KR20090090328A - Avian derived erythropoietin - Google Patents

Abstract

Erythropoietin obtained from eggs laid by transgenic avians having avian N-linked and O-linked glycosylation patterns. ® KIPO & WIPO 2009

Description

Algae-derived erythropoietin {AVIAN DERIVED ERYTHROPOIETIN}

Related Application Information

This application discloses US Provisional Application No. 60 / 857,896, filed November 9, 2006; 60 / 877,601, filed December 28, 2006; And 60 / 918,504, filed March 16, 2007, as priority.

Field of invention

The present invention relates to the introduction of exogenous genetic material into algal cells and the expression of exogenous genetic material in algal cells. The present invention relates in particular to transgenic avian species, including chickens, quails and turkeys, and to birds that produce exogenous proteins, eg eggs containing pharmaceutical proteins.

background

Many natural and synthetic proteins are used in diagnostic and therapeutic applications; Many other proteins are under development or in clinical trials. Current protein production methods include isolation from natural sources and recombinant production in bacterial and mammalian cells. However, due to the complexity and cost of these protein production methods, efforts are underway to develop alternative methods. For example, methods have been reported for producing exogenous proteins in the milk of pigs, sheep, goats and cattle. This method has certain limitations, including variable generation levels due to the long generation period between the founder and the production transgenic group, the cost of predatory breeding and veterinary medicine, and the location of transgene insertion sites in the genome. Has Proteins are also produced using milling and malting processes from barley and rye. However, post-translational modifications of plants are different from post-translational modifications of vertebrates, which often have a significant effect on the function of exogenous proteins such as pharmaceutical proteins.

Like tissue culture and mammary gland bioreactors, algal fallopian tubes can potentially function as bioreactors. Successful methods of modifying algal genetic material that allow high levels of exogenous proteins to be secreted into the fallopian tubes and packaged into eggs will enable low cost production of large amounts of protein. Several advantages of this method include: a) rapid establishment of the transgenic herd via short generation periods (24 weeks) and artificial insemination; b) easily increased production by increasing herd size to meet the required production; c) post-translational modification of the expressed protein; 4) automated feeding and egg collection; d) natural sterile whites; And e) reduced processing costs due to the high concentration of protein in the whites.

The reproductive system of birds, including chickens, is widely described. Eggs consist of several layers that are secreted from egg yolk as they pass through the fallopian tubes. Egg production begins with the formation of a huge egg yolk in the hen's ovary. The unfertilized oocytes are then placed on top of the yolk sac. Upon ovulation or release of egg yolk from the ovary, oocytes pass through the infuundibulum of the fallopian tube, where it is fertilized if semen is present. The oocytes then migrate to the magnum of the fallopian tube in which the tubular gland is lined. These cells secrete white protein, including egg albumin, lysozyme, ovomucoid, conalbumin, and ovomucin, into the lumen of the bulge, where they are deposited in avian embryos and egg yolks.

The egg albumin gene encodes a 45 kD protein that is specifically expressed in coronary cells of the bulge of the fallopian tube (Beato Cell 56: 335-344 (1989)). Egg albumin is a very abundant white protein that exceeds 50 percent of the total protein produced by coronary cells or contains about 4 grams of protein per large Class A egg (Gilbert, "Egg albumen and its formation" in Physiology and Biochemistry of the Domestic Fowl, Bell and Freeman, eds., Academic Press, London, NY, pp. 1291-1329). The egg albumin gene and each flanking region greater than 20 kb were cloned and analyzed (Lai et al., Proc. Natl. Acad. Sci. USA 75: 2205-2209 (1978); Gannon et al., Nature 278: 428-424 (1979); Roop et al., Cell 19: 63-68 (1980); and Royal et al., Nature 279: 125-132 (1975).

Much attention has been focused on the regulation of egg albumin genes. The gene is a steroid hormone, e.g. estrogen, glucocorticoid, which induces the accumulation of about 70,000 egg albumin mRNA transcripts per coronary cell of immature chickens and 100,000 egg albumin mRNA transcripts per coronary cell of mature laying hens. And progesterone (Palmiter, J. Biol. Chem. 248: 8260-8270 (1973); Palmiter, Cell 4: 189-197 (1975)). DNAse hypersensitivity analysis and promoter-reporter gene analysis in transfected coronary cells identified a region of 7.4 kb that contained sequences required for egg albumin gene expression. This 5 'flanking region contains four DNAseI-sensitive sites concentrated at -0.25, -0.8, -3.2 and -6.0 kb from the transcription start site. Such sites are referred to as HS-I, -II, -III and -IV, respectively. This region reflects alterations in chromatin structure and is specifically related to the expression of egg albumin genes in fallopian cells (Kaye et al., EMBO 3: 1137-1144 (1984)). The hypersensitivity of HS-II and -III is induced by estrogen, which supports the role of this region in hormone-induced egg albumin gene expression.

Both HS-I and HS-II are required for steroid induction of egg albumin gene transcription, and the 1.4 kb portion of the 5 'region containing these elements drives steroid-dependent egg albumin expression in explanted coronary cells. (Sanders and McKnight, Biochemistry 27: 6550-6557 (1988)). HS-I is referred to as a negative-responsive element (“NRE”) because HS-I contains several negative regulatory elements that inhibit egg albumin expression in the absence of hormones (Haekers et al., Mol. Endo 9: 1113-1126 (1995)). Protein factors, including several factors found only in the fallopian nucleus, bind to this element, suggesting a role in tissue-specific expression. HS-II is referred to as a steroid-dependent response element (“SDRE”) because HS-II requires steroid induction of transcription. HS-II binds to a protein or protein complex known as Chirp-I. Chirp-I is induced by estrogen and is rapidly converted in the presence of cyclohexamide (Dean et al., Mol. Cell. Biol. 16: 2015-2024 (1996)). Experiments with an explanted coronary cell culture system have identified a further set of factors that bind to SDRE in a steroid-dependent manner including NFκB-like factors (Nordstrom et al., J. Biol. Chem. 268: 13193-). 13202 (1993); Schweers and Sanders, J. Biol. Chem. 266: 10490-10497 (1991)).

Less is known about the function of HS-III and -IV. HS-III contains a functional estrogen response element and confers estrogen inducibility to the egg albumin adjacent promoter or heterologous promoter when co-transfected with HeLa cells with the estrogen receptor cDNA. These data indicate that HS-III can play a functional role in the overall regulation of the egg albumin gene. Little is known about the function of HS-IV except that HS-IV does not contain a functional estrogen-responsive element (Kato et al., Cell 68: 731-742 (1992)).

Much attention has been paid to modifying the eukaryotic genome by introducing foreign genetic material and / or destroying certain genes. Certain eukaryotic cells may prove to be good hosts for the production of exogenous eukaryotic proteins. The introduction of genes encoding specific proteins also allows the generation of new phenotypes with increased economic value. In addition, some genetically induced disease states can be treated by the introduction of foreign genes that allow genetically defective cells to express proteins that otherwise would not be able to produce. Finally, alteration of the animal genome by insertion or removal of genetic material allows for a basic study of gene function, ultimately allowing the introduction of genes that can be used to treat disease states, or for improved animal phenotypes. Can be generated.

Transgenesis has been achieved in animals by several different methods. First, in animals including mice, pigs, goats, sheep and cows, the transgene is microinjected into the pronucleus of the fertilized egg, and then implanted in the womb of the foster mother and trans Produces gin-containing prototype animals. Transgenes are designed to contain promoters with specific regulatory sequences that direct expression of foreign proteins to specific cell types. Since the transgene is randomly inserted into the genome, the effect of position at the insertion site of the transgene into the genome can variably reduce the level of transgene expression. This method also requires the characterization of the promoter to ensure that the sequences required to express the transgene in the desired cell type are included and included in the transgene vector (Hogan et al. Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY (1988).

A second method for performing animal transfection is targeted gene disruption wherein a target vector containing the sequence of the target gene flanked by the selectable marker gene is introduced into an embryonic stem ("ES") cell. By homologous recombination, the target vector is inserted into an internal sequence that replaces the target gene sequence at the chromosomal locus or prevents the expression of the target gene product. Clones of ES cells with appropriately disrupted genes are selected and then injected into early stage follicles, resulting in chimeric prototypes, some of which have transgenes in the dot line. When a transgene deletes a target locus, it consists of DNA encoding the target locus, a selection marker useful for detecting transfected ES cells in culture, which then encodes a foreign protein inserted in place of the deleted gene. The DNA sequence is replaced with a foreign DNA borne in the transgene vector, which may further contain, and the target gene promoter drives the expression of the foreign gene (US Pat. Nos. 5,464,764 and 5,487,992 (MP Capecchi and KR Thomas) ). This method has the limitation that ES cells are not available in many mammals, including goats, cattle, sheep and pigs. Moreover, such methods are not useful where the deleted gene is necessary for the survival or proper development of the organism or cell type.

Recently developed algal transgenics allow modification of the algal genome. Pointline transgenic chickens can be produced by injecting a retrovirus that is defective in replication into the dorsum of a new chick blastocyst (US Pat. No. 5,162,215; Bosselman et al., Science 243: 533-534). (1989); Thoraval et al., Transgenic Research 4: 369-36 (1995). Retroviral nucleic acids with foreign genes are randomly inserted into the chromosomes of embryonic cells, resulting in transgenic animals, some of which carry a transgene in the dot line. The use of an insulator element inserted in the 5 'or 3' region of a fused gene construct to overcome the site effect at the insertion site is described (Chim et al., Cell 74: 504-514 (1993) )).

In another method, transgenes were microinjected into embryonic discs of fertilized eggs to produce stable transgenic progenitor algae capable of delivering genes to the F1 generation (Love et al., Bio / Technology 12: 60-63 (1994)). . However, this method has several disadvantages. Hens must be sacrificed to harvest fertilized eggs, fewer fractions of transgenic ancestors are needed, and injected eggs require labor-intensive in vitro culture in a surrogate shell.

In another method, blastocyst cells containing putative primordial germ cells (“PGCs”) are excised from donor eggs, transfected with a transgene, and introduced into the embryonic embryo. Transfected donor cells are injected into receptor embryos to produce transgenic embryos, some of which are expected to have transgenes in the dot line. Transgenes are randomly inserted at chromosomal sites by nonhomologous recombination. However, transgenic precursor algae have not yet been produced by this method.

See Lui, Poult. Sci. 68: 999-1010 (1995) used a targeting vector containing the contiguous DNA sequence of the vitelogenin gene to delete a portion of the gene present in chicken hemiblastoma cells in culture. However, it has not been demonstrated whether these cells can contribute to this point line to produce transgenic embryos. In addition, this method is not useful where the deleted gene is necessary for the survival or proper development of the organism or cell type.

Thus, it can be appreciated that there is a need for a method by which foreign DNA operably linked to appropriate promoters can be introduced into the avian genome, whereby effective expression of the exogenous gene can be achieved. Moreover, there is a need to generate pointline modified transgenic birds that express exogenous genes in the fallopian tubes and secrete the expressed exogenous protein into eggs.

When interferon was discovered in 1957, it was welcomed as a significant antiviral agent. In the late 1970s, interferon was involved in recombinant gene technology. Today, interferon is a symbol of the complexity of biological processes in cancer and has become a worthy entity of persistence and survival against this complexity.

Abnormal genes that cause cancer include three or more types: First, there are oncogenes that promote abnormal growth and division that characterize cancer upon alteration. Second, there are tumor suppressor genes that fail to control abnormal growth and division upon alteration. Third, there are DNA repair genes that fail to repair mutations that can cause cancer by alteration. The researchers speculate that there are about 30 to 40 tumor suppressor genes in the body, each of which produces a protein. Such proteins can be controlled by "master" tumor suppressor proteins such as Rb (first related to retinoblastoma) and p53 (associated with many different tumors). Evidence from the experiments suggests that returning only one of the tumor suppressor genes to its normal function can significantly reduce the aggressiveness of the malignancy.

When scientists found that interferon could inhibit cell growth, they became interested in interferon. In addition, interferons have been found to have certain positive effects on the immune system. Currently, interferon is considered similar to tumor suppressor protein; Inhibit the growth of cells, especially malignant cells; Block the effects of multiple oncogenes and growth factors; Unlike other biological agents, it is considered to inhibit cell movement important for the process of metastasis.

