspect of the present invention to provide non-human transgenic mammalian mammals that are capable of producing human glycosyltransferases that are secreted extracellularly through the mammary tissue into the milk produced by these mammalian species. In addition, it is an aspect of the present invention to provide non-human transgenic mammalian species having milk, which are capable of producing human glycosylated proteins and oligosaccharides secreted extracellularly through the mammary tissue into the milk produced by these mammalian species. The present invention also relates to non-human transgenic mammalian species having milk, which are capable of producing glycoelated human milk proteins and lipids, in the milk of those transgenic animals. It is also an aspect of the present invention to provide non-human transgenic mammalian species that have milk, which are capable of producing human oligosaccharides in the milk of those transgenic animals. The present invention also relates to food formulations containing human glycosylated proteins, lipids, and oligosaccharides from that tranegenic milk. The present invention also relates to pharmaceutical, medical, diagnostic, and agricultural formulations containing glycosylated proteins, lipids, and oligoeaccharides obtained from the milk of transgenic animals. It is also an aspect of the present invention to provide transgenic bovine species that are capable of producing glycosylated proteins, such as glycosylated human milk proteins and lipids, in their mammary glands. It is a further aspect of the present invention to provide transgenic bovine species that are capable of producing human oligoeaccharides in the milk of those transgenic cows. The present invention also relates to food formulations containing glycosylated proteins, lipids, and oligoeaccharide from that transgenic bovine milk. The present invention also relates to pharmaceutical, medical, diagnostic, and agricultural formulations containing glycosylated proteins, lipids, and oligoeaccharides obtained from tranegenic cow milk.
Description of the Invention The present invention utilizes transgenes encoding a heterologous catalytic entity to produce secondary gene products in the milk of non-human transgenic mammals. More particularly, the present invention uses transgenes encoding heterologous glycosyltransferases to produce heterologous oligoeaccharides and glycosylated glycoconjugates in the milk of transgenic non-human mammals. A milk is described from a non-human transgenic mammal, this milk being characterized in that it contains heterologous components produced as the secondary gene products of at least one heterologous gene contained in the genome of the non-human transgenic mammal. Also described is a product produced in the milk of transgenic non-human mammals, wherein the product results from the action of a catalytic entity selected from the group consisting of heterologous enzymes and heterologous antibodies, and wherein the non-human transgenic mammal contains in its genome when menoe a heterologous gene that codes for that catalytic entity. Examples of the aforementioned product are oligosaccharide and glucoconjugate. The production of transgenic milk containing human oligosaccharides and / or glycosylated proteins with certain oligosaccharides is desirable, since it provides a milk matrix where little or no additional purification is needed for human consumption, and where the transgenic milk resembles biochemically to human milk. Humanized milk is described wherein the milk is produced by a non-human transgenic mammal, wherein the genome of the non-human transgenic mammal contains at least one heterologous gene encoding a human catalytic entity. The catalytic entity produces oligosaccharides and glucoconjugates that are present in the milk of the non-human transgenic mammal. A method for obtaining a humanized milk is also described, this method comprising the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene that encodes the production of a human catalytic entity, where this catalytic entity produces a secondary gene product in non-human mammalian milk; and (b) milking the non-human mammal.
Also disclosed is a method for obtaining a biological product from humanized milk, this method comprising the steps of: (a) inserting into the genome of a non-human mammal, a heterologous gene encoding the production of a heterologous catalytic entity, in where the catalytic entity produces a secondary gene product in the milk of the non-human mammal; and (b) milk the non-human mammal; and (c) isolate the biological product from the milk. Also disclosed is a non-human transgenic mammal characterized in that the mammalian genome contains at least one heterologous gene which codes for the production of the heterologous catalytic entity selected from the group consisting of enzymes and antibodies, and wherein the catalytic entity produces a second heterologous product in the milk of the mammal. Also described is a transgenic cow characterized in that the cow genome contains at least one heterologous gene that codes for the production of a heterologous glycosyltraneferase selected from the group consisting of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferase, glucuronylpimerae, sialiltraneferasas, manoeiltraneferasas, sulfotransferasas, β-acetilgalactoeaminiltraneferasas, and N - acetilglucoeaminiltransferasae, and in where milk of the cow contains heterologous oligosacáridos and glucoconjugadoe produced by said glucosiltransferasa. The representative non-human mammals useful in the present invention are mice, rats, rabbits, pigs, goats, sheep, horses, and cows. Representative heterologous genes useful in the present invention are the genes encoding human enzymes and human antibodies. -? (Human enzymes and human antibodies are also referred to herein and in the claims as a catalytic entity). Examples of human enzymes useful in the present invention are enzymes selected from the group consisting of glucosyltransferases, phosphorylase,hydroxylases, peptidase, and sulfotransferases. Glucosyltransferases are especially useful in the practice of the present invention. Illustrative glycosyltransferases are especially useful in the practice of the present invention, with the enzymes selected from the group'20 consists of fucosyltransferase, galactosyltransferase, glucosyltransferase, xylosyltransferase, acetylases, glucuronyltransferases, glucuronilepimerasae, sialyltransferase, mannosyltraneferases, sulfotransferases, β-acetylgalactosaminyltransferases, and N-25 acetylglucosaminyltraneferases.
Examples of the desired heterologous gene gene products of the present invention are oligosaccharides and glucoconjugates. (Heterologous secondary gene products are also referred to herein and in the claims as "a biological product," or simply as a "product"). The representative heterologous oligosaccharides produced as secondary gene products are lactose, 2-fucosyl lactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentase I, lacto-N-fucopentase II, lacto-N-fucopentaose III, lacto-N-difucopentaoea I, eialyl-lactoea, 3-sialyl-lactose, sialyl-tetrasaccharide-a, sialyl-tetrasaccharide-b, sialyl-tetra-saccharide-c, disialyl-tetra-trac-acid, and eialyl-lacto-N-fucopentase. Exemplary heterologous glycoconjugates produced as secondary gene products described herein, are glycosylated homologous proteins, heterologous glucoeiladae proteins, and glycosylated lipids. Representative or desirable glycosylated heterologous proteins according to the practice of the present invention are the proteins selected from the group of proteins consisting of human serum proteins and human milk proteins. Examples of human milk proteins are proteins selected from secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and lipase stimulated by the bile salt. An enteral nutrition product containing humanized milk useful in the nutritional maintenance of an animal is also described. Also disclosed is a pharmaceutical product containing the product of the present invention, useful in the treatment of an animal. In addition, a medical diagnosis containing the product of the invention, useful in the diagnosis of an animal, is described. Also described are agricultural product containing the product of the invention, useful in the maintenance of crops. Also disclosed is a method for producing a non-human transgenic mammal species, capable of producing heterologous secondary gene products in the milk of said species, the method comprising the steps of: (a) preparing a transgene, this transgene consenting at least an expression regulation DNA sequence, functional in