Intercellular signaling is based on the proper functioning of all structural components of the tissue to which a message is delivered, such as the substrate, cell membrane, cytoskeleton and the tubule itself. In cancer, the signaling network between cells is disrupted. When the cytoskeleton divides, the message does not reach the nucleus, and the nucleus begins to function abnormally. Since the nucleus is the site where the oncogene or tumor suppressor gene is opened and closed, this abnormal function can lead to malignancy. When this happens, the cells grow irregularly and start not to differentiate. It also migrates and begins to divide other cells. Perhaps in cooperation with other extracellular and cellular materials, interferon is believed to restore balance and homeostasis, ensuring that the message arrives correctly. Interferon stops growth, stops movement, and enhances the cell's ability to respond to the environment through adhesion molecules. It also corrects defects and damage in the cytoskeleton. Interferon has been shown to block angiogenesis, the first step in the formation of new blood vessels essential for the growth of malignancies. Moreover, it blocks fibrosis, a response to damage that stimulates many different types of cells and promotes cell growth (Kathryn L. Hale, Oncolog, Interferon: The Evolution of a Biological Therapy, Taking a New Look at Cytokine Biology) .

Interferons are produced by animal cells when they are invaded by a virus and are released into the bloodstream or tissue fluid to induce healthy cells to produce enzymes that fight infection. For many years, the supply of human interferon for research has been limited due to expensive extraction techniques. However, in the 1980s, higher amounts of protein were made available through genetic engineering (ie, recombinant forms of proteins). The scientists also decided that the body made three distinct types of interferon, referred to as α- (alpha), β- (beta) and γ- (gamma) interferons. Although interferon was initially thought to be very species-specific, it is presently known that individual interferons can have a wide range of activity in other species. Alpha interferon (α-IFN) has been approved for the treatment of hairy cell leukemia and hepatitis C. α-IFN has also been found to be effective in treating chronic hepatitis B, a major cause of liver cancer and cirrhosis, as well as genital warts and some rare cancers of the blood and bone marrow. Intranasal sprays containing α-IFN provide constant protection against colds caused by rhinoviruses. Human α-IFN belongs to the family of extracellular signaling proteins with antiviral, antiproliferative and immunomodulatory activity. IFN-α protein is encoded by a polygenic family comprising 13 genes clustered on human chromosome 9. Most of the IFN-α genes are expressed at the mRNA level in leukemia caused by Sendai virus. In addition, at least nine different subtypes have also been found to be produced at the protein level. The biological significance of the expression of several similar IFN-a proteins is unknown, but they are believed to have quantitatively individual patterns of antiviral, growth inhibition and killer cell-stimulating activity. Currently, two IFN-α variants, IFN-α2a and IFN-α2b, are produced in large quantities in E. coli by recombinant technology and are commercially available as drugs.

Unlike native IFN-α, this recombinant IFN-α product has been found to be immunogenic in some patients, which may be due to the unnatural form of IFN-α protein. Thus, for the development of IFN-α drugs, it is necessary to identify IFN-α subtypes and variants expressed in normal human leukocytes, as well as to characterize their possible post-translational modifications (Nyman et al. (1998) Eur. J. Biochem. 253: 485-493).

Nyman et al. (Supra) studied glycosylation of native human IFN-α. They found that two of the nine subtypes produced by leukocytes after Sendai-virus induction, that is, glycosylated IFN-α14c and IFN-α2b, which is consistent with previous studies. IFN-α14 is the only IFN-α subtype with the potential N-glycosylation sites Asn2 and Asn72, but only Asn72 was actually glycosylated. IFN-α2 was O-glycosylated at threonine 106 (Thr106). Interestingly, other IFN-α subtypes do not contain Thr at this position. In this study, Neiman et al. Liberated and separated oligosaccharide chains and analyzed their structures by mass spectrometry and specific glycosidase digestion. Both IFN-α2b and IFN-α14c were analyzed with three peaks in reverse phase high performance liquid chromatography (RP-HPLC). Electrospray ionization mass spectrometry (ESI-MS) analysis of IFN-α2b fractions from RP-HPLC showed differences in their molecular masses, suggesting that they have different protein glycoforms. This was confirmed by mass spectrometry of the free O-glycans of each fraction. IFN-α2b was estimated to contain about 20% core type-2 pentasaccharide, and about 50% isialylated core type-1 glycan and 30% temporarily allylated core type-1 glycan . Neman et al. Data are consistent with previous characterization of IFN-α2b glycosylation (Adolf et al. (1991) Biochem. J. 276: 511-518). The role of glycosylation in IFN-α14c and IFN-α2b is not clearly established. According to Neiman et al. (See above), carbohydrate chains are not essential for biological activity, but glycation can affect the pharmacokinetics and stability of the protein.

There are more than 15 functional genes encoding proteins of the IFN-α family in the human genome. Amino acid sequence similarity is generally present in about 90% of the region, so these molecules are structurally closely related. The IFN-α protein contains 166 amino acids (except IFN-α2 with 165 amino acids) and features four conserved cysteine residues that form two disulfide bonds. IFN-α species are characteristically slightly acidic and lack a recognition site for asparagine-related glycosylation (except IFN-α14, which contains a recognition site for asparagine-related glycosylation). IFN-α2a (Lys-23, His-34), which is a variant of three IFN-α2 with different amino acids at positions 23 and 34; IFN-α2b (Arg-23, His-34); And IFN-α2c (Arg-23, Arg-34) are known. IFN-α2a and IFN-α2c are thought to be allelic variants of IFN-α2b. See, Gewert et al (1993) J. Interferon Res. vol 13, p 227-231]. Small differences in amino acid content of IFN-α2 species are not expected to affect the glycosylation of interferon. That is, the glycosylation pattern is expected to be essentially the same for each of IFN-α2a, 2b and 2c. Two other human IFN species, IFN-ω1 and IFN-β, are N-glycosylated and are more indirectly related to IFN-α. IFN-α, -β, and -ω, collectively referred to as Class I IFNs, bind equally high affinity to cell membrane receptors (Adolf et al. (1991) Biochem. J. 276: 511-518).

Adolf et al. (Supra) used the specificity of monoclonal antibodies for the isolation of native IFN-α2 from human leukocyte IFN. They obtained 95% pure protein via immunoaffinity chromatography and confirmed the expected antiviral activity of IFN-α2. Analysis of native IFN-α2 by reversed phase HPLC showed that the native protein could be broken down into two components that are more hydrophilic than E. coli derived IFN-α2. SDS / PAGE indicated that the protein was also heterogeneous in molecular weight, producing three bands, all of which showed lower electrophoretic mobility than equivalent E. coli derived proteins.

Adolf et al. (See above) also speculate that native IFN-α2 has O-linked carbohydrate moieties. Their hypothesis was confirmed by the degradation of putative peptide-carbohydrate bonds with alkalis; The resulting protein was homologous and showed the same molecular weight as the recombinant protein. Further comparison of native and recombinant proteins after proteolysis and subsequent isolation and analysis of the resulting fragments made it possible to identify candidate glycopeptides. Sequencing of these peptides identified Thr-106 as an O-glycosylation site. Comparison of the amino acid sequences of all published IFN-α2 species showed that these threonine residues were unique for IFN-α2. All other proteins had glycine, isoleucine or glutamic acid at the corresponding site 107.

The preparation of IFN-α2 produced in E. coli lacks O-glycosylation and is registered as a medicament in many countries. However, the immunogenicity of E. coli-derived IFN-α2 therapeutically applied may be affected by a lack of glycosylation. Studies showed that 4 out of 16 patients receiving recombinant human granulocyte-macrophage colony-stimulating factors produced in yeast developed antibodies to the protein. Interestingly, such antibodies have been found to be protected by O-linked glycosylation at endogenous granulocyte-macrophage colony-stimulating factors but react with epitopes exposed at recombinant factors (Adolf et al., Supra).

Similarly, induction of antibodies to recombinant E. coli-derived IFN-α2 after long-term treatment of patients has been described and it has been speculated that native IFN-α2 may be less immunogenic than recombinant IFN-α2 proteins (Galton et al. (1989) ) Lancet 2: 572-573).

There is a need for improved methods of producing therapeutic or pharmaceutical proteins such as cytokines and antibodies comprising interferon, G-CSF and erythropoietin.

Summary of the Invention

The present invention provides vectors and methods for stably introducing exogenous nucleic acid sequences into the genome of algae by expressing exogenous sequences to alter the phenotype of the algae or to produce the desired protein. In particular, transgenic birds are produced that express exogenous sequences in the alveolar canal and deposit exogenous proteins, such as pharmaceutical proteins, on the eggs of the birds. Eggs of algae containing the exogenous protein are included in the present invention. The present invention is a novel form of therapeutic protein (eg, human cytokine), including interferon, G-CSF, G-MCSF and erythropoietin, which are effectively expressed in the oviducts of transgenic birds and are deposited with algal eggs. Provides additional.

In one aspect, the present invention relates to a protein (eg, a human protein) such as a cytokine produced in algae. In one particular embodiment, the present invention relates to human erythropoietin (eg, poultry derived erythropoietin) with a glycosylation pattern, wherein the erythropoietin is a transgenic chicken, transgenic quail or transgenic turkey. Obtained from. Human proteins, including cytokines, such as erythropoietin, which are produced in algae in isolated or purified form and are present in pharmaceutical compositions, are also included in the present invention. Isolation of recombinant proteins of the present invention comprising erythropoietin can be accomplished by methods that are readily apparent to those skilled in the art of protein purification. The preparation of formulations useful for producing pharmaceutical compositions is also well known in the art. In one embodiment, the proteins of the invention comprising erythropoietin have a glycosylation pattern obtained from poultry or avian fallopian cells, such as coronary cells (eg, chicken coronary cells).

One aspect of the invention provides a method of producing exogenous protein in certain tissues of algae. Exogenous proteins can be expressed in algae fallopian tubes, blood and / or other cells and tissues. In one embodiment, the transgene is introduced into the blastocyst cells of the embryo near stage X, for example, to produce a transgenic alga, whereby the protein of interest is expressed in the tubular cells of the bulge of the fallopian tube and secreted into the lumen, It is deposited on the egg white of the hard shell egg. The resulting transgenic alga can have a transgene in its dot line. Thus, exogenous genes can be delivered to birds by artificial introduction of exogenous genes into avian embryonic cells and stable delivery of exogenous genes to avian descendants by the Mendelian mode.

The present invention includes a method for producing exogenous protein in an algae fallopian tube. Such methods may provide a vector containing a coding sequence and a promoter operably linked to the coding sequence, such that the promoter may comprise a first step that may affect the expression of the nucleic acid in avian fallopian tubes. Next, transgenic cells and / or tissues can be generated, wherein the vector is introduced into a newly isolated avian embryonic germ leaf cell in culture or embryo, which vector sequence is randomly inserted into, for example, the algal genome. do. Finally, mature transgenic birds expressing exogenous proteins in the fallopian tubes can be derived from transgenic cells and / or tissues. This method also contains an exogenous protein, such as a pharmaceutical protein (eg, cytokine), when the exogenous protein expressed in the fallopian tube is also secreted into the fallopian lumen and deposited on the egg, eg the white of the hard shell egg. Can be used to produce algal eggs.

In one aspect, the generation of a transgenic bird by chromosomal insertion of the vector into the algal genome may comprise DNA transfection of embryonic germ leaf cells which are then optionally injected into the dorsum below the receptor hemisphere. The vector used in this method may have a promoter that is fused to the exogenous coding sequence and drives the expression of the coding sequence in the coronary cells of the fallopian tube.

In another embodiment of the invention, random chromosomal insertion and generation of transgenic birds are performed using embryos using replication-deficient or replication-qualified retrovirus particles with the transgene genetic code, between the 5 'and 3' LTRs of the retroviral vector. Achieved by transduction of blastocyst cells. For example, an avian leukemia virus (ALV) retroviral vector or a murine leukemia virus (MLV) retroviral vector comprising a modified pNLB plasmid containing an exogenous gene inserted downstream of a segment of a promoter region can be used. . RNA copies of modified retroviral vectors packaged into viral particles can be used to infect embryonic germ layers and develop transgenic birds. Alternatively, helper cells that produce retroviral transducing particles are delivered to embryonic germ cells.