the mammary secretory cells of the transgenic species, a secretory DNA sequence, functional in mammary secretory cells of the tranegenic species, and a recombinant DNA sequence encoding a recombinant heterologous catalytic entity , the secretory DNA sequence being operably linked to the recombinant DNA sequence to form a secretory-recombinant DNA sequence, and the at least one expression regulation sequence being operably linked to a secretory-recombinant DNA sequence, wherein the transgen is capable of directing the expreation of the DNA sequence sec retora-recombinante in mammary secretory cells of the transgenic species that contains the transgene, to produce a recombinant heterologous catalytic entity that, when expressed by mammary secretory cells, catalyzes the production of the secondary gene products in the milk of the species transgenic; (b) introducing the transgene into the objective embryonic cell; transplant the objective transgenic embryonic cell formed by the same, or the embryo formed from the same, in a receiving female mother; and (c) identifying at least one female progeny that is capable of producing the secondary gene products in the milk of the progeny. A useful method for producing large non-human transgenic mammals such as pigs, goats, sheep, horses, and cows, capable of producing heterologous secondary gene products in their milk is also disclosed. The method described comprises the steps of: (a) preparing a transgene capable of conferring said phenotype when incorporated into the cells of the transgenic non-human mammal; (b) methylation of this transgene; (c) introducing the methylated tranegen into fertilized oocytes of the non-human mammal, to allow the integration of the transgene into the genomic DNA of the fertilized oocytes. (d) cultivate the individual oocytes formed by the same, for the preimplantation of embryos, thus replicating the genome of each of the fertilized oocytes; (e) removing at least one cell from each of the preimplantation embryos, and removing the at least one cell to release the DNA contained therein; (f) contacting the released DNA with a restriction endonuclease capable of dissociating the methylated transgene, but unable to dissociate the non-methylated form of this transgene, formed after integration into, and replication of, the genomic DNA; and (g) detecting which of the cells from the embryos of the preimplantation contain a transgene that is resistant to dissociation, by means of the restriction endonucleae, as an indication of which embryos of the preimplantation have integrated the transgene. In accordance with the above method, the removal of the first semi-embryos that are used and analyzed according to steps (d) to (f) are also described, including this method: (g) cloning when one of the seconde-embryos; and (h) forming a multiplicity of transgenic embryos. Transplantation of more than one of the transgenic embryos in female host mothers is also described, to produce a population that contains at least two non-human transgenic mammals that have the same genotype, and to transplant the rest of the pre-implantation embryos containing a genomically integrated transgene, in a recipient female mother, and identify at least one progeny having the desirable phenotype, this phenotype having the ability to produce a hete ulogo secondary gene product in the milk of that species, with heterologous secondary gene products being selected from the group consisting of oligosaccharides and glucoconjugates. The DNA sequence forming the useful transgene in the present invention comprises at least three functional parts: (a) A portion encoding the human glucosyltransferaea. This portion of the transgene is referred to later in the preamble as the "recombinant portion" or the "recombinant sequence"; (b) A portion of signal; and (c) A portion of expiry regulation. The recombinant portion of the transgene comprises a DNA sequence encoding the desired glycosyltransferase enzyme. The signal portion may be naturally present, or it may be genetically engineered into the DNA sequence. This signal encodes a secretory sequence that ensures that the glycosyltransferase is transported to the Golgi apparatus of the cell. In the present invention, the signal DNA sequence is functional in mammary secretory cells. These sequences are operably linked to form a recombinant-expression-DNA sequence. The expression sequence ensures that the transgene is expressed in certain types of tissue only. In the present invention, the expression is regulated towards the mammary secretory tissue. At least one expression regulation sequence, functional in the mammary secretory cells of the transgenic species, is operably linked to the recombinant signal DNA sequences. The transgene thus constructed is capable of directing the expression of the recombinant signal DNA sequence in the mammary secretory cells containing the transgene. This expression results in the production of the glycosyltransferase secreted from the mammary secretory cells into the milk of the transgenic species. In addition to the functional parts described above, the transgene can also comprise additional elements. For example, the recombinant portion can code for more than one protein. Therefore, in addition to encoding the glycosyltraneferaea, it can also code for one or more human proteins. Also, multiple transgenes encoding other glycosyltraneferasae and other heterologous proteins can be transfected simultaneously. All additional transgenes are also operably linked to the secretory and expression regulation sequences of the glycosyltransferase transgene. The expression of multiple transgenes results not only in the production of the glycosyltransferase, but also in the other proteins, all of which are secreted from the mammary secretory cells into the milk of the transgenic species. In the presence of suitable substrate materials, the glucosyltransferase will convert the onosaccharide unitsindividual in the desired oligosaccharides. The desired oligosaccharides will be present in the milk of the transgenic species. The same glycosyltransferase enzyme will also covalently link the monosaccharides to the proteins via the available glycosylation sites. TheseProtein glycoelated with the desired oligosaccharides will also be present in the milk of the transgenic species. The advantages of the present invention will come to be better understood by reference. to the following detailed description, when taken in conjunction with the accompanying Figures.
Brief Description of the Drawings Figure 1. Nucleotide and amino acid sequence of human alpha-l, 2-fucosyltransferaea. Figure 2. Iluetration of the protocol to achieve 5 the amplification and expression of the fucosyltransferase cDNA. Figure 3. Illustration of the construction of the plasmid pWAP-polyA, using the regulatory sequence (promoter) of the whey acid protein (WAP). Figure 4. Illustration of pWAP- * jj fucosyltransferase plasmid used for microinjection in mouse embryos. Figure 5. Photograph of a Western blot illustrating the presence of human alpha-l, 2-fucosyltransferase in the milk of transgenic mice. 15 Figure 6A to 6F. High-pressure liquid chromatography profiles of milk samples obtained from normal or non-transgenic mice (Frames A and B) and transgenic, expressing human alpha-1, 2-fucosyltransferase (Frames C, D, E, and F) . Figure 7. Photograph of a fluorophore-assisted carbohydrate electrophoresis gel of an oligosaccharide material pooled after separation by high pressure liquid chromatography. Figure 8. Photograph of a gel of electrophoresis 25 of carbohydrate acetylated by fluorophore following the digestion of samples of oligosaccharide with a specific fucosidase for alpha-1,2-fucose bonds. Figure 9. Photograph of a gel of electrophoresis of carbohydrate aeietide by fluorophore, which removes the composition of the monosaccharide from the samples of oligosaccharide alieladae from milk, followed by an exhaustive digestion with a mixture of fucosidaea and β-galactoeidase. The released monoeaccharide units were labeled with 8-aminonaphthalen-2,3,6-trisulfonic acid (ANTS) to facilitate detection. Figure 10. Photograph of a Western blot of milk protein isolated from normal (non-transgenic) and transgenic mice expressing human alpha-1,2-fucosyltransferase. The glycosylation of the stained proteins was detected by immunofluorescence using a specific lectin for the alpha-1,2-fucose bond. The figure tests the presence of glycosylated milk proteins with the H antigen product of the transgenic enzyme. Figures 1 to 10 are provided in accordance with title 37 of the Federal Code of Regulations, section 1.81.
Detailed Description of the Invention The present invention relates to the expression in vivo in mammary tissue of non-human mammals, of catalytically active heterologous glycosyltransferases that control the production of secondary gene product resulting from the activity of the specific glucosyltransferase enzyme. These glycosyltransferase enzymes control the synthesis of the free oligosaccharides, or the covalent attachment of the oligosaccharides with proteins or lipid. This expression is achieved in a cell by using genetic engineering to instruct the cell to produce specific heterologous glycosyltransferases (primary gene product), and then employs the specific catalytic activity associated with each glycosyltransferase, to produce a specific product, the secondary gene product. In the case of glycosyltransferases, the secondary gene product includes not only the synthesized oligosaccharides, but also the glycosylated proteins and lipids. Oligoeacáridoc and glycosylated proteins / lipids are secreted and found in the free form in the milk of the transgenic mammalian species. As used in this, and in the claims, the term "glycosylation" is understood to mean modification after translation of a protein or lipid by an enzymatic process facilitated by expressed glycosyltransferase, which results in the covalent attachment to the protein or lipid of a or more monosaccharide units. This glycosylation is done instructing the cell to produce both the glucosyltransferases and the protein or lipid of interest. The protein or lipid of interest can be a homologous or heterologous entity. As used herein and in the claims, the term "homologous" is understood to refer to a composition or molecular form normally produced by the host cell or animal. As used herein and in the claims, the term "heterologous" is understood to refer to a composition or molecular form not normally produced by the host cell or animal. Genetic engineering techniques are used to incorporate foreign genetic material into the genome of the host animal, that is, genetic material derived from another species. As used herein and in the claims, the terms "transgenic cell" or "transgenic animal" are intended to refer to a host cell line or an animal that contains those transformed genomes. As used herein and in the claims, "transgenic products" are intended to refer to products derived from those transgenic entities.; for example, milk derived from a transgenic cow, referred to as tranegenic milk. The present invention is based, in part, on the production of a transgenic non-human mammal, wherein the cells comprising the mammary gland contain a transgene that expresses a desired glycosyltransferase. (The genome of the tranegenic mammary cell can also be transfected with a gene that encodes a human protein). The resulting glycosyltransferase, when expressed in the transgenic host mammary cells, is useful to produce free soluble oligosaccharides in the milk produced by this transgenic animal. The glycosyltransferase also expressed is useful in the glycosylation of homologous milk proteins, or heterologous human proteins, when the transgenic mammary cell also expresses those proteins. The same concept can be applied to the modification of lipids. The present invention has a broad application in x O synthesis of oligosaccharides by different glucosyltransferaeae, talee as fucosyltransferase, galactosyltransferase, glucosyltransferase, sialyltransferases, mannosyltransferaeae, xylosyltransferases, sulfotransferase, glucuronyltransferases, β-acetylgalactosaminyltransferase and N-15 acetylglucosaminyltransferase. The products of other classes of Golgi apparatus enzymes, such as acetylases, glucuronylepimerases, glucosidases, acetyltransferases, mannosidases, and phosphotransferases, can also be synthesized by the method described. BEST MODE FOR CARRYING OUT THE INVENTION The following describes the incorporation of DNA encoding the production of a fucosyltransferase, particularly human alpha-l, 2-fucosyltraneferase (referred toalso later in the preeent as Fuc-T), in the genome of cells that form the non-human mammary glands. An example of a Fuc-T product is 2 '-fucosyl-lactoea. This is one of the oligosaccharides in human milk, and has the chemical formula of fucose-alpha-1,2-Gal-β-l, 4-Glc. Other Fuc-T products will include glycoprotein containing β-linked terminal galactose reeiduoe, which can be fucosylated by Fuc-T. The resulting carbohydrate carbohydrate is fucose-alpha-1, 2-galactose-β-R, wherein R is selected from the group consisting of β-1,3-GlcNAc, β-1,4-GlcNAc, and the like , they are known in the field of blood group serology as the "H Antigen". It is well recognized by those skilled in the art that other glycosyltraneferases and Golgi processing enzymes may also be used in accordance with the present invention. In the non-limiting examples described below, transgenic mice were employed. Mouse genomes do not contain or express the DNA encoding Fuc-T. Accordingly, in the transgenic mice producing either Fuc-T, 2 '-fucosyl-lactose, or H antigen, then a successful incorporation of the gene encoding Fuc-T must have been presented in the murine genome. It is well known in the art that it is possible to insert the DNA encoding the glycosyltransferases into the genome of the transgenic host cells. Some of the cell lines that could be used for the transgenic expression of glycosyltransferases are Chinese hamster's ovary (CHO) cells, mouse L cells, A9 mouse cells, baby Hamster kidney cells, C-127 cells, cells PC8, insect cells, yeast, and other eukaryotic cell lines. In a preferred embodiment of the present invention, the host cells are mammary cells, these cells comprising the tissue of the mammary glands of the non-human transgenic mammals. Preferred embodiments of the present invention utilize mice, rats, rabbits, pigs, sheep, goats, horses, or transgenic cows. Particularly preferred embodiments use sheep, goats, or transgenic cows. A particularly preferred embodiment of the present invention is the use of bovine mammary tissue in lactating transgenic cows. The precise procedure used to introduce the altered genetic material into the host cell is not critical. Any of the well-known procedures for introducing the foreign * nucleotide sequences into the host cells can be employed. These include the use of plasmid vectors, viral vectors, and any other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into the host cell. It is only necessary that the particular genetic engineering method used be able to successfully introduce at least one transgene in the host cell, which is then capable of expressing the desired glycosyltransferase. A preferred technique in the practice of the present invention is transfection of an objective embryonic cell, transplanting the objective transgenic embryonic cell formed by the same to a recipient surrogate mother, and identifying at least one female progeny that is capable of producing the oligosaccharides. free human or recombinant human glycosylated protein in their milk. A more preferred embodiment of the present invention comprises the steps of transfecting an embryonic cellObjective IJ of a coil species, transplant the objective transgenic embryonic cell, formed by the same to a recipient bovine mother, and identify at least one female bovine progeny that is capable of producing the free human oligosaccharides or the recombinant proteinhomologous or heterologo glycosylated in their milk. The following examples demonstrate the alteration of the genome of non-human mammalian host cells by inserting therein heterologous DNA encoding specific glycosyltransferases. The transgenic hostthen expresses catalytically active specific glycosyltransferases that facilitate the production of a desirable secondary gene product, more specifically, a specific oligosaccharide. The glycosylation of milk proteins is also demonstrated. If in addition toAs the DNA encoding the oligosaccharides, heterologous DNA encoding human milk proteins in the host genome is also inserted, then that host will also express human milk protein. Since the human host will also express the glycosyltraneferase, the glucoeilation of human milk proteins will be pre-prepared with certain specific oligosaccharides. Human milk proteins of interest include secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, lactoperoxidase, alpha-lactalbumin, β-lactalbumin, and lipase stimulated by the bile salt. This approach to oligosaccharide synthesis and protein / lipid glycosylation has several advantages over other currently available methods. This approach is supported by the novel combination of: (a) the use of tranegenic mammary cells for the production of sugar nucleotides from natural carbon sources, such as glucose; (b) the expregation of heterologous recombinant glycosyltransferase gene in transgenic mammalian cells; (c) the production of heterologous oligosaccharides of the desired structure by the natural lactating mammary glands of transgenic animals, this production being the result of the enzymatic activity of the expressed heterologous glycosyltransferase enzymes; and (d) the use of the heterologous glycosyltransferase enzyme to glycosylate homologous or heterologous proteins or lipids. The experiments described below illustrate the following points: (a) A human alpha-1, 2- fucosyltransferase gene was isolated and cloned from a human epidermal carcinoma cell line. This enzyme is responsible for the synthesis of the oligosaccharide 2 '-fucosyl-lactose, and the glycosylation of proteins with the specific antigen H of the x blood group; (b) The functional nature of the gene was demonstrated by its ability to express catalytically active alpha-l, 2-fucosyltraneferaea in cultured non-human cell lines. The presence of 1,2-fucosyltransferase was demonstratedby enzymatic activity assays specific for this enzyme. The presence of catalytically active alpha 1, 2-fucosyltransferase was also demonstrated using the immunofluorescence technique, to show the presence of the H antigen on the surface of the expressing cells.the enzyme; (c) The utility of this gene in the formation of non-human transgenic animals capable of expressing the alpha-l, 2-fucosyltransfeiaea gene product was demonstrated by the successful development of transgenic mice carrying thehuman alpha-1, 2-fucosyltraneferae gene which is capable of expressing the catalytically active alpha-l, 2-fucosyltransferase; (d) The expression in the breast tissue of a non-human transgenic animal of catalytically active human alpha-l, 2-fucosyltransferase. The presence of the enzyme was established through tests of direct enzymatic activity and immunofluorescence using antibodies that exhibit binding specificity for the enzyme. (e) The formation of secondary gene products resulting from the catalytic activity of human alpha-1, 2-fucosyltransferase expressed in non-human milk. These products include the release of human oligosaccharide, 2'-fucosyl lactose, in milk, and the glycosylation of milk proteins with the H antigen product of the enzyme. The presence of the secondary gene products was established through the biochemical analysis of the compounds and immunofluorescence using lectins exhibiting binding specificity for the H antigen. The following examples are provided as representative of the scope of the invention, and should not be considered to limit the invention claimed herein. Examples 1 and 2 use tissue culture systems. These in vitro experiments were undertaken to prove that expulsion of enzymatically active heterologous glycosyltransferases was possible. Examples 1 and 2 are not critical to enable the present invention, and are provided exlusively for the purpose of ensuring an understanding and appreciation of the invention. Examples 3, 4, 5, and 6 prove that the production of heterologous gene gene products in the milk of transgenic non-human mammals is possible in vivo. Examples 3 to 6 are provided for the purpose of making the teachings, scope, and claims of the invention possible. In light of the foregoing, the Requesters believe that a deposit of biological material is not required in accordance with Title 37 of the Code of Federal Regulations, section 1.802.