Another aspect of the invention provides a vector comprising coding sequences and promoters of operative and positional relationships in which the coding sequence is expressed in avian fallopian tubes. Such vectors include, but are not limited to, avian leukemia virus (ALV) retroviral vectors, murine leukemia virus (MLV) retroviral vectors, and lentiviral vectors. The vector can also be a nucleic acid sequence comprising the LTR of an avian leukemia virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, or a lentiviral vector. The promoter is sufficient to influence the expression of the coding sequence of the algae fallopian tube. The coding sequence encodes an exogenous protein that is deposited into the egg white of the hard shell egg. As such, the coding sequences are exogenous proteins such as transgenic poultry derived proteins such as interferon-α2b (TPD IFN-α2b) and transgenic poultry derived erythropoietin (TPD EPO) and transgenic poultry derived granulocyte colonies. Encode stimulation factor (TPD G-CSF). In one embodiment, the vectors used in the methods of the invention contain promoters that are particularly suitable for the expression of exogenous proteins in algae and eggs thereof. As such, expression of the exogenous coding sequence can occur in the fallopian tubes of transgenic birds and in the whites of blood and eggs thereof. Promoters include cytomegalovirus (CMV) promoter, MDOT promoter, Raus-sarcoma virus (RSV) promoter, β-actin promoter (e.g. chicken β-actin promoter), murine leukemia virus (MLV) promoter, mouse breast cancer virus ( MMTV) promoter, egg albumin promoter, lysozyme promoter, cornalbumin promoter, ovomucoid promoter, ovomucin promoter, and ovotransferrin promoter. Optionally, the promoter may be a segment of one or more promoter regions, such as eggs of albumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and ovotransferin promoter region. In one embodiment, the promoter is a combination or fusion of one or more promoters or fusions of one or more promoter moieties such as eggalbumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and ovotransferin promoter.

One aspect of the invention truncates and / or shrinks an egg albumin promoter to ensure that it is small enough to be readily incorporated into a vector while still retaining the necessary sequence for expression in the coronary cells of the bulge of the fallopian tube. Or reduce important regulatory elements of the egg albumin promoter. For example, segments of egg albumin promoter regions can be used. This segment contains the 5'- flanking region of the egg albumin gene. The total length of the egg albumin promoter segment may be between about 0.88 kb and about 7.4 kb, preferably between about 0.88 kb and about 1.4 kb. The segment preferably comprises both steroid-dependent regulatory elements and negative regulatory elements of the egg albumin gene. Optionally, the segment also includes residues from the 5 'untranslated region (5'UTR) of the egg albumin gene. Alternatively, the promoter may be a segment of the promoter region of lysozyme-, conalbumin-, ovomucin-, ovomucoid-, and ovotransferrin gene. An example of such a promoter is a synthetic MDOT promoter consisting of elements from the ovomucoid (MD) and ovotransferrin (OT) promoters.

In another aspect of the invention, a vector integrated into the avian genome contains a constant expression promoter operably linked to an exogenous coding sequence (eg, cytomegalovirus (CMV) promoter, Raus-sarcoma virus (RSV) promoter) , And murine leukemia virus (MLV) promoter). Alternatively, non-expressed promoters can be used, such as the mouse breast cancer virus (MMTV) promoter.

Another aspect of the invention provides a transgenic bird having a transgene in the genetic material of the dot line tissue. More particularly, transgenes include promoters of operative and positional relationships for expressing exogenous genes and exogenous genes. Exogenous genes may be expressed in the algal fallopian tubes and blood of transgenic birds. Exogenous genes encode exogenous proteins, such as pharmaceutical proteins, including cytokines such as TPD IFN-α (eg IFN-α2) and TPD EPO and TPD G-CSF. Exogenous protein is deposited on the egg white of the hard shell egg.

Another aspect of the invention provides eggs of algae containing proteins that are exogenous to algal species. The use of the present invention allows for the expression of exogenous proteins in fallopian cells and the secretion of proteins into the lumen of the fallopian tube bulges and the deposition of algae eggs into the whites. The eggs packaged protein may be present in an amount of 1 gram or more per egg. Exogenous proteins include, but are not limited to, TPD IFN-α2 and TPD EPO and TPD G-CSF.

Another aspect of the invention provides a recombinant transgenic poultry derived interferon-α2b coding sequence encoding an optimized coding sequence of human interferon-α2b (IFN-α2b), ie a transgenic poultry derived interferon-α2b (TPD IFN-α2b). It provides an isolated polynucleotide sequence comprising. The invention also includes an isolated protein comprising the polypeptide sequence of TPD IFN-α2b, wherein the protein is N-acetyl-galactosamine, galactose, N-acetyl-glucosamine, sialic acid, and their at Thr-106. O-glycosylated into the combination.

The present invention further contemplates pharmaceutical compositions comprising the polypeptide sequence of TPD IFN-α2b, wherein the protein is N-acetyl-galactosamine, galactose, N-acetyl-glucosamine, sialic acid, and these at Thr-106. O-glycosylated with the combination of.

One aspect of the invention provides a coding sequence for an exogenous protein produced as described herein, wherein the coding sequence is a codon optimized for expression in birds, eg chickens. Codon optimization may be determined from codon usage of proteins expressed in at least one, preferably one or more algal cells (eg, chicken cells). For example, codon usage may be determined from nucleic acid sequences encoding the protein egg albumin, lysozyme, ovomucin and ovotransferin in chickens. For example, the DNA coding sequence for an exogenous protein is a Wisconsin package, version 9.1 (Wisconsin) with codon usage tables compiled from egg albumin, lysozyme, ovomucoid, and ovotransferin proteins from chickens (Gallus gallus). Package, version 9.1) (Genetics Computer Group, Inc., Madison, Wis.) May be optimized codons using the BACKTRANSLATE® program.

One aspect of the invention comprises an optimized coding sequence of human erythropoietin (EPO), ie a recombinant transgenic poultry derived erythropoietin coding sequence encoding transgenic poultry derived erythropoietin (TPD EPO). It provides an isolated polynucleotide sequence.

Another aspect of the invention provides a vector comprising a promoter of an operative and positional relationship to the first and second coding sequences and the first and second coding sequences for expressing the first and second coding sequences of avian fallopian tubes. In such embodiments, the vector may comprise an internal ribosome entry site (IRS) element located between the first and second coding sequences, where the first coding sequence encodes protein X and the second coding sequence is protein Y And one or both of protein X and protein Y are deposited on hard shell eggs (eg, whites).

For example, protein X can be the light chain (LC) of a monoclonal antibody and protein Y can be the heavy chain (HC) of a monoclonal antibody. Alternatively, a protein (eg, an enzyme) encoded by a second coding sequence can provide post-translational modifications of the protein encoded by the first coding sequence. The vector optionally comprises additional coding sequences and additional IRES elements, each coding sequence in the vector being classified from another coding sequence by an IRES element. Other examples of using IRES contemplated for use in the present invention are described, for example, in US patent application Ser. No. 11 / 047,184, filed January 31, 2005, which is incorporated herein by reference in its entirety. .

The present invention also contemplates a method of producing algal eggs containing proteins such as pharmaceutical proteins, including monoclonal antibodies, enzymes and other proteins. Such methods provide a vector with a promoter, coding sequence, and one or more IRES elements; Introducing the vector into avian embryonic germ leaf cells, wherein the vector sequence is randomly inserted into the algal genome to produce transgenic cells or tissues; Inducing mature transgenic algae from a transgenic cell or tissue. The induced transgenic bird can express a coding sequence in its fallopian tube, and the resulting protein is secreted into the fallopian lumen so that the protein is deposited on the white of the hard shell egg. The invention also encompasses progeny of transgenic birds that produce eggs containing recombinant proteins. Typically, the progeny will contain transgene in essentially all of the cells of the bird, or the cells of the progeny bird will not contain the transgene.

One important aspect of the present invention relates to algal hard shell eggs (eg, chicken hard shell eggs) containing exogenous peptides or proteins, including but not limited to pharmaceutical proteins. Exogenous peptides or proteins can be encoded by the transgene of a transgenic bird. In one embodiment, the exogenous peptide or protein (eg, pharmaceutical protein) is glycosylated. Proteins can be present in any useful amount. In one embodiment, the protein is present in an amount ranging from about 0.01 μg per hard-shell egg to about 1 gram per hard-shell egg. In another embodiment, the protein is present in an amount ranging from about 1 μg per hard-shell egg to about 1 gram per hard-shell egg. For example, the protein may range from about 10 μg per hard-shell egg to about 1 gram per hard-shell egg (eg, from about 10 μg per hard-shell egg to about 400 milligrams per hard-shell egg). May be present in amounts.

In one embodiment, the exogenous protein of the invention, for example exogenous pharmaceutical protein, is in the egg white. In one embodiment, the protein is present in an amount ranging from about 1 ng per milliliter of white to about 0.2 grams per milliliter of white. For example, the protein may be present in an amount ranging from about 0.1 μg per milliliter of white to about 0.2 grams per milliliter of white (for example, the protein may be from about 1 μg per milliliter of white to about 100 milligrams per milliliter of white) May be present in an amount in the range). In one embodiment, the protein is present in an amount ranging from about 1 μg per milliliter of white to about 50 milligrams per milliliter of white. For example, the protein may be present in an amount ranging from about 1 μg per milliliter of white to about 10 milligrams per milliliter of white (eg, the protein may be from about 1 μg per milliliter of white to about 1 milligram per milliliter of white) May be present in an amount in the range). In one embodiment, the protein is present in an amount of at least about 0.1 μg per milliliter of egg white. In one embodiment, the protein is present in an amount of at least 0.5 μg per milliliter of egg white. In one embodiment, the protein is present in an amount of at least 1 μg per milliliter of egg white. In one embodiment, the protein is present in an amount of at least 1.5 μg per milliliter of egg white.

The present invention contemplates the production of hard shell eggs containing any useful protein, including one or more pharmaceutical proteins. Such proteins include, but are not limited to, hormones, immunoglobulins or portions of immunoglobulins, cytokines (eg, GM-CSF, G-CSF, erythropoietin and interferon) and CTLA4. The invention also encompasses the generation of hard shell eggs comprising fusion proteins, including but not limited to, portions of immunoglobulins or translational globulins fused to certain useful peptide sequences. In one embodiment, the present invention provides for the generation of hard shell eggs containing antibody Fc fragments. For example, the egg may contain an Fc-CTLA4 fusion protein according to the present invention.

Algae developed from blastocyst cells into which vectors have been introduced are the G0 generation and are referred to as "eponymous". Archeopteryx is typically a chimera for each inserted transgene. That is, only some of the cells of the G0 transgenic bird contain the transgene (s). Typically, the G0 generation is also semiconjugated to the transgene (s). The GO generation can be crossed with non-transgenic animals to generate G1 transgenic offspring that are also semiconjugated to the transgene and essentially contain the transgene (s) in all of the bird's cells. G1 semiconjugate offspring can be crossed with non-transgenic animals to generate G2 semiconjugate offspring or can be crossed with G1 semiconjugate offspring to generate G2 progeny homozygous for the transgene. Substantially all cells of algae positive for transgenes derived from G1 progeny will contain the transgene (s). In one embodiment, a semiconjugate G2 progeny from the same line can be crossed to produce a G3 progeny homozygous for the transgene. In one embodiment, the semiconjugate G0 animals can be crossed together to generate a homozygous G1 progeny containing two copies of the transgene (s) in each cell of the animal. This is merely an example of a particular useful breeding plan and the present invention contemplates the use of any useful breeding plan as is known to those skilled in the art.

One aspect of the invention relates to a composition containing a protein produced according to the invention having a poultry derived glycosylation pattern, for example a chicken derived glycosylation pattern. One aspect of the invention relates to a composition containing a protein produced according to the invention having an algal derived glycosylation pattern, for example a chicken derived glycosylation pattern. For example, the present invention includes pharmaceutical proteins having one or more of the poultry derived glycosylation patterns, such as those described herein. The present invention also includes human proteins having one or more of poultry derived glycosylation patterns, eg, glycosylation patterns described herein.