Example 1; Isolation of the Gene for Alpha-2, 2- Human Fucosyltransferase from the Human Epidermal Carcinoma Cell Line. The cDNA encoding alpha-l, 2-fucosyltransferase, was isolated from a CADN library of an epidermal carcinoma cell line (A 431), since alpha-1,2-fucosyltransferase was previously cloned from this source (VP Rajan et al., J. Biological Chemistry, volume 264, pages 11158-11167, 1989). This reference is incorporated herein by reference. After amplification mediated by polymerase chain reaction (PCR) of the protein encoding the sequence, the cDNA was cloned into a bacterial vector to determine the cDNA sequence of the amplified gene. The DNA sequence was determined from each of six independently isolated clones of human alpha-1,2-fucoeiltraneferase. Eeta nucleotide sequence and the corresponding amino acid sequence are shown in Figure 1. In order to determine the sequence of cDNA previously noted, two alpha-1, 2-fucosyltransferase preparers were designed, each containing 31 nucleotides (31- mers), based on the published alpha-1, 2-fucosyltraneferase cDNA sequence. The BigNH2 preparer contained the methionine initiator residue at position 27, where transcription of Fuc-T (start of open reading frame) begins. The second preparer, BigCOOH, contained a stop codon at position 10. The primers are indicated in Figure 2. The polymerase chain reaction included approximately lμM of each preparator, 1 microgram of template with polymerase chain reaction regulator , and Taq polymerase. The polymerase chain reaction was performed in a thermal cycler (Perkin and Elmer, Model 840) using a temperature cycle of 94 ° C for 1 minute, 60 ° C for three minutes, 72 ° C for three minutes, for 30 cycles , followed by an extension of 5 minutes at 72 ° C. The product of the polymerase chain reaction was electrophoresed, about 0.8 weight percent / volume of low melting point agarose. A 1.1 kilobaee fragment was detected. This fragment was eeparated and subcloned into the PCR II cloning vector. One of the transformants, hereinafter referred to as the selectant, was selected and characterized both by restriction analysis and by nucleotide sequence analysis. DNA sequencing was performed using an Applied Biosystems Model 373A automatic DNA sequencer. The restriction pattern of the insert indicated similarity with the coding region of alpha-1,2-fucosyltransferase. The nucleotide sequence of this candidate clone was identical to the published sequence, except at position 640. In vitro site-directed mutagenesis was employed to correct this single defective bae, thereby forming the wild-type sequence that was used in the transfection experiments described later.
Example 2: Expression in the Host Cell of Human Glucosyltransferases. This example describes the transfection of cultivated mouse L cells and Chinese Hamster Ovary (CHO) cells with a gene capable of expressing the specific human glucosyltransferase, alpha-1, 2-fucosyltransferase or Fuc-T. These cell lines were selected for transfection, since their natural genomes do not carry the DNA encoding the Fuc-T. If following the transfection, it is shown that the cell lines produce either Fuc-T or the enzymatic product thereof (2 '-fucosyl-lactose or H antigen bound to the glycoproteins), then a transfection of success. This is demonstrated by the immunofluorescence technique, using specific antibodies and / or a specific lectin that binds selectively with the H antigen. The Fuc-T gene used for transfection was obtained as described in Example 1. Transfection and the materials used in it are described below.-. Ú Phenyl-β-D-galactoside was obtained from Sigma ChemicalCo. The nucleotide sugar, GDP-L- (U-14C) fucose, with a specific activity of 278 mCi / millimole, was purchased from Amersham Corporation. The A431 human epidermal carcinoma cDNA library was obtained as a present from Dr. NevisFrigien, The University of Miami, Oxford, Ohio. The PCR II vector was purchased from Invitrogen Corporation. The expiration vector pQEll was purchased from Qiagen Inc. Plasmid pSV2- neo was obtained from Pharmacia Fine Chemicals Corporation. Plasmid pMet-FucT-bGH was obtained from Drs. Xhou Chen and BruceKelder at the University of Ohio, Athens, Ohio. This connection contains the cADM that encodes Fuc-T. The primers were synthesized by Operon Technology or Fischer Scientific Corporation. The mouse monoclonal antibody for H antigen was purchased from Dako Corporation. Thegoat-to-mouse antibodies labeled with fluorescein-isothiocyanate were purchased from Sigma Chemical Company. Rabbit polyclonal antibodies to alpha-1, 2-fucosyltransferaea were cultured as a means to detect the expreration of this enzyme. In order to cultivate enoughenzyme to act as the antigen in the induction of• antibody, the insert of the selector was subcloned into an inducible expression vector in the frame with a 6XHis label(pQEll). A protein labeled with 6XHis was purifiedM. easily with a nickel affinity chromatography column. To avoid possible cellular toxicity, the hydrophobic region of alpha-1, 2- fucoeiltraneferaea was suppressed. To make this, two new trainers were built. The first, a BamHI-NH2, is hybridized in the template at position 60; the second preparer, Salí COO, extends into the stop codon. The BamHI site and Sal I were designed on the upstream and downstream preparators. The product of the polymerase chain reaction was subcloned into the frame at a BamHI / Sal I site of the expression vector pQEll, allowing fusion with the 6XHis tag. Tree milligramoe of the fusion protein (alpha-1, 2-fucoeiltransferaea-6XHie) were purified using a Ni-agarose affinity column. This material was used to culture polyclonal rabbit antibodies that exhibited specificity against Fuc-T.
Cell Line and Culture; The mouse L cells and lae cells of Chinese hamster ovary were obtained from the American Tissue Culture Collection (ATCC) in Washington, D.C. Cells were cultured in an alpha minimal essential medium (alpha-MEM, GIBCO, Gran Island, New York) supplemented with 10 percent fetal calf serum (GIBCO), penicillin, 80 micrograms / milliliter (Sigma), streptomycin 80 micrograms / milliliter (Sigma), and L-glutamine (Sigma), hereinafter referred to as alpha-MEM / 10 percent FCS. The transfected L cells were cultured on alpha-MEM containing G418 (GIBCO) at 400 micrograms / milliliter. The transfected Chinese Hamster Ovary cells were cultured on alpha-MEM containing G418 (GIBCO) at 1000 micrograms / milliliter.
Transient Transfection; L cells were cultured on 8-well chamber slides (Lab-Tek) haeta at a confluence level of 75 percent. A tranefection cocktail was added to each DNA chamber pMet-Fuc-bGH (2 micrograms), lipofection (2 microliters), and 200 microliters of Opti-MEM medium (GIBCO). After 6 hours of incubation at 37 ° C, 200 microliters of alpha-MEM / 10 percent FCS were added, and after 48 hours of another incubation at 37 ° C, the slides were processed for indirect immunofluorescence as described above. ahead . The ability of the cloned cDNA fragment to encode functional alpha-1, 2-fucosyltransferase was tested by demonstrating the presence of the catalytic product of this enzyme, i.e., the H antigen, on the cell surface of the cultured mouse L cells. (L cells do not normally have the H antigen on their membrane). The wild-type insert of the selector noted in Example 1, was subcloned into the pMet-bGH plasmid in an EcoRI site. In this construct, the expression of alpha-1,2-fucosyltransferase activity is under the control of the metallothionein promoter. This promoter is zinc-inducible. The mouse L cells were tranefectively tranected with the pMet-Fuc-bGH linkage, and the presence of the structure of the H antigen on the cell surface was confirmed using the immunofluorescence technique with mouse monoclonal antibodies against the primary H antigen as described. later. Secondary antibodies labeled with fluorescein were goat against mouse antibodies. The preemption of the H ee antigen was further confirmed using the fluorescein-labeled lectin, Ulex europaeus agglutinin 1, which specifically binds to the fucose-alpha-1,2-galactose structures.
Indirect immunofluorescence. Successful transfection was demonstrated by the presence on the cell surface of the H antigen. Indirect immunofluorescence assays were performed using 8-well tissue culture chamber slides. The cells were coated in each chamber to an appropriate density, incubated overnight at 37 ° C, and then assayed for H antigen. The chamber slides were washed with phosphate buffered serum (PBS), fixed with 100 microliters. of a 2 percent solution of formalin in Hanke's balanced salt solution (HBSS), and permeabilized with saponin (2 milligrams / milliliter; Sigma) in 1 percent FCS, and incubated with a dilution of 1 1,000 of the anti-H antibody for 60 minutes in a humid chamber at room temperature. Subsequently, the slides were washed three times with PBS, and incubated for an additional 60 minutes with a 1: 1000 dilution of goat anti-mouse antibody labeled with FITC at room temperature in a humid chamber. The humidification prevented the drying of the mueetra. The efficiency of transfection of L cells, or the percentage of transformed L cells expressing the H antigen, based on immunofluorescence, was approximately 30 percent.
The previously noted results clearly demonstrate successful transfection of non-human mammalian cell lines with DNA encoding Fuc-T. The transfected cultured cell lines produce not only the primary gene product, Fuc-T, but also the modified glycoproteins. As a result of the activity of Fuc-T, the modified proteins carry the H antigen.
These results prove that the cloned cDNA fragment encoding Fuc-T is capable of expressing the enzymatically active Fuc-T. Accordingly, this cDNA was used for the production of transgenic animals as described below.
Example 3; Non-Human Transgenic Mammal that Has the Gene that Encodes a Specific Human Glucosyltransferase. This experiment proves that transgenic non-human mammals are capable of producing a catalytically active heterologous glycosyltransferase. More specifically, production of transgenic animals of human alpha-l, 2-fucosyltransferase is tested. Transgenic mice were produced by microinjection of human Fuc-T cDNA into the genome of mouse embryos. The fertilized mouse eggs were isolated in the single cell stage, and the male pronuclei were injected with the transgenic construct containing the human alpha-1, 2-fucoeyltransferase gene as shown in Example 1. These embryos were implanted then in pseudopregnant mice that had previously been coupled with sterile males. The founder pups of transgenic mice were identified after approximately 25 days after birth, using polymerase chain reaction amplification to analyze the chromosomal DNA obtained from a tail fragment with probes specific for the inserted human gene. Standard techniques were used in this field to achieve the desired transformation. These details have been described more fully in the following references, which are incorporated herein by reference, and which were also discussed earlier in the preamble. (a) International Patent Application NumberPCT / US90 / 06874; (b) International Patent Application Number PCT / DK93 / 00024; (c) International Patent Application Number PCT / GB87 / 00458; and (d) International Patent Application Number PCT / GB89 / 01343. One aspect of the present invention relates to the expression of a catalytically active human glycosyltransferase in the milk of a non-human mammal, and the use of that glycosyltransferase to effect the formation of the desired secondary gene product. In order to achieve the specific expression by the mammary gland of the human gene during the lactation of the transgenic mice, the regulatory sequence (promoter) of the serum acid protein (WAP) of a mouse was used to generate a tranegenic construct for the expression of human alpha-l, 2-fucosyltraneiferase. The murine serum acid protein promoter was received as a present from Dr. L. Henninghauser of the National Institutes of Health, Bethesda, Maryland. This material was used to construct the plasmid pWAP-polyA shown in Figure 3. This plasmid contains the polyadenylation signal sequence (polyA) of bovine growth hormone at the 3 'end of the fusion gene, which results in the expression effective, processing, and stability of messenger RNA. The human alpha-1, 2-fucosyltraneiferase (Fuc-T) gene was inserted into this plasmid to result in the formation of plasmid pWAP-polyA-Fuc-T illustrated in Figure 4. This plasmid was used for the microinjection of mouse embryos as described above. Using microinjections of DNA in concentrations of 2 and 4 micrograms / milliliter, a total of 85 pups were obtained from 16 injections. Only two injections did not give pregnancy as a result. The size of the bait of a single injection was normal, averaging three to ten pups per litter. Tail biopsies were performed on all 85 mouse pups. It was determined, by the tail biopsy assay, that nine of the founder population, hereinafter referred to as F0, possessed the gene encoding human alpha-1, 2-fucosyltransferase. This corresponds to a production efficiency of transgenic mice of approximately 11 percent. This falls within the scale of expected production efficiency of between 5 and 25 percent. The F0 progeny comprised eight males and one female. Six of the founders were then bred with normal mice, resulting in a total of one progeny out of a total of 98. Progeny 38, (hereinafter referred to as Fl) possessed the gene encoding alpha-1, 2-fucosyltransferase human, as determined from tail biopsies and polymerase chain reaction analysis. This corresponds to an efficiency Fl of approximately 36 percent. The Fl generation is comprised of 19 males and 19 females. Table 2 summarizes the results obtained.
TABLE 2 EFFICIENCY OF PRODUCTION OF THE GENERATION Fl FROM SIX FOUNDING MICE Founder # Total Number Progeny Number Efficiency of Trans-Male Procrenie Trans-Male Female genesis (%)6 16 4 2 37.528 18 2 2 22.229 18 4 6 55.634 13 3 6 69.254 15 2 1 20.072 18 4 2 33.3Fifteen of the bra he Fl (second generation) were allowed to mature, and were reared with normal mice. Pregnant females were allowed to give birth. The milk of four of these Fl mothers was harvested ten days after birth.
The collection of the milk was done using one of two techniques that are standard in this field: (a) breast suction using a vacuum line connected to a trap flask and a suction cup; or (b) anesthetizing and sacrificing the animal, and then piercing the nipples to release the fluid content of the mammary gland. The milk samples were kept frozen on dry ice until they were subjected to the analytical procedures described below. The collected milk samples were prepared to initially separate the oligosaccharides from the milk proteins and lipids. This was achieved using the methods described by A. Kobata (Methods in Enzymology, chapter 24, volume 28, pages 262-271, 1972), and a. Kobata et al. (Methods in Enzymology, chapter 21, pages 211-226, 1978). The milk samples were treated as follows. Samples typically of 90 to 100 microliters, obtained from control animals (non-transgenic), and transgenic, were centrifuged at 10,000 relative centrifugal force (RCF) for 20 minutes in centrifuge tubes of polypropylene conical. The centrifugation resulted in the preparation of the milk in two layers: an upper layer of cream, consisting mostly of lipids, and a lower layer. The lower layer, which contained soluble material, was removed and transferred to a new centrifugal tube. Two volumes of ice-cold ethanol were added, mixed by vortexing, and centrifuged at 10,000 RCF The soluble supernatants in ethanol were recovered and concentrated by evaporating the alcohol using a Speed-Vac concentrator. Insoluble in ethanol was kept frozen at -70 ° C until another analysis.After the concentration, the extracts containing the oligosaccharide were resuspended in water to the exact volume of the original milk bottle.Eetae resuependidae samples were retained. 4 ° C in an autosampler, refrigerated until further use When appropriate, these samples were subjected to compositional analysis, as described in Examples 4 to 7. One aspect of the present invention is the transgenic expression of heterologous glycosyltransferases in the mammary gland of non-human mammals that have milk The expression of heterologous glycosyltransferases is p You can show two- 1 / ways: (a) directly, by determining the presence of the enzyme (primary gene product) itself; and (b) indirectly, by determining the presence of the enzymatic product (secondary gene product:oligosaccharide or glycosylated protein) in the milk of the transgenic animal. As noted above, the murine genome does not encode the specific alpha-l, 2-fucosyltraneiferase specific to the synthesis of H antigen. Therefore, ifIf either Fuc-T or the products of Fuc-T are present in the milk of the transgenic mice, then a successful transgenesis has been presented, which provides a unique means to synthesize, and consequently, to obtain secondary gene products. . An important aspect of the present invention isthe production of heterologous secondary gene products in the milk of non-human animalee. As noted above, the secondary gene products may comprise not only the immediate product of the enzyme, the oligosaccharide, but also the protein or the heterologous or heterologous glycoeilated lipids, which are glycolized through the covalent attachment of the oligosaccharide with the protein or the lipid. The milk harvested from Example 3 was analyzed for the presence of human alpha-1, 2-fucosyltransferase, and also for the presence of secondary gene products, specifically 2 '-fucosyl lactose and proteins glycosylated with the H antigen. Examples 4 , 5, 6, and 7 test the production of human Fuc-T and Fuc-T products in the milk of non-human animals.