In one aspect, the invention comprises G-CSF, which has a poultry derived glycosylation pattern, ie, transgenic poultry derived G-CSF or TPD G-CSF. In one embodiment, the invention comprises G-CSF, which has a transgenic algae derived glycosylation pattern, ie, a transgenic algae derived G-CSF. In one embodiment, the glycosylation pattern is different from the pattern of G-CSF produced in human cells and / or CHO cells. That is, the composition has G-CSF molecules with poultry or algae derived carbohydrate chains (ie, glycosylated structures) and such carbohydrate chains or glycosylated structures are not found in G-CSF obtained from human cells and / or CHO cells. However, the composition may also comprise G-CSF molecules having the same glycosylation structure as found in G-CSF obtained from CHO cells and / or human cells. Glycosylation of human G-CSF produced in CHO cells is described in Holloway, CJ., European J. of Cancer (1994) vol 3 OA, pS2-S6, which is incorporated herein by reference in its entirety; Oheda et al (1988) J. Biochem., V 103, p 544-546, which is hereby incorporated by reference in its entirety; And Andersen et al (1994) Glycobiology, vol 4, p 459-467, the entire contents of which are incorporated herein by reference. The structures such as A and G shown in Example 20 appear to be identical or similar to the glycosylated structures reported for G-CSF generated in CHO cells. In one embodiment, the glycosylation pattern of G-CSF produced according to the present invention is different from the pattern of G-CSF produced in mammalian cells.

In one embodiment, the present invention provides isolated G-CSF. That is, the G-CSF contained in the composition may be an isolated G-CSF. For example, G-CSF can be separated from the whites. An isolated G-CSF can be a G-CSF molecule with a different glycosylation structure in the G-CSF molecule, or an isolated G-CSF is an isolation of a G-CSF molecule with only one specific glycosylation structure of the species of the G-CSF molecule. May be individual species.

In one embodiment, the G-CSF of the composition of the present invention is present in the hard shell egg. For example, G-CSF may be present in the whites of the hard shell eggs produced by the transgenic birds of the present invention. That is, in one embodiment, the present invention relates to algae (eg chicken) whites containing the G-CSF of the present invention. In one embodiment, G-CSF is present in the whites in an amount greater than about 1 microgram per ml of whites. For example, G-CSF may be present in an amount greater than about 2 micrograms per ml of whites (eg, in an amount of about 2 micrograms to about 200 micrograms per ml of whites).

In one particular embodiment of the invention, G-CSF is glycosylated in alveolar fallopian cells, eg, in chicken fallopian cells. For example, G-CSF can be produced and glycosylated in fallopian cells. In one embodiment, G-CSF is glycosylated in coronary cell (eg, G-CSF is produced and glycosylated in coronary cell).

G-CSF is thought to be glycosylated at threonine 133. However, the present invention is not limited to glycosylation at any particular site on the G-CSF molecule.

Typically, the G-CSF of the present invention is human G-CSF. In one embodiment, mature G-CSF has the amino acid sequence of FIG. 18C.

In one embodiment, the compositions of the present invention comprise G-CSF molecules glycosylated with the following glycosylation structure:

In addition, the present invention relates in particular to compositions containing G-CSF molecules having one of the above specific glycosylation structures. Such compositions may also include one or more G-CSF molecules with one or more other glycosylation structures.

That is, in one embodiment, the invention specifically comprises a composition containing a G-CSF molecule having the following glycosylation structure:

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A composition containing a G-CSF molecule having the following glycosylation structure:

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A composition containing a G-CSF molecule having the following glycosylation structure:

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A composition containing a G-CSF molecule having the following glycosylation structure:

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A composition containing a G-CSF molecule having the following glycosylation structure:

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A composition containing a G-CSF molecule having the following glycosylation structure:

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To a composition containing a G-CSF molecule having the following glycosylation structure;

here,

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

The invention also relates to a method of increasing leukocyte cell number in a patient, comprising administering to the patient a therapeutically effective amount of G-CSF produced according to the invention. Typically, the therapeutically effective amount is the amount of G-CSF that increases the leukocyte cell number of the patient to the desired amount.

One aspect of the invention relates to a composition containing EPO, ie the EPO molecules produced according to the invention. In one particularly useful embodiment, the EPO is purified or isolated. For example, EPO is isolated from the contents of the hard shell eggs laid by transgenic birds. In one particularly useful embodiment, the EPO is human EPO. In one embodiment, the EPO of the present invention has a glycosylation pattern resulting from EPO produced in alveolar fall cells. Another aspect of the invention relates to a composition containing an EPO having a glycosylation pattern, wherein the glycosylation pattern is different from the glycosylation pattern of EPO produced in human cells or CHO cells, wherein the EPO is produced in chicken fallopian cells. . In one aspect, the present invention provides compositions containing isolated EPO (eg, human EPO) having an algal or poultry derived glycosylation pattern. For example, the composition may contain a mixture of EPO molecules produced according to the invention in algae, for example chicken, and isolated from egg whites. In one useful embodiment, the EPO containing composition is a pharmaceutical formulation.

In one embodiment, the oligosaccharides present on the EPO of the present invention do not contain fucose. In another embodiment, at least about 90% of the N-linked oligosaccharides present on the EPO of the present invention do not contain fucose. In another embodiment, at least about 80% of the N-linked oligosaccharides present on the EPO of the present invention do not contain fucose. In another embodiment, at least about 70% of the N-linked oligosaccharides present on the EPO of the present invention do not contain fucose. In another embodiment, at least about 60% of the N-linked oligosaccharides present on the EPO of the present invention do not contain fucose. In another embodiment, at least about 50% of the N-linked oligosaccharides present on the EPO of the present invention do not contain fucose.

In one embodiment, at least about 95% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid. In another embodiment, at least about 90% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid. In another embodiment, at least about 80% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid. In another embodiment, at least about 70% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid. In another embodiment, at least about 60% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid. In another embodiment, at least about 50% of the N-linked oligosaccharides present on the EPO of the present invention do not contain sialic acid.

In one embodiment, at least about 95% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 90% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 80% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 70% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 60% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 50% of the N-linked oligosaccharides present on the EPO of the present invention contain terminal N-acetyl glucosamine.

In one embodiment, the N-linked oligosaccharide structure type present on the EPO molecules of the present invention is essentially free of fucose. In another embodiment, at least about 90% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain fucose. For example, if there are 20 oligosaccharide structure types, at least 18 of the structure types do not contain fucose. In another embodiment, at least about 80% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain fucose. In another embodiment, at least about 70% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain fucose. In another embodiment, at least about 60% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain fucose. In another embodiment, at least about 50% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain fucose.

In one embodiment, the N-linked oligosaccharide structure type present on the EPO molecules of the present invention is essentially free of sialic acid. In another embodiment, at least about 90% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain sialic acid. For example, if there are 20 oligosaccharide structure types, at least 18 of the structure types do not contain sialic acid. In another embodiment, at least about 80% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain sialic acid. In another embodiment, at least about 70% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain sialic acid. In another embodiment, at least about 60% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain sialic acid. In another embodiment, at least about 50% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention do not contain sialic acid.

In one embodiment, all of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 90% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine. For example, if there are 20 oligosaccharide structure types, at least 18 of these structure types will contain terminal N-acetyl glucosamine. In another embodiment, at least about 80% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 70% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 60% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine. In another embodiment, at least about 50% of the N-linked oligosaccharide structure types present on the EPO molecules of the present invention contain terminal N-acetyl glucosamine.

In one aspect, the present invention relates to an EPO obtained from a transgenic bird, such as a transgenic chicken, containing a transgene encoding an EPO. In one embodiment, the EPO is produced in avian fallopian cell, eg, coronary cell. In one embodiment, the EPO is contained in hard shell eggs, eg eggs in hard shells laid by birds, such as chickens. For example, EPO can be present in the contents of an intact hard shell egg. In one particularly useful embodiment, the EPO of the present invention is human EPO.

In one aspect, the invention relates to a composition containing an isolated EPO molecule, for example a human EPO molecule, wherein the EPO is produced in an alga containing a transgene that encodes the EPO. In one embodiment, the EPO is produced in fallopian cells (eg, coronary cells) of a transgenic bird (eg, a transgenic chicken), and the EPO is isolated from the whites of the transgenic bird. In one embodiment, the EPO of the present invention has the amino acid sequence of Figure 19A. EPO is considered to be N-glycosylated and / or O-glycosylated. In one embodiment, the EPO is glycosylated in the fallopian cells (eg, coronary cell) of a bird, eg, a chicken.

In one aspect, the invention relates to a composition, eg, a pharmaceutical formulation, containing isolated EPO, eg, human EPO, having an algal derived glycosylation pattern. In one aspect, the invention relates to a composition, eg, a pharmaceutical formulation, containing isolated EPO, eg, human EPO, with a poultry derived glycosylation pattern. In one aspect, the present invention relates to a composition, eg, a pharmaceutical formulation, containing isolated EPO, eg, human EPO, produced according to the present invention. In one embodiment, the EPO in the composition of the present invention contains a glycosylation pattern that is different from the glycosylation pattern of EPO produced in mammalian cells. In one embodiment, the EPO in the composition of the present invention contains a glycosylation pattern that is different from the glycosylation pattern of EPO produced in CHO cells and human cells. In one embodiment, the EPO of the present invention is attached to one or more N-linked oligosaccharide structures described herein (eg, the structure shown in FIG. 21). In one embodiment, the EPO of the present invention is attached to one or more O-linked oligosaccharide structures described herein (eg, the structure shown in FIG. 20).

One aspect of the invention relates to a method of treating a patient comprising administering to the patient an EPO obtained from a therapeutically effective amount of a transgenic bird. In one embodiment, the therapeutically effective amount is an amount that increases the red blood cell number to the desired amount. It is contemplated that the EPO produced according to the present invention can be used to treat chronic kidney disease, eg, a disease in which tissues do not maintain erythropoietin production.

One aspect of the invention relates to a composition containing an isolated glycosylated human protein molecule produced in the fallopian tube of a transgenic alga, wherein the transgenic algae (e.g., a transgenic chicken) comprises a transgene that encodes a human protein. And the human protein contains chicken derived oligosaccharides that are not generally present on human proteins. In one embodiment, the human protein is attached to one or more N-linked oligosaccharide structures (structures shown in FIG. 21) described herein. In one embodiment, the human protein is attached to one or more O-linked oligosaccharide structures described herein (eg, the structure shown in FIG. 20).

In one embodiment, the invention relates to an isolated protein molecule produced in the fallopian tube of a transgenic chicken, for example as described herein, wherein said transgenic chicken contains a transgene encoding a protein molecule, said Protein molecules contain chicken derived oligosaccharides. For example, protein molecules include GM-CSF, interferon β, fusion protein, CTLA4-Fc fusion protein, growth hormone, cytokines, structural interferon, lysozyme, β-casein, albumin, α-1 antitrypsin, Antithrombin III, collagen, factor VII, VII, VII (etc.), fibrinogen, lactoferrin, protein C, tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin, immunoglobulins, antibodies, immunity Toxin, factor VII, b-domain deletion factor VII, factor VIIa, factor VII, anticoagulant; Hirudin, alteplase, tpa, reteplase, tpa, tpa lacking three of the five domains, insulin, insulin lispro, insulin aspart, insulin glargine, Persistent insulin analogs, glucagon, tsh, polytropin-beta, fsh, pdgh, inf-beta 1b, ifn-beta 1a, ifn-gamma 1b, il-2, H-11, hbsag, ospa, dornase ) -Alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diphtheria toxin fusion protein, tnfr-Igg fragment fusion protein laronidase, dnaases, alefacept, tocitumomab (tositumomab), murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (adagumab) (Igg1) , Anakinra, biological modulators, nesiritide, human b-type sodium urinary excretion Peptide (hbnp), colony stimulating factor, pegvisomant, human growth hormone receptor antagonist, recombinant activating protein c, omalizumab, immunoglobulin e (Ige) blocker, ibritumomab thiuxetane Ibritumomab tiuxetan), ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigment hormone, somatomedin, luteinizing hormone, chorionic gonadotropin, hypothalamic hormone, etanercept, antidiuretic hormone , Prolactin and thyroid stimulating hormone, immunoglobulin polypeptide, immunoglobulin polypeptide D region, immunoglobulin polypeptide J region, immunoglobulin polypeptide C region, immunoglobulin light chain, immunoglobulin heavy chain, immunoglobulin heavy chain variable region, immunoglobulin light chain variable region and linker Glycosylated forms of the peptide. Proteins that are not normally glycosylated can be engineered to contain glycosylation sites to be glycosylated in algal systems, as is understood by those skilled in the art. In one embodiment, the isolated protein is attached to one or more N-linked oligosaccharide structures described herein (eg, the structure shown in FIG. 21). In one embodiment, the isolated protein is attached to one or more O-linked oligosaccharide structures described herein (eg, the structure shown in FIG. 20).