Example 4; Analysis that Tests the Production of a Specific Glucosiltransferase in the Milk of Non-Human Transgenic Mammals. This example demonstrates the feasibility of obtaining human alpha-1, 2-fucosyltransferase in tranegenic mouse milk. As previously noted, the murine serum acid protein promoter was employed to ensure expression by the site-specific mammary gland of human alpha-1, 2-glucosyltransferase. The ethanol-insoluble milk protein precipitate, obtained from the mice as described above in Example 3, was re-suspended in a polyacrylic amide gel electrophoresis regulator (PAGE) containing sodium dodecyl sulfate (SDS). The SDS-PAGE regulator volume used to re-suspend the protein granule was exactly equal to the original volume of the milk sample. The reconstituted samples were assayed for the presence of alpha-1,2-fucosyltraneferase. This presence was determined using immunoblot technology as described below. More specifically, Western blots were employed. Five microliter samples of the protein pellet re-suspended in SDS-PAGE were electrophoresed on a 12.5 percent polyacrylic amide gel. Electrophoresis was performed at 150 volts. Following the electrophoresis, the resolved proteins were transferred to a nitrocellulose membrane. Transmigration was performed for one hour at 100 volts using a 12.5 mM Tris-HCL buffer, pH 7.5, containing 96 mM glycine, 20 percent methanol, and 0.01 percent SDS. Following the transfer, the remaining unbound reactive groups on the nitrocellulose membranes were blocked by incubation in a 50 mM Tris-HCL buffer, pH 7.5, containing 0.5 M NaCl and 2 percent gelatin, referred to hereinbelow. as TBS. Then, the membranes were washed three times in TBS containing 0.05 percent Tween-20.
The membranes were incubated for 18 hours in 1 percent gelatin / TBS containing a 1: 500 dilution of rabbit polyclonal antibody with a specificity against alpha-l, 2-fucoeiltraneferasa. This polyclonal antibody was obtained as described in Example 2. After rinsing with TBS-Tween, the membrane was then incubated with a solution of 1% gelatin-TBS containing goat anti-rabbit IgG previously conjugated with horseradish peroxidase. . Then the membrane was washed with TBS-Tween. The presence and position of the proteins on the nitrocellulose membrane were visualized by incubating the membrane in a volume of 50 milliliters of TBS containing 0.018 percent hydrogen peroxide and 10 milliliters of methanol containing 30 milligrams 4-chloro-naphthol . Figure 5 shows the results of this experiment for milk samples obtained from a control animal (non-transgenic) and two transgenic animals. The transgenic animals are referred to in Figure 5 as 28-89 and 29-119. Non-transgenic animals are referred to in Figure 5 as the control. Figure 5 indicates that there is clearly a very intens band present in the milk samples obtained from the two transgenic animals, but it is absent from the milk obtained from the non-transgenic control animal. Clearly there are intense bands present at a relative molecular weight of approximately 46 kilodaltons, corresponding to the predicted molecular weight of alpha-1,2-fucosyltransferase. There are also intense bands present in the positions corresponding to the lowest relative molecular weights on the scale of approximately 30 to 25 kilodaltons. These bands are absent in the sample of milk derived from the non-transgenic sample. Without forcing the inventors it is speculated that these lower molecular weight bands probably correspond to Fuc-T fragment. This is proof that milk samples from the transgenic samples contain Fuc-T, while milk samples from the non-transgenic animal do not contain Fuc-T.
Example 5; Analysis that Tests the Production of Specific Heterologous Secondary Gene Products in the Milk of Non-Human Transgenic Mammals. This example proves the feasibility of obtaining heterologous secondary gene products in the milk of non-human transgenic mammals. More specifically, this example demonstrates the ability to obtain the secondary gene product of Fuc-T in the milk of a non-human animal. More specifically, the presence of the secondary gene product, 2 '-fucosyl lactose, was demonstrated in the transgenic milk. Control non-transgenic mouse milk does not contain 2 '-fucosyl lactose.
The evaporated oligosaccharide extracts, obtained as described in Example 3, were analyzed and separated using a combination of high pressure liquid chromatography and ion exchange chromatography on a Dionex apparatus as previously described by Reddy and Bush.
(Analytical Biochemistry, volume 198, pages 278-284, 1991) and Towneend and Hardy (Glycobiology, volume 1, pages 139-147,1991). These techniques are well known and standard in this field. The specifics of the experimental establishment, the elution profiles and the conditions for the separation and analysis of the oligosaccharide extracts, were as follows: The Dionex apparatus was adapted with a degasser to remove the C02 from the elution regulators, a suppressor of ions to remove ions from the eluents in the column, and an online conductivity meter to ensure the removal of ions by the ion suppressor. The parameters of the chromatography were as follows:Test time: 45 minutes Peak Width: 50 seconds Peak Peak: 0.500 Peak Area Rejection: 500 Injection Volume: 20 liters Flow Rate: 1.0 milliliters / minute.
The gradient elution program, presented in Table 3, comprised the following tree eluyentee:Eluent 1: 600 mM eodium acetate in 100 mM sodium hydroxide 5.
Eluent 2: Water Milli-Q NanoPureEluent 3: Sodium hydroxide 200 M *? 0TABLE 3 ELUTION GRADIENT PROGRAM Time (minute) Flow (ml / min)% # 1% # 2% # 30.0 1.0 0 50 50 12.0 1.0 0 50 50 12.1 1.0 7 46 47 20.0 1.0 7 46 47 20.1 1.0 10 45 45 20 27.0 1.0 10 45 45 27.1 1.0 50 25 25 32.0 1.0 50 25 25 32.0 1.0 0 50 50 45.0 1.0 0 50 50 25 90.0 0.1 0 50 50 The eluted fractions were collected every 0.5 minutes.
The chromatographic profiles of the milk samples obtained from two control mice and four transgenic animals expressing alpha-1, 2-fucosyltransferase, are shown in Figures 6A to 6F. It was determined that 2'-fucosyl lactose (which is the oligosaccharide product synthesized by the enzyme encoded by the transgene) is eluted after the lactose. A review of the profiles revealed that only the transgenic animals produce milk containing a carbohydrate that co-elutes with the standard 2 '-fucosyl lactose.
Based on the chromatographic peak areas, it was possible to calculate the concentrations of 2 '-fucosyl-lactoea preeente in the milk diaetrates from tranegenic animals using eetándaree technique. The data is summarized in Table 4.
TABLE 4 i Concentration of 2 • -fucosyl lactose in different samples of non-human milk. Donor Concentration of 2 '-fucosyl lactose (mcr / L)1. Control (non-transgenic) 0 2. Transgenic 28-29 711 29-119 468 34-34 686 72-66 338These data prove, in accordance with the present invention, that a secondary gene product, ie, a 2 '-fucosyl-lactoea, can be produced in the milk of non-human tranegenic mammals. To further characterize the oligosaccharide, a different method was used for the analysis of carbohydrates. The electrophoresis of carbohydrate aeietide by fluorophore (FACE) is a technology first described by P. Jackson, J. Chromatography, volume 270, page 705-713, 1990. The FACE technique was employed to unequivocally demonstrate that the carbohydrate that co-elutes with the 2'-fucoeil-lactoea has the same mobility as the authentic 2'-fucoeil-lactoea standards in an electrophoresis system. This provides further confirmation that the identity of the oligosaccharide contained in the transgenic milk sample is 2 '-fucosyl-lactoea. In order to conduct FACE experiments with the putative 2 '-fucosyl lactose, the separated fractions were pooled during Dionex-HPLC chromatography. The fractions between the arrows (indicated on the left) in Figures 6A to 6F were grouped from each sample. Portions of each group (1/8) were obtained from the control and from two transgenic mice, and labeled for 3 hours at 45 ° C using 8-aminonaphthalen-2,3,6-trisulfonic acid (ANTS) from Glyko Inc. (Novato, California). The secae samples were resuspended in 5 microliters of the labeling reagent, and 5 microliters of reducing reagent solution (sodium cyanoborohydride) was incubated at 45 ° C for 3 hours. The resulting labeled samples were dried and re-suspended in 6 microliters of deionized water. From this solution, a 2 microliter aliquot was transferred to a fresh microcentrifuge tube. Two microliters of charge buffer containing glycerol was added, and then the mixture was mixed vigorously. Then the total mixture (4 microliters) was subjected to electrophoreation in an "O-linked oligosaccharide gel" (Glyko). Electrophoresis was conducted at a constant current of 20 milliamps and at 15 ° C. The profile of the migrated gel pattern thus obtained is imaged using a Millipore imaging apparatus. Figure 7 shows the image of the gel obtained in this way. The sample from a control mouse (lane 2) shows a single band that migrates at the position of a standard lactose marker. The samples obtained from transgenic mice (lanes 3 and 4) contained an additional band of a higher molecular weight. This band indicated in the figure with an arrow, migrates in the position of a standard 2 '-fucosil-lactoea (pieta 6). Another characterization of the oligosaccharide was performed by incubating aliquots equivalent to 1/8 of the groups illustrated in Figures 6A to 6F in the. presence of the enzyme fucosidase, which is specifically dissociated in the alpha-1,2-fucose bond. This enzyme used was derived from Corynebacterium sp. and was purchased at Panvera Corp. Madison, WI. The dried oligosaccharides were incubated overnight in the presence of 20 microliters of sodium phosphate buffer, pH 6.0, containing 20 milliunits of the enzyme at 37 ° C. Then the digests were labeled with ANTS, and subjected to electrophoresis as described above. The gels in Figure 8 show the results of this experiment. It can be easily seen that the material that is co-eluted with 2 '-fucosyl lactose in the chromatography of Dionex-HPLC and that co-migrates with the same molecule after being labeled with ANTS and electrophoresis, is also susceptible to action of the specific hydrolytic enzyme, alpha-1,2-fucosidae. The 3 '-fucoeil-lactoea (which is the most similar isomer of 2' -fucosyl lactose) is not affected by the enzyme. This experiment further confirms the identity of the oligosaccharide in the transgenic milk sample as 2 '-fucosyl-lactoea. In contrast, the milk extract obtained from non-tranegenic control animals (tracks 6 and 14) in the wake of the hydrolysis, produce only one band (lanes 7 and 15) that migrates at the position of the galactose standard.
Example 6; Analysis to Test the Identity of the Oligosaccharide Produced in the Milk of Non-Human Transgenic Mammals. This experiment evaluated the monosaccharide units comprising the oligosaccharide. For this purpose, the pooled milk samples obtained from the control and the transgenic mice were thoroughly treated with a mixture of glucosidases. The aliquots (1/8 of the total in 20 microliters of water) of the groups illustrated in Figures 6A to 6F were dried by evaporation in conical tubes. The dry content was resuspended in 20 microliters of a solution containing 20 milliunidadee of alpha 1, 2-fucosidase (Panvera, Madison, Wisconein) and 20 microliter of a suspension containing 30 units of E. coli β-galactosidase ( Boehringer Mannheim, Indianapolis, Indiana). The resulting suspensions were incubated for 18 hours at 37 ° C under an atmosphere of toluene. In this way, only those oligoeaccharide susceptible to sequential actions of fucosidase and β-galactosidase in their corresponding monosaccharide units were hydrolyzed. After incubation, the mixtures were dried in a Speed Vac concentrator. The oligoeaccharides resulting from this hydrolysis were labeled as described above in Example 5. The labeled monosaccharides were subjected to electrophoresis in a "monosaccharide gel" (Glyko). Electrophoresis was performed at a constant voltage of 30 milliamps for 1 hour and 10 minutes. Figure 9 shows the results of this experiment. Milk samples obtained from transgenic animals (lanes 2 and 4) contain three bands that correspond to fucoea, galactoea, and glucoea. The monosaccharides released from a standard 2 '-fucosyl-lactose (lane 1) are identical to the monosaccharides released from the oligosaccharide groups obtained from two transgenic animals (lanes 2 and 4). The 3 '-fucosyl-lactoea is not affected by the enzymatic action of the glucosidases (lane 3). This result unequivocally establishes the identity of the oligosaccharide in the tranegenic milk as 2 '-fucosyl lactose.
Collectively, these results discussed above prove that the invention, as described and claimed, makes possible the production of secondary gene products in the milk of transgenic animals. More specifically, the experimental data prove the feasibility of obtaining oligosaccharides in the milk of transgenic animals that contains a transgene comprised, in part, of DNA encoding glycosyltransferases. To further corroborate the invention, it was decided to demonstrate the presence of other glucoconjugates, such as glycoproteins in the milk of the transgenic animals. These glycoproteins are covalent adducts of protein and oligosaccharide, wherein the oligosaccharide is the product of the glucosyltransferase. The oligosaccharide is covalently linked to the protein by the glycosyltransferase.
Example 7; Analysis that Tests the Production of Glucoconjugates in the Milk of Non-Human Transgenic Mammals. This example demonstrates the feasibility of obtaining glycoprotein in the milk of non-human tranegenic animals. The oligosaccharide fraction is the same oligoeaccharide produced as a result of the activity of the primary gene product, glycosyltraneferase. The resulting glycosylated protein is an example of a secondary gene product.
Western blots were prepared from the transgenic and control animal milk proteins in the manner described in Example 4. However, instead of incubating the transferred membrane with polyclonal rabbit antibodies, the membrane was incubated with the Ulex lectin europaeus agglutinin 1 (UEA 1). Eeta lectin specifically binds to the alpha-l, 2-fucose bond. For this purpose, the protein granules described in Example 3 were centrifuged at 13,000 x g for 10 minutes, the supernatant (excess of ethanol and water) was removed, and the resulting granules were resuspended in a sample regulator volume of SDS-PAGE equal to the original volume of milk. Five microliters of these extracts were electrophoresed on 12.5 percent polyacrylic amide SDS-PAGE, as described in detail in Example 3. Following the electrophoresis, the proteins were transferred to nitrocellulose membranes for 1 hour at 100 minutes. volts in 12.5 mM Tris-HCl, 96 mM glycine, 20 percent methanol, 0.01 percent SDS, pH 7.5. Lae nitrocellulose membranes were blocked for 1 hour with 2 percent gelatin in TBS (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl), and washed 3 x 5 minutes in TBS containing 0.05 percent Tween-20. . The membranes were then incubated for 18 hours in 1% gelatin / TBS containing a 1: 500 dilution of peroxidase-labeled UEA-1 (Sigma, St. Louis Mo.). Then the resulting membrane was washed, and the proteins were visualized by incubation in a mixture of 50 milliliters of TBS containing 0.018 percent hydrogen peroxide, and 10 milliliters of methanol containing 30 milligrams of 4-chloro-naphthol (Bio Rad, Richmond, California). It is clear that only the tranegenic animals produced milk containing fucoeilated proteins specifically recognized by the lectin UEA-1, these proteins migrated with a relative molecular weight of 35-40 kilodaltons, and These results indicate that glycoproteins carrying the 2 'oligosaccharide -fucosyl lactose (H antigen) have been produced in the milk of the animal. e transgenics carrying a transgene encoding alpha-1, 2-fucosyltransferase. The milk of non-transgenic control animals did not contain glycoproteins carrying 2 '-fucosyl lactose. Examples 3 to 7 have proven that it is possible to produce non-human transgenic mammals capable of synthesizing secondary gene products in their milk. More specifically, it is possible to produce non-human transgenic mammals that express human glycosyltraneferases in mammary tissue, resulting in the presence of human oligoeaccharide and glycosylated glycoconjugates in the milk of these animals.
Industrial ApplicabilityThe invention as described and claimed herein, solves a long-felt need in that it provides a means to obtain large quantities of oligoeaccharide and desired glycoconjugates. The desired oligosaccharides and glycoconjugates can be isolated from the- or milk of the transgenic mammals, and can be used in the preparation of pharmaceutical products, diagnostic kits, nutritional products, and the like. Whole tranegenic milk can also be used to formulate nutritional products that provide special benefits.
Tranegenic milk can also be used in the production of specialized nutritional food products. The invention as described and claimed, eliminates the laborious organic chemistry and the synthesis of immobilized enzymatic chemistry of these very important materials that have usein pharmaceutical, research, diagnostic, nutritional, and agricultural formulas.