Features contemplated specifically for a particular protein described herein, eg, EPO (eg, composition, glycosylation structure), are also contemplated for other specific proteins described herein that may be produced in accordance with the present invention.

The present invention also provides a method of making a glycosylated protein described herein, such as erythropoietin, including generating a transgenic alga containing a transgene that encodes a protein (eg erythropoietin). And the protein is packaged in the eggs of the hard husk laid by the algae. Also included are eggs laid by algae containing proteins (eg erythropoietin).

The present invention also provides compositions containing a separate mixture of useful protein molecules, e.g., individual types of proteins described herein, wherein one or more of the protein molecules contained in the mixture can be produced by a transgenic algae. Which have particular oligosaccharide structures attached to the oligosaccharide structures described herein. For example, the present invention provides an isolated mixture of EPO molecules, such as human EPO molecules (eg, EPO of SEQ ID NO: 50), containing EPO molecules glycosylated with one or more of the following structures: Each of the other oligosaccharide structures is shown in FIGS. 20 and 21:

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Any useful combination of the features described herein is within the scope of the present invention, provided that the features included in any of the above combinations are not mutually inconsistent, as will be apparent from the context of the present invention, the specification, and the knowledge of those skilled in the art. .

Further objects and aspects of the invention will become more apparent upon an overview of the detailed description set forth below in conjunction with the accompanying drawings, which are briefly described below.

Brief description of the drawings

1A and 1B show an egg albumin promoter expression vector comprising an egg albumin promoter segment and a coding sequence of gene X encoding exogenous protein X. X is any exogenous gene or exogenous protein of interest.

2A, 2B, 2C and 2D show retroviral vectors of the present invention comprising an egg albumin promoter and a coding sequence of gene X encoding exogenous protein X. X is any exogenous gene or exogenous protein of interest.

2E depicts a method for amplifying exogenous genes for insertion into vectors of 2A and 2B.

2F depicts a retroviral vector comprising an egg albumin promoter that regulates the expression of the coding sequence of gene X, and an internal ribosomal binding site (IRES) element that enables expression of the second coding sequence of gene Y. X and Y are any genes of interest.

3A and 3B show graphical representations of ALV-derived vectors pNLB and pNLB-CMV-BL, respectively. Since NLB was not fully sequenced, measurements of bp (base pairs) were estimated from published data (Cosset et al., 1991; Thoraval et al., 1995) and the data discussed herein. The vector was shown to be both integrated into the chicken genome.

4A and 4B show the amount of β-lactamase (lactamase) in the blood serum of chimeric and transgenic chickens. In FIG. 4A, the concentration of viable lactamase in the serum of G0 chickens transduced with NLB-CMV-BL transgene was measured 8 months after hatching. Generation, gender and number of wing bands are shown. Lactamase serum concentrations were measured for G1 transgenic chickens after 6-7 months of hatching. Arrows indicate G1 chickens crossed from rooster 2395. In FIG. 4B, lactase serum concentrations were measured for G1 and G2 transgenic chickens. Arrows indicate G2 crossed from hen 5657 or rooster 4133. Samples from chickens 4133, 5308 and 5657 are the same as the samples of FIG. 4A. Samples from G2 birds crossed from 5657 were harvested 3 to 60 days after hatching. Samples from G2 birds crossed from 4133 were harvested 3 months after hatching.

FIG. 5 shows a family tree of a chicken comprising a transgenic locus covered by hen 5657 (FIG. 5A) or rooster 4133 (FIG. 5B). 2395 is a rooster with a number of transgenic loci. 2395 was crossed with non-transgenic hens, creating three offspring, each with a transgene at a unique location in the chicken genome. Briefly, not only did transgenic offspring appear in the expression data, but non-transgenic offspring were omitted from the pedigree. The number of bands is indicated by the following symbols: hen; □ rooster; Hens with NLB-CMV-BL transgenes; ■ Rooster with NLB-CMV-BL transgene.

6 depicts β-lactamase (lactamase) in the whites of hen 5657 and its descendants. In FIG. 6A, egg white from hen 5657 and its transgenic offspring was analyzed for active lactamase. The control is an untreated hen and the clutchmate is a non-transgenic G2 crossed from hen 5657. Collected in March 2000. Arrows indicate G2 crossed from hen 5657. In FIG. 6B, white samples from G2 transgenic hens (semiconjugates) with one copy of the transgene were compared to white samples of G3 hen 6978 (homozygotes) with two copies. The eggs were harvested in February 2001. Generation and blade width are shown on the left.

7 depicts β-lactamase (lactamase) in the eggs of G2 and G3 hens crossed from rooster 4133. In FIG. 7A, egg whites from four representative semiconjugate transgenic hens crossed from rooster 4133 were analyzed for active lactamase. The eggs were harvested in October 1999, March 2000 and February 2001 and at least four eggs per hen were analyzed one month after each set was collected. The control is white from untreated hens. The number of bands is shown on the left. An average of 4 hens was counted during each period. In FIG. 7B, the whites from the semiconjugate G2 transgenic hens were compared to the whites of the semiconjugate and homozygous transgenic G3 hens. The eggs were harvested in February 2001. Generation and transgene copy numbers are shown as data bars for each hen. Average concentrations for hens with one or two copies are shown at the bottom of the chart.

8A and 8B show pNLB-CMV-IFN vectors for expressing IFN-α2b in chickens; And pNLB-MDOT-EPO vectors used to express erythropoietin (EPO) in chickens, respectively.

FIG. 9 shows novel glycosylation patterns of transgenic poultry derived interferon-α2b (TPD IFN-α2b) comprising all six bands.

10 shows a comparison of human peripheral blood leukocyte derived interferon-α2b (PBL IFN-α2b or native hIFN) and transgenic poultry derived interferon-α2b (TPD IFN-α2b or egg white hIFN).

11A shows optimized human interferon-α2b (IFN-α2b), ie the synthetic nucleic acid sequence of recombinant TPD IFN-α2b (cDNA, residues 1-498) (SEQ ID NO: 1). 11B depicts the synthetic amino acid sequence (residues 1-165) (SEQ ID NO: 2) of transgenic poultry derived interferon-α2b (TPD IFN-α2b).

12A depicts optimized human erythropoietin (EPO), ie the synthetic nucleic acid sequence of recombinant TPD EPO (cDNA, residues 1-579) (SEQ ID NO: 3). FIG. 12B depicts the synthetic amino acid sequence (residues 1-193) (SEQ ID NO: 4) of transgenic poultry derived erythropoietin (TPD EPO) (see NCBI Accession Number NP 000790 for natural human EPO).

Figure 13 shows synthetic MDOT promoters linked to IFN-MM CDS. MDOT promoters include the chicken ovomucoid gene (obomucoid promoter) in the range of -435 to -166 bp (see NCBI Accession Number J00894) and the chicken conalbumin gene (obotransferrin promoter) in the range of -251 to +29 bp (NCBI Accession). Elements from Numbers Y00497, M11862 and X01205).

14 provides a summary of main white protein.

15A and 15D show the pCMV-LC-emcvIRES-HC vector, wherein the light chain (LC) of human monoclonal antibodies by placement of IRES from brain myocarditis virus (EMCV) to test expression of monoclonal antibodies. ) And heavy chains (HC) were expressed from the single vector. In comparison, FIGS. 15B and 15C show separate vectors pCMV-HC and pCMV-LC, respectively, which vectors were also used to test the expression of monoclonal antibodies.

FIG. 16 shows silver stained SDS PAGE of Neuupogen® (lane A) and TPD G-CSF (lane B).

FIG. 17 depicts the increase in total neutrophil levels (ANC) of TPD G-CSF relative to bacterial derived human G-CSF over a 14 day period.

FIG. 18A (SEQ ID NO: 39) shows the nucleotide sequence encoding the amino acid sequence of FIG. 18B. 18B (SEQ ID NO: 40), corresponding to NCBI Accession No. NP 7577373, shows the amino acid sequence of G-CSF, including the natural signal sequence, which degrades during cell secretion to form mature G-CSF. 18C (SEQ ID NO: 41) shows the amino acid sequence of mature G-CSF protein produced according to the present invention.

19A depicts a nucleotide encoding the sequence used to generate the 165 amino acid form of human erythropoietin in transgenic birds. 19B shows amino acid sequences in the 165 amino acid form of human erythropoietin produced in transgenic birds.

20 shows representative O-linked glycosylation structures determined for erythropoietin produced in accordance with the present invention.

21A and 21B show exemplary N-linked glycosylation structures determined for erythropoietin produced according to the present invention. Parentheses before the sugar residue group mean that the designated sugar (s) can be attached to the sugar in any parenthesis. For example, in structure E-n, the designated galactose molecule attached to sialic acid may be attached to any one of the five terminal n-acetyl glucosamines. Assumed bonds are also shown for the structure, which is understood in the art. For each of the structures designated C-n, E-n, F-n and H-n, it is contemplated that the two terminal NAcGlu residues linked to one mannose may be 2,6-bonded instead of 2,4-linked to mannose.

22 shows in vitro activity of purified transgenic chicken derived EPO. ED 50 = 0.44 ng / ml.

details

Specific definitions are described herein to illustrate and define the meaning and scope of various terms used to describe the present application.

“Nucleic acid or polynucleotide sequence” includes, but is not limited to, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, including native nucleoside base adenine, guanine, cytosine, thymidine and uracil. . The term also includes sequences having one or more modified bases.

As used herein, the term “algae” includes, but is not limited to, chickens, turkeys, ducks, geese, quails, pheasants, parrots, finch, falcons, crows and liquors such as ostrichs, emus and cassowary By any species, subspecies or variety of an organism of the upper class Ava. The term refers to known strains of Gallus gallus, or chickens (eg, White Leghorn, Brown Leghorn, Barred-Rock, Sussex). , New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray, as well as turkey, pheasant, quail, duck These include ostrich and other poultry systems commercially available and raised. It also includes individual algal organisms at all stages of development, including embryonic and fetal stages.

A "therapeutic protein" or "pharmaceutical protein" includes amino acid sequences that make up the drug in whole or in part.

A “coding sequence” or “open reading frame” is a polynucleotide or nucleic acid that can be transcribed and translated (for DNA) or translated (for mRNA) in vitro or in vivo when placed under the control of appropriate regulatory sequences Refers to the sequence. The range of coding sequences is determined by the translation start codon at the 5 '(amino) terminus and the translation stop codon at the 3' (carboxy) terminus. The transcription termination sequence will usually be located 3 'to the coding sequence. Coding sequences can be flanked at the 5 'and / or 3' ends by untranslated regions.

"Exon", when transcribed into a nuclear transcript, refers to the portion of a gene that "expresses" in cytoplasmic mRNA after removal of the intron or intervening sequence by nuclear splicing.

Nucleic acid “control sequences” or “regulatory sequences” are necessary and sufficient promoter sequences, translation start and stop codons, ribosome binding sites, adenylpolymerization signals, transcription termination sequences, for transcription and translation of provided coding sequences in defined host cells, Upstream regulatory domain, enhancer, and the like. Examples of regulatory sequences suitable for eukaryotic cells are promoters, adenylpolymerization signals, and enhancers. All of these regulatory sequences need not be present in the recombinant vector as long as there are sufficient regulatory sequences necessary for transcription and translation of the desired gene.