Having described the preferred embodiments of the present invention, it will be obvious to the ordinarily skilled in this field, that various modifications may be made to the modalities described, and that it is intended that these modifications be within the scope of the present invention.
SEQUENCE LIST (1) GENERAL INFORMATION (i) APPLICANT: Abbott Laboratories, ROSS Products Division (ii) TITLE OF INVENTION: Humanized Milk (iii) SEQUENCE NUMBER: 1 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Donald O. Nickey ROSS Products Division Abbott Laboratories (B) STREET: 625 Cleveland Avenue (C) CITY Columbus (D) STATE Ohio (E) COUNTRY: United States of America (F) POSTAL CODE: 43215 (v) LEGIBLE FORM BY COMPUTER: (A) TYPE OF MEDIUM: 3.5-inch floppy disk, 1.44 Mb storage. (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) DATA FROM THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: Not applicable (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (614) 624- 7080 (B) TELEFAX: (614) 624-3074 (C) TELEX: none (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1,155 base pairs. (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple. (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cloned cDNA representing the product of a segment of human genomic DNA. (A) DESCRIPTION: GDP-L-fucoea-ß-D-galactoeide 2-alpha-fucosyltransferase. (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) TYPE OF FRAGMENT: The entire amino acid sequence is provided. (vi) ORIGINAL SOURCE: Cell Line of Human Epidermal Carcinoma. (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL INSULATED: (D) DEVELOPMENT STAGE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ) ORGANELO: (vii) IMMEDIATE SOURCE: Cell Line of Human Epidermal Carcinoma (A) LIBRARY: (B) CLON: (viii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: 19 (B) POSITION OF THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis. (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID NO: 1: ee the enzyme, GDP-L-fucose-β-D-galactoside 2-alpha-fucosyltraneferase, having the amino acid sequence described in SEQ. ID NO: 1 :. Eeta enzyme is responsible for the synthesis of 2 '-fucoeil-lactose.(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:GAATTCGGCT TATCTGCCAC CTGCAAGCAG CTCGGCC ATG TGG CTC CGG AGC CAT 55 Met Trp Leu Arg Ser His 1 5 CGT CAG CTC TGC CTG GCC TTC CTG CTA GTC TGT GTC CTC TCT GTA? TC 103 Arg Gln Leu Cys Leu Ala Phe Leu Leu Val Cys Val Leu Ser Val lie 10 15 20 TTC TTC CTC CAT ATC CAT CA GAC AGC TTT CCA CAT GGC CTA GGC CTG 151 Phe Phe Leu His lie His Gln Asp Ser Phe Pro His Gly Leu Gly Leu 25 30 35 TCG ATC CTG TGT CCA GAC CGC CGC CTG GTG ACÁ CCC CCA GTG GCC ATC 199 Ser lie Leu Cys Pro Asp Arg? Rg Leu Val Thr Pro Pro Val Ala He 40 45 50TTC TGC CTG CCG GGT ACT GCG ATG GGC CCC AAC GCC TCC TCT TCC TGT ¿< . < Phe Cys Leu Pro Gly Thr Wing Met Gly Pro Asn Wing Ser Ser Cys 55 60 65 70 CCC CAG CAC CCT GCT TCC CTC TCC GGC ACC TGG ACT GTC TAC CCC AAT 2? Pro Gln His Pro Wing Ser Leu Ser Gly Thr Trp Thr Val Tyr Pro Asn 75 80 85 GGC CGG TTT GGT AAT CAG ATG GGA CAG TAT GCC ACG CTG CTG GCT CTG 3-13 Gly Arg Phe Gly Asn Gln Met Gly Gln Tyr Ala Thr Leu Leu Ala Leu 90 95 '100 GCC CAG CTC AAC GGC CGC CGG GCC TTT? TC CTG CCT GCC ATG CAT GCC 391 Ala Gln Leu Asn Gly Arg Arg Ala Phe He Leu Pro Ala Met His Ala 105 110 115 GCC CTG GCC CCG GTA TTC CGC ATC ACC CTG CCC GTG CTG GCC CCA GAA 3 Wing Leu Wing Pro Val Phe Arg He Thr Leu Pro Val Leu Wing Pro Glu 120 125 130 GTG GAC AGC CGC ACG CCG TGG CGG GAG CTG CAG CTT CAC GAC TGG ATG 487 Val Asp Be Arg Thr Pro Trp Arg. Glu Leu Gln Leu His Asp Trp Met 135 140 145 150 TCG GAG GAG TAC GCG GAC TTG AGA GAT CCT TTC CTG AAG CTC TCT GGC 535 Ser Glu Glu Tyr Ala Asp Leu Arg Asp Pro Phe Leu Lys Leu Ser Gly 155 '160 165 TTC CCC TGC TCT TGG ACT TTC TTC CAC CAT CTC CGG GAA CAG ATC CGC 583 Phe Pro Cys Ser Trp Thr Phe Phe His His Leu Arg Glu Gln He? Rg 170 175 180 AGA GAG TTC ACC CTG CAC GAC CAC CTT CGG GAA GAG GCG CAG AGT GTG 631 Arg Glu Phe Thr Leu His Asp His Leu Arg Glu Glu Wing Gln Ser Val 185 190 195 CTG GGT CAG CTC CGC CTG GGC CGC ACA GGG GAC CGC CCG CGC ACC TT 679 Leu Gly Gln Leu Arg Leu Gly Arg Thr Gly Asp Arg Pro Arg Thr Phe 200 205 210 GTC GGC GTC CAC GTG CGC CGT GGG GAC TAT CTG CAG GTT ATG CCT CAG? 7 Val Gly Val His Val Arg Arg Gly Asp Tyr Leu Gln Val Met Pro Gln 215 220 225 230 CGC TGG AAG GGT GTG GTG GGC GAC AGC GCC TAC CTC CGG CAG GCC ATG 775 Arg Trp Lys Gly Val Val Gly Asp Ser Ala Tyr Leu Arg Gln Wing Met 235 240 245 GAC TGG TTC CGG GCA CGG CAC G? A GCC CCC GTT TTC GTG GTC? CC? GC 0? 1 Asp Trp Phe Arg Wing Arg His Glu Wing Pro Val Phe Val Val Thr Ser 250 255 260 AAC GGC ATG GAG TGG TGT AAA GAA AAC ATC GAC ACC TCC CAG GGC GAT 871 Asn Gly Met Glu Trp Cys Lys Glu Asn He Asp Thr Ser Gln Gly Asp 265 270 275 GTG ACG TTT GCT GGC GAT GGA CAG GAG GCT ACÁ CCG TGG AAA GAC TTT 9i_Q Val Thr Phe Wing Gly Asp Gly Gln Glu Wing Thr Pro Trp Lys Asp Phe 280 285 290 GCC CTG CTC ACA CAG TGC AAC CAC ACC ATT ACC ATT GGC ACC TTC 967 Ala Leu Leu Thr Gln Cys Asn His Thr He Met Thr He Gly Thr Phe 295 300 305 310 GGC TTC TGG GCT GCC TAC CTG GCT GGC GGA G? C ACT GTC TAC CTG GCC 1P1 '. Gly Phe Trp Wing Wing Tyr Leu Wing Gly Gly Asp Thr Val Tyr Leu Wing 315 320 235 AAC TTC ACC CTG CCA GAC TCT GAG TTC CTG AAG ATC TTT AAG CCG GAG ICU Asn Phe Thr Leu Pro Asp Ser Glu Phe Leu Lys He Phe Lys Pro Glu 330 335 340 GCG GCC TTC CTG CCC GAG TGG GTG GGC ATT AAT GCA GAC TTG TCT CCA My wing Wing Phe Leu Pro Glu Trp Val Gly He Asn Wing Asp Leu Ser Pro 345 350 355 CTC TGG ACÁ TTG GCT AAG CCT TGAGAGCCAG GGAAGCCGAA TTC nS5Leu Trp Thr Leu Wing Lys Pro 360 365