"Operably or operably linked" means the placement of coding and regulatory sequences to perform a desired function. Thus, a regulatory sequence operably linked to a coding sequence can achieve expression of the coding sequence. The coding sequence is operably linked to or located under the control of a transcriptional regulatory sequence in the cell such that the DNA polymerase binds to the promoter sequence and the coding sequence is transcribed into mRNA which can be translated into the encoded protein. Regulatory sequences need not be contiguous with the coding sequence as long as they operate to drive expression of the coding sequence. Thus, for example, an intervening yet untranslated transcriptional sequence may be present between the promoter sequence, and the coding sequence and the promoter sequence may still be considered "operably linked" to the coding sequence.

The term “heterologous” and “exogenous” when referring to nucleic acid sequences, such as coding sequences and regulatory sequences, usually refers to sequences that are not associated with a region of a recombinant construct or a particular chromosomal locus and / or usually with no particular cell. Thus, an “exogenous” region of a nucleic acid construct is a identifiable segment of nucleic acid that is present in or attached to another nucleic acid molecule that is not found to be associated with other molecules in nature. For example, the exogenous region of the construct may comprise coding sequences flanking sequences that are not found to be associated with coding sequences in nature. Another example of an exogenous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a synthetic sequence with a codon different from the native gene). Similarly, host cells transformed with a construct or nucleic acid generally not present in the host cell will be considered exogenous for the purposes of the present invention.

As used herein, the terms "oligosaccharide", "oligosaccharide structure", "glycosylation pattern" and "glycosylation structure" have essentially the same meaning, each of which has one or more structures formed from sugar residues and attached to glycated proteins. it means.

As used herein, an “exogenous protein” is a protein that is not naturally present in a particular tissue or cell, a protein that is an expression product of an exogenous expression construct or transgene, or a protein that is not naturally present in an amount provided to a particular tissue or cell. Means. Proteins that are exogenous to eggs are proteins that are not usually found in eggs. For example, a protein exogenous to the egg may be a protein present in the egg as a result of expression of the coding sequence present in the egg's transgene.

"Endogenous gene" means a naturally occurring gene or fragment thereof that is usually associated with a particular cell.

"EPO" means "erythropoietin" and these two terms are used interchangeably throughout this specification.

The expression products described herein may be composed of proteinaceous materials with defined chemical structures. However, it depends on various factors, especially chemical modifications that are common to proteins. For example, since all proteins contain ionizable amino and carboxyl groups, the proteins can be obtained in acidic or basic salt form, or in neutral form. Primary amino acid sequences can be derived using sugar molecules (glycosylation) or by other chemical derivatization reactions, including covalent or ionic bonds with lipids, phosphates, acetyl groups, and the like, which often occur through association with sugars. Such modifications can occur in vitro or in vivo, and in vivo modifications are performed by the host cell via a post-translational processing system. Such modifications can increase or decrease the biological activity of the molecule, and such chemically modified molecules are also within the scope of the present invention.

Alternative methods of cloning, amplification, expression and purification will be apparent to those skilled in the art. Representative methods are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

"Vector" means a polynucleotide consisting of single stranded, double stranded, circular or supercoiled DNA or RNA. Conventional vectors may consist of the following elements operably linked to appropriate distances to allow functional gene expression: origin of replication, promoter, enhancer, 5 'mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and adenyl Polymerization sites, and selective labeling sequences. One or more of these elements may be omitted in certain applications. The nucleic acid cassette may comprise a restriction site for insertion of the expressed nucleic acid sequence. In functional vectors, nucleic acid cassettes contain expressed nucleic acid sequences that include translation initiation and termination sites. Optionally, introns can be included in the construct, eg 5 'to the coding sequence. The vector is constructed such that a particular coding sequence is located in a vector having the appropriate regulatory sequence, and the positioning and orientation of the coding sequence relative to the regulatory sequence is such that the coding sequence is transcribed under the "regulation" of the control or regulatory sequence. Modifications of the sequences encoding a particular protein of interest may be desirable to achieve this purpose. For example, in some cases, it attaches to a controlled column in the proper orientation; It may be necessary to alter the sequence to maintain the translation framework. Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains a control sequence and an appropriate restriction site present in the translational framework with or under regulatory control of the control sequence.

A "promoter" is a site of DNA to which an RNA polymerase binds to initiate transcription of a gene. In some embodiments, the promoter will be modified by addition or deletion of sequences, or replacements using alternative sequences, including sequences that can be natural and synthetic sequences as well as sequences of synthetic and native sequences. Many eukaryotic promoters contain two types of cognitive sequences: a TATA box and an upstream promoter element. The TATA box located upstream of the transcription initiation site is involved in causing RNA polymerase to initiate transcription at the correct site, and the upstream promoter element appears to determine the rate of transcription and is located upstream of the TATA box. Enhancer elements can also stimulate transcription from related promoters, but many of these enhancers function exclusively in certain cell types. Many enhancer / promoter elements derived from viruses such as the SV40 promoter, cytomegalovirus (CMV) promoter, the Raus-sarcoma virus (RSV) promoter, and the murine leukemia virus (MLV) promoter are all active in a wide range of cell types, It is referred to as "ubiquitous". Alternatively, non-expressed promoters, such as the mouse breast cancer virus (MMTV) promoter, may also be used in the present invention. The nucleic acid sequence inserted at the cloning site may have any open reading frame that encodes the polypeptide of interest, provided that the coding sequence encodes the polypeptide of interest, which may block the production of appropriate mRNA molecules and / or abnormally splices. There must be a lack of latent splice sites that produce a mRNA or abnormal mRNA molecule.

The term “poultry derived” means a composition or substance produced by or obtained from poultry. "Poultry" means birds that can be raised as domestic animals, including but not limited to chickens, ducks, turkeys, quails and liquors. For example, “poultry derived” may mean chicken derived, turkey derived and / or quail derived.

A "labeled gene" is a gene that encodes a protein that allows for the identification and isolation of correctly transfected cells. Suitable label sequences include, but are not limited to, green, yellow and blue fluorescent protein genes (GFP, YFP and BFP, respectively). Other suitable labels include thymidine kinase (tk), dihydrofolate reductase (DHFR), and aminoglycoside phosphotransferase (APH) genes. Aminoglycoside phosphotransferases confer resistance to aminoglycoside antibiotics such as kanamycin, neomycin and geneticin. These and other marker genes, such as chloramphenicol acetyltransferase (CAT), marker genes encoding β-lactamase, β-galactosidase (β-gal), together with genes expressing the desired protein The selection marker may be incorporated into the primary nucleic acid cassette or the selection label may be contained in a separate vector and cotransfected.

A "reporter gene" is a marker gene that "reports" its activity in a cell by the presence of a protein encoded thereby.

"Retroviral particles", "particles that introduce traits" or "transduction particles" refer to replication-deficient or replication-competent viruses capable of transducing non-viral DNA or RNA into cells.

The terms "transformation", "transduction" and "transfection" all refer to the introduction of polynucleotides into avian blastocyst cells. The "bulge" is part of the fallopian tube between the papilla and the isthmus, which contain coronary cells that synthesize and secrete the egg whites.

As used herein, “MDOT promoter” is a synthetic promoter that is active in the coronary cells of the bulge of the fallopian tube in other tissues. MDOT consists of elements from the ovomucoid (MD) and ovotransferrin (OT) promoters (FIG. 13).

The term "optimized" is used in connection with "optimized coding sequence" and the most frequently used codons for each particular amino acid found in the egg white proteins egg albumin, lysozyme, ovomucoid and ovotransferrin are present invention It is used in the design of optimized human interferon-α2b (IFN-α2b) polynucleotide sequences that are inserted into a vector of. More specifically, the DNA sequence for optimized human IFN-α2b is based on hen oval optimization codon usage and codons aggregated from chicken (Gallus gallus) egg albumin, lysozyme, ovomucoid and ovotransferrin proteins It is created using the BACKTRANSLATE program of the Wisconsin Package, Version 9.1 (Genetics Computer Group Inc., Madison, Wis.) With a frequency table. For example, the percent usage for the four codons of amino acid alanine in the four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCU is used as codon for most alanines in optimized human IFN-α2b coding sequences. Vectors containing genes for optimized human IFN-α2b are used to generate transgenic birds expressing transgenic poultry derived IFN-α2b (TPD IFN-α2b) in tissues and eggs of poultry. Similarly, the method is used in the design of other coding sequence proteins such as human erythropoietin (EPO) or other proteins that can be produced according to the present invention.

By the methods of the present invention, transgenes can be introduced into avian embryonic germ leaf cells to produce transgenic chickens, transgenic turkeys, transgenic quails and other algal species with the transgene in the genetic material of the dotline tissue. Typically the blastocyst cells are stage VII-XII cells, or cells of equivalent stage thereof, and in one embodiment are near stage VII. Cells useful in the present invention include embryonic germ (EG) cells, embryonic stem (ES) cells & primordial germ cells (PGCs). Embryonic germ leaf cells may be freshly isolated, maintained in culture, or present in embryos.

Vectors useful for carrying out in the methods of the invention are described herein. Such vectors can be used for the stable introduction of exogenous coding sequences into the genome of birds. Alternatively, vectors can be used to produce exogenous proteins in certain tissues of algae, such as alveolar fallopian tissues. Vectors can also be used in methods for producing algal eggs containing exogenous proteins. In one embodiment, both the coding sequence and the promoter are located between 5 'and 3' LTRs prior to introduction into the germ leaf cells. In one embodiment, the vector is a retrovirus and both the coding sequence and the promoter are located between 5 'and 3' LTRs of the retroviral vector. In one useful embodiment, the LTR or retroviral vector is derived from avian leukemia virus (ALV), murine leukemia virus (MLV), or lentivirus.

In one embodiment, the vector comprises a signal peptide coding sequence operably linked to the coding sequence, such that, after translation in the cell, the signal peptide induces the secretion of the exogenous protein expressed by the vector into the egg whites. something to do. The vector may comprise a label gene, which is operably linked to a promoter.

In some cases, the introduction of the vectors of the invention into embryonic germ leaf cells is carried out using newly isolated or cultured embryonic germ leaf cells. Subsequently, transgenic cells are usually injected subcutaneously beneath the receptor ventricle in the egg. In some cases, however, the vector is delivered directly to the cells of the blastocyst embryo.

In one embodiment of the invention, the vector used to transfect the blastocyst cells and create a stable integration into the algal genome is operative and positional for expressing the coding sequence in the coronary line of the bulge of the algae fallopian tube. Containing a coding sequence and a promoter in relation, the coding sequence encodes an exogenous protein that is deposited on the egg white of the hard shell egg. Optionally, the promoter may be a segment of an egg albumin promoter region that is large enough to drive expression of the coding sequence in the coronary cell. The present invention includes truncating the egg albumin promoter and / or condensing an important regulatory element of the egg albumin promoter, which is small enough to be easily incorporated into a vector, but in coronary cells of the bulge of the fallopian tube. Retains the sequence necessary for expression of In one embodiment, segments of egg albumin promoter regions can be used. This segment contains the 5'- flanking region of the egg albumin gene. The total length of the egg albumin promoter segment may be between about 0.88 kb and about 7.4 kb in length, preferably between about 0.88 kb and about 1.4 kb in length. The segment preferably comprises both steroid-dependent regulatory elements and negative regulatory elements of the egg albumin gene. Optionally, the segment also includes residues from the 5 'untranslated region (5' UTR) of the egg albumin gene. Thus, the promoter may be derived from the promoter region of the egg albumin-, lysozyme-, conealbumin-, ovomucoid-, ovotransferrin- or ovomucin gene (FIG. 14). An example of such a promoter is a synthetic MDOT promoter consisting of elements from the ovomucoid and ovotransferrin promoter (FIG. 13). The promoter may also be a promoter specific to the bulge that is large but not very large, for example a lysozyme promoter. The promoter may also be a mouse breast cancer virus (MMTV) promoter. Alternatively, the promoter may be a constant expression promoter (eg, cytomegalovirus (CMV) promoter, Raus-sarcoma virus (RSV) promoter, murine leukemia virus (MLV) promoter, etc.). In one preferred embodiment of the invention, the promoter is a cytomegalovirus (CMV) promoter, an MDOT promoter, a Raus-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse breast cancer virus (MMTV) promoter, an egg albumin promoter , Lysozyme promoter, conealbumin promoter, ovomucoid promoter, ovomucin promoter, and ovotransferin promoter. Optionally, the promoter may be one or more segments of the promoter region, such as eggs of albumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and ovotransferin promoter region. In one embodiment, the promoter is a CMV promoter.

1A and 1B illustrate examples of egg albumin promoter expression vectors. Gene X is the coding sequence that encodes an exogenous protein. Curved arrows indicate transcription start sites. In one example, the vector contains 1.4 kb of the 5 ′ flanking region of the egg albumin gene (FIG. 1A). The sequence of the “-1.4 kb promoter” of FIG. 1A starts at about 1.4 kb upstream (1.4 kb) of the egg albumin transcription start site and extends about 9 residues into the 5 ′ untranslated region of the egg albumin gene. Corresponding. A segment of about 1.4 kb-length has two important regulatory elements, the steroid-dependent regulatory element (SDRE) and the negative regulatory element (NRE). NRE was named because it contains several negative regulatory elements that block the expression of genes in the absence of hormones (eg estrogen). The shorter 0.88 kb segment also contains both elements. In another example, the vector contains a 5 'flanking region of the egg albumin gene of about 7.4 kb, and has two additional elements (HS-III and HS-IV), one for the induction of the gene by estrogen It is known to contain functional regions that make it possible (FIG. 1B). The shorter 6 kb segment also contains all four elements and can optionally be used in the present invention.

Each vector used for random integration according to the invention preferably comprises at least 1.2 kb of elements from the chicken β-globin locus which isolates the gene from both active and inactive at the insertion site into the genome. In one embodiment, two insulator elements are added to one end of the egg albumin gene construct. At the β-globin locus, an insulator element is provided to prevent the distant locus regulatory region (LCR) from activating upstream genes from the globin gene domain and has been found to overcome positional effects in transgenic flies, This indicates that they can protect against both positive and negative effects at the insertion site. The insulator element (s) are only needed at the 5 'or 3' end of the gene because the transgene is incorporated into multiple inline copies that effectively produce a series of genes adjacent to the insulator side of the adjacent transgene. In another embodiment, the insulator element is not linked to the vector but is cotransfected with the vector. In this case, the vectors and elements are connected in line in the cell by the process of random integration into the genome.

Optionally, each vector also includes a marker gene that allows for the identification and enrichment of cell clones that are stably integrated into the expression vector. Expression of marker genes is driven by ubiquitous promoters that drive high levels of expression in a wide range of cell types. In one embodiment of the invention, the marker gene is human interferon driven by the lysozyme promoter. In another embodiment, the green fluorescent protein (GFP) reporter gene (Zolotukhin et al., J. Virol 70: 4646-4654 (1995)) is a Xenopus elongation factor 1-α (ef-1-α) Driven by a promoter (Johnson and Krieg, Gene 147: 223-26 (1994)). Xenopus ef-1-α promoter is a potent promoter expressed in a wide range of cell types. GFP enhances its fluorescence and contains mutations that are humanized or modified so that the codon matches the codon frequency profile of the human gene. Since avian codon usage is actually the same as human codon usage, the humanized form of the gene is also highly expressed in algal blastocyst cells. In alternative embodiments, the marker gene is operably linked to one of the ubiquitous promoters of HSV tk, CMV, β-actin or RSV.

Human and avian codon usage matches well, but when a non-vertebrate gene is used as a coding sequence in a transgene, the non-vertebral gene sequence is appropriate so that the codon usage is similar to that of human and avian. It can be modified to change.

Transfection of germ leaf cells can be mediated by any number of methods known to those skilled in the art. The introduction of the vector into the cell can be facilitated by first mixing the nucleic acid with the polylysine or cationic lipid to help facilitate passage across the cell membrane. However, the introduction of the vector into the cell is preferably achieved through the use of a delivery vehicle such as liposomes or viruses. Viruses that can be used to introduce the vectors of the invention into blastocyst cells include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and vaccinia viruses.

In one method of transfecting germ leaf cells, the packaged retrovirus-based vector is used to deliver the vector to embryonic germ leaf cells, allowing the vector to integrate into the algal genome.

As an alternative to delivering retroviral transducing particles to embryonic germ germ cells of the embryo, helper cells that produce retroviruses can be delivered to the germ leaf.

Retroviruses useful for randomly introducing transgenes into the avian genome are replication-deficient avian leukemia virus (ALV), replication-deficient murine leukemia virus (MLV) or lentiviruses. To generate a suitable retroviral vector, the pNLB vector is modified by inserting an egg albumin promoter region and one or more exogenous genes between the 5 'and 3' long terminal repeats (LTRs) of the retroviral genome. The present invention contemplates that any coding sequence located downstream of a promoter active in coronary cell will be expressed in coronary cell. For example, since the egg albumin promoter drives the expression of egg albumin protein and is active in tubal coronary cells, the egg albumin promoter will be expressed in the coronary cells of the tubal bulge. Although the 7.4 kb egg albumin promoter has been found to produce the most active constructs when analyzed in cultured fallopian blastocyst cells, the egg albumin promoter is preferably reduced in length for use in retroviral vectors. In one embodiment, the retroviral vector comprises a 1.4 kb segment of the egg albumin promoter; 0.88 kb segment is also sufficient.

Any vector of the invention also includes a coding sequence that encodes a signal peptide that optionally induces secretion of the protein expressed by the coding sequence of the vector from the coronary cells of the fallopian tube. This aspect of the invention effectively extends the range of exogenous proteins that can be deposited on algal eggs using the methods of the invention. If the exogenous protein is not otherwise secreted, the vector containing the coding sequence is modified to include a DNA sequence comprising about 60 bp encoding a signal peptide from the lysozyme gene. The DNA sequence encoding the signal peptide is inserted into the vector and located at the N-terminus of the protein encoded by the DNA.

2A-2D illustrate examples of suitable retroviral vector constructs. Vector constructs are inserted into the algal genome along with the 5 'and 3' flanking LTRs. Neo is a neomycin phosphotransferase gene. Curved arrows indicate transcription start sites. 2A and 2B show oviduct transcripts with sequences encoding LTR and lysozyme signal peptide (LSP), while FIGS. 2C and 2D show transcripts without these sequences. There are two parts to the retroviral vector method. Any protein containing the signal peptide of the eukaryote can be cloned into the vectors shown in Figures 2B and 2D. Any protein that is not normally secreted can be cloned into the vectors shown in FIGS. 2A and 2B to allow secretion from coronary cells.

2E shows a method for cloning exogenous genes with lysozyme signal peptide vectors. Polymerase chain reaction using oligonucleotide primer pairs containing restriction enzyme sites that allow insertion of amplified genes into plasmids after digestion with two enzymes to amplify a copy of the coding sequence, gene X, is used. 5 'and 3' oligonucleotides contain Bsu36I and XbaI restriction sites, respectively.

Another aspect of the invention provides for the use of an internal ribosome binding site (IRES) in any vector of the invention that allows translation of two or more proteins from disistronic or polycistronic mRNA. (Example 15). The IRES units are then fused to the 5 'end of one or more additional coding sequences that are inserted into the vector at the end of the original coding sequence so that the coding sequences are distinguished from each other by the IRES (Figures 2F, 15A and 15D). According to this aspect of the invention, one coding sequence is capable of encoding an enzyme that can modify other coding sequence products, thereby facilitating post-translational modification of the product. For example, the first coding sequence can encode collagen that is hydroxylated and activated by an enzyme encoded by the second coding sequence. In the example of the retroviral vector of FIG. 2F, the internal ribosomal binding site (IRES) element is located between two exogenous coding sequences (gene X and gene Y). IRES allows both Protein X and Protein Y to be translated from the same transcript of transcription driven by a promoter such as an eggalbumin promoter. Curved arrows indicate transcription start sites. The expression of the protein encoded by gene X is expected to be highest in coronary cell, which is specifically expressed but not secreted. The protein encoded by gene Y is also specifically expressed in coronary cell, but since it is secreted effectively, protein Y is packaged into eggs. In the example of the retroviral vectors of FIGS. 15A and 15D, the light chain (LC) and heavy chain (HC) of human monoclonal antibodies are a single vector pCMV-LC-emcvIRES-HC by placement of IRES from brain myocarditis virus (EMCV). Is expressed from. Transcription is driven by the CMV promoter (Murakami et al. (1997) "High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site" Gene 202: 23-29; Chen et al. (1999) "Production and design of more effective avian replication-incompetent retroviral vectors" Dev. Biol. 214: 370-384; Noel et al. (2000) "Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts" J. Invest Dermatol. 115: 740-745).

In another aspect of the invention, the coding sequence of the vector used in any of the methods of the invention is provided with a 3 'untranslated region (3' UTR) that imparts stability to the resulting RNA. If 3 'UTR is added to the retroviral vector, the orientation of promoter, gene X and 3' UTR are reversed in the construct, so that addition of 3 'UTR should not interfere with transcription of full length genomic RNA. In one embodiment, the 3 'UTR can be a 3' UTR of an egg albumin or lysozyme gene, or any 3 'UTR that functions in bulge cells, ie, the SV40 subregion.

In one alternative embodiment of the invention, a coexpression promoter (eg CMV) is used to express the coding sequence of the transgene in the bulge of the bird. In this case, expression is not limited to the bulge portion; Expression also occurs in other tissues of the algae (eg blood). The use of such transgenes, which include a constant expression promoter and coding sequences, is particularly suitable for achieving or driving protein expression in the fallopian tubes and subsequent secretion of proteins into egg whites (pNLB-CMV for expressing IFN-α2b in chickens). See FIG. 8A for an example of a CMV driven construct, such as an -IFN vector.

3A shows a schematic of a replication-deficient avian leukemia virus (ALV) -based vector pNLB, a vector suitable for use in the present invention. In the pNLB vector, most of the ALV genome is replaced with the neomycin resistance gene (Neo) and the lacZ gene encoding b-galactosidase. 3B shows the vector pNLB-CMV-BL with lacZ replaced by the CMV promoter and β-lactamase coding sequence (β-La or BL). Construction of vectors is reported in specific examples (Example 1, see below). β-lactamase is expressed from the CMV promoter and utilizes an adenylpolymerization signal (pA) at the 3 ′ long terminal repeat (LTR). β-lactamase protein has a natural signal peptide; Accordingly, it is found in blood and whites.

Avian embryos are transduced with pNLB-CMV-BL vector (Example 2, see below). The egg whites of the resulting stably transduced hens contained up to 60 micrograms (μg) of secreted active β-lactamase per egg (Examples 2 and 3, see below).

8A and 8B illustrate the pNLB-CMV-IFN vector used to express interferon-α2b (IFN-α2b) and the pNLB-MDOT-EPO vector used to express erythropoietin (EPO), respectively. Both exogenous proteins (EPO, IFN) are expressed in algae, preferably chickens and turkeys.

The pNLB-MDOT-EPO vector is generated by replacing the EPO encoding sequence with the BL encoding sequence (Example 10, see below). In one embodiment, a synthetic promoter referred to as MDOT is used to drive expression of EPO. MDOT contains urea from both ovomucoid and ovotransferin promoters. The DNA sequence for human EPO is shown in Wisconsin Package, Version 9.1 (Genetics Computer Group, Inc., with codon usage tables compiled from chicken (Gallus gallus) egg albumin, lysozyme, ovomucoid, and ovotransferin protein). Based on hen oval tube optimization codon usage as generated using the BACKTRANSLATE program of Madison, Wis. The EPO DNA sequence is synthesized, cloned into a vector, and the resulting plasmid is pNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2). In one embodiment, the transgenic particles are titrated to determine the appropriate concentration that can be used to introduce the particles (ie transduced particles) into the vector and to inject into the embryo. The eggs then hatch about 21 days after the transduced particles are injected.

Exogenous protein levels, such as EPO levels, can then be determined by ELISA analysis from serum samples collected from chicks one week after hatching. Male birds are selected for mating, where such birds are screened for G0 roosters containing EPO transgenes in their sperm. Preferably, the rooster with the highest level of transgene in the sperm sample is crossed with the non-transgenic hen by artificial insemination. Blood DNA samples are screened for the presence of the transgene. Many chicks are usually found to be transgenic (G1 birds). Chick serum is tested for the presence of human EPO (eg, ELISA assays). Egg whites of eggs from G1 hens are also tested for the presence of human EPO. EPO present in the eggs of the invention (ie, EPO derived from an optimized coding sequence of human EPO) is biologically active (Example 11).

Similarly, pNLB-CMV-IFN vector (FIG. 8A) is generated by replacing IFN encoding sequence with BL encoding sequence (Example 12, see below). In one embodiment, the always-expressing cytomegalovirus (CMV) promoter is used to drive the expression of IFN. More particularly, IFN coding sequences are regulated by cytomegalovirus (CMV) immediate early promoter / enhancer and SV40 polyA site. 8A depicts pNLB-CMV-IFN used to express IFN in algae such as chickens and turkeys. Coding sequences optimized for human IFN-α2b are generated, and the most frequently used codons for each specific amino acid found in the egg white proteins egg albumin, lysozyme, ovomucoid and ovotransferrin are inserted into the vector of the present invention. Is used in the design of human IFN-α2b sequences. More specifically, the DNA sequence for optimized human IFN-α2b (FIG. 11A) is based on hen falloptic optimization codon usage and is based on chicken (Gallus gallus) egg albumin, lysozyme, ovomucoid and ovotransferrin proteins. It is generated using the BACKTRANSLATE program (see above) with codon usage tables compiled from. For example, the percent usage for the four codons of amino acid alanine in the four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCU is used as codon for most alanines in optimized human IFN-α2b sequences. Vectors containing genes for optimized human IFN-α2b sequences are used to generate transgenic birds expressing TPD IFN-α2b in the tissues and eggs of the birds.

Particles that introduce the trait (ie transduced particles) are generated for the vector and titrated to determine the appropriate concentration that can be used for injecting into the embryo (Example 2, see below). Thus, chimeric algae are produced (see Example 13, see below). Algae eggs are observed according to the Speksnijder method (U.S. Pat. No. 5,897,998), transduced particles are injected into eggs, and eggs hatch about 21 days after injection. HIFN levels are measured from serum samples collected from chicks one week after hatching (eg, ELISA assays). Male birds with EPO (see above) are selected for mating. To screen G0 roosters containing IFN transgenes in sperm, DNA is extracted from rooster sperm samples. The G0 rooster with the highest level of transgene in the sperm sample is crossed with non-transgenic hens by artificial insemination. Blood DNA samples are screened for the presence of the transgene. The serum of transgenic roosters is tested for the presence of hIFN (eg, ELISA assays). If exogenous protein is identified, the sperm of the transgenic cock is used for artificial insemination of the non-transgenic hen. Thereafter, a certain percentage of descendants will contain the transgene (eg, at least 50%). If IFNs (ie, IFNs derived from optimized coding sequences of human IFNs) are present in the eggs of the present invention, IFNs can be tested for biological activity. Such eggs with EPO usually contain a biologically active IFN, for example TPD IFN-α2b (FIG. 11B).

The method of the present invention provided for the production of exogenous protein in an algae fallopian tube and for the production of eggs containing exogenous protein provides a suitable vector and further introduces the vector into the embryonic germ germ cell to integrate the vector into the algae genome. Subsequent steps. Subsequent steps include inducing mature transgenic algae from the transgenic blastocyst cells produced in the previous step. Inducing mature transgenic algae from blastocyst cells optionally involves transferring the transgenic blastocyst cells to the embryo and allowing the embryo to fully develop, thus integrating the cells into the algae as the embryo develops. The resulting chicks then grow and mature. In one embodiment, the cells of the blastocyst embryos are directly transfected or transduced into the embryo using a vector (Example 2). The resulting embryos develop and the chicks mature.

In any case, transgenic algae generated from transgenic blastocyst cells are known as epochs. Some precursors will have a transgene in the coronary cells in the bulge of their fallopian tubes. Such algae will express exogenous proteins encoded by the transgene of its fallopian tubes. Exogenous proteins may also be expressed in other tissues (eg, blood) in addition to fallopian tubes. If the exogenous protein contains the appropriate signal sequence (s), it will be secreted into the lumen of the fallopian tube and the egg white of the egg. Some prototypes are pointline prototypes (Examples 8 and 9). Jumline precursors are precursors that carry a transgene to the genetic material of the dotline tissue and can also carry a transgene in fallopian bulge coronary cells expressing exogenous proteins. Thus, according to the present invention, the transgene alga will have coronary cells expressing exogenous protein, and the descendants of the transgene alga will also have tubal bulge coronary cells expressing exogenous protein. Alternatively, offspring exhibit a phenotype determined by expression of exogenous genes in specific tissue (s) of the bird (Example 6, Table 2). In one embodiment of the invention, the transgene bird is a chicken or turkey.

The present invention can be used to express high yields and low costs of desired proteins, including those used as human and animal pharmaceuticals, diagnostics, and animal feed additives. For example, the present invention includes eggs from transgenic algae that produce the protein, and, for example, transgenic algae containing the protein in the whites. The present invention contemplates use in the production of any desired protein, including pharmaceutical proteins, which requires that the coding sequence of the protein can be introduced into the fallopian cells according to the present invention. In fact, all proteins tested for exogenous production according to the present invention, including interferon α2b, GM-CSF, interferon β, erythropoietin, G-CSF, CTLA4-Fc fusion proteins and β-lactamases, are herein It was successfully created using the described method.

The production of human proteins as described herein is of particular interest. Human forms of each of the proteins described herein are contemplated for production in accordance with the present invention.

Proteins contemplated for production described herein include fusion proteins, growth hormones, cytokines, structural proteins and enzymes such as human growth hormones, interferons, lysozyme, and β-casein, albumin, α-1 antitrypsin, antithrombin Ⅲ, collagen, factor VII, Ⅸ, Ⅹ (브리), fibrinogen, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor ( GM-CSF), tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin. Modified immunoglobulins and antibodies, including immunotoxins that bind to and destroy surface antigens on human tumor cells, can also be generated as described herein.

Other specific examples of therapeutic proteins that may be produced as described herein include factor VII, factor VII depleted in b-domain, factor VIIa, factor VII, anticoagulant; Hirudin, alteplase, tpa, reteplase, tpa, tpa-3 missing 5 domains, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogue, hgh, glucagon, tsh, polytropin-beta, fsh, gm-csf, pdgh, ifn alpha2, ifn alpha2a, ifn alpha2b, inf-alpha, inf-beta1b, ifh-beta 1a, ifh-gamma 1b, il-2 , il-11, hbsag, ospa, murine mab specific for t-lymphocyte antigen, murine mab specific for tag-72, tumor-associated glycoprotein, chimeric specific for platelet surface receptor gpII (b) / III (a) fab fragment derived from mab, murine mab fragment specific for tumor-associated antigen cal25, murine mab fragment specific to human cancer embryo antigen, cea, murine mab fragment specific to human heart myosin, murine specific to tumor surface antigen psma mab fragment, murine mab fragment specific for hmw-maa (fab / fab2 mixture), murine mab fragment (fab) specific for carcinoma-associated antigen, nca 90 Specific mab fragment (fab), surface granulocyte nonspecific cross-reactive antigen, chimeric mab specific for cd20 antigen found on the surface of b lymphocytes, humanized mab specific for alpha chain of il2 receptor, alpha chain specific for il2 receptor Chimeric mab, tnf-alpha specific chimeric mab, humanized mab specific for epitopes on the surface of respiratory syncytial virus, humanized mab specific for her 2, human epidermal growth factor receptor 2, cytokeratin tumor-associated antigen anti chima mab, beta glucocerebrosidase, tnf-alpha, il-2-diphtheria toxin fusion protein, tnfr-Igg, specific for cd 20 surface antigen of human mab, b lymphocyte dornase-alpha dnase specific for ctl4- Fragment fusion proteins laronidase, DNAase, alefacept, darbepoetin alpha (colonal stimulating factor), tositumomab, murine mab, alemtuzumab, Rasburicaase, agalidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (Igg1), anakinra, biological modulators, nesiritide, human b- Type sodium excretion promoter peptide (hbnp), colony stimulating factor, pegvisomant, human growth hormone receptor antagonist, recombinant activating protein c, omalizumab, immunoglobulin e (Ige) blocker, ibritumomab Lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigment hormone, somatomedin, erythropoietin, progesterone, chorionic gonadotropin, hypothalamic releasing factor, Etanercept, antidiuretic hormone, prolactin and thyroid stimulating hormone.

The present invention includes a method for producing a polymer protein comprising an immunoglobulin, for example an antibody, and an antigen binding fragment thereof. Thus, in one embodiment of the invention, the polymer protein is an immunoglobulin, wherein the first and second heterologous polypeptides are immunoglobulin heavy and light chains, respectively.

In certain embodiments, an immunoglobulin polypeptide encoded by a transcription unit of one or more expression vectors may be an immunoglobulin heavy chain polypeptide comprising a variable region or variant thereof, which may be a D region, a J region, a C region, or a combination thereof. It may further comprise water. The immunoglobulin polypeptides encoded by the expression vector may also be immunoglobulin light chain polypeptides comprising a variable region or variant thereof, which may further comprise a J region and a C region. The present invention also contemplates multiple immunoglobulin regions derived from a mixture of the same animal species or species, including but not limited to humans, mice, rats, rabbits and chickens.

In other embodiments, immunoglobulin polypeptides encoded by one or more expression vectors include immunoglobulin heavy chain variable regions, immunoglobulin light chain variable regions, and linker peptides, thereby allowing single-chain antibodies to selectively bind antigens. Is formed.

Examples of therapeutic antibodies that may be produced in the methods of the invention include HERCEPTIN ™ (trastuzumab) (Genentech, CA), a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO ™ (abciximab) (Centocor), an anti-glycoprotein IIb / IIIa receptor on platelets to prevent clot formation; ZENAPAX ™ (daclizumab), an immunosuppressive humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection (Roche Pharmaceuticals, Switzerland); PANOREX ™ (Glaxo Wellcome / Centocor), a murine anti-17-IA cell surface antigen IgG2a antibody; ImClone System (BEC2), a murine anti-object specific (GD3 epitope) IgG antibody; IMC-C225 (ImClone System), a chimeric anti-EGFR IgG antibody; VITAXIN ™ (Applied Molecular Evolution / Medlmmune), a humanized anti-αVβ3 integrin antibody; Campath; Campath 1H / LDP-03 (Leukosite), a humanized anti CD52 IgG1 antibody; Smart M 195 (Protein Design Lab / Kanebo), a humanized anti-CD33 IgG antibody; RITUXAN ™ (IDEC Pharm / Genentech, Roche / Zettyaku), a chimeric anti-CD2O IgG1 antibody; LYMPHOCIDE ™ (Immunomedics), a humanized anti-CD22 IgG antibody; ICM3 (ICOS Pharm), a humanized anti-ICAM3 antibody; IDEC-114 (IDEC Pharm / Mitsubishi), a primate anti-CD80 antibody; ZEVALIN ™ (IDEC / Schering AG), a radiolabeled murine anti-CD20 antibody; IDEC-131 (IDEC / Eisai), a humanized anti-CD40L antibody; IDEC-151 (IDEC), a primateized anti-CD4 antibody; IDEC-152 (IDEC / Seikagaku), a primatized anti-CD23 antibody; SMART anti-CD3 (Protein Design Lab) which is a humanized anti-CD3 IgG; 5G1.1 (Alexion Pharm), a humanized anti-complement factor 5 (CS) antibody; Humanized anti-TNF-α antibody D2E7 (CATIBASF); CDP870 (Celltech), a humanized anti-TNF-α Fab fragment; IDEC-151 (IDEC Pharm / SmithKline Beecham), a primatized anti-CD4 IgG1 antibody; MDX-CD4 (Medarex / Eisai / Genmab), a human anti-CD4 IgG antibody; CDP571, a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 (LeukoSite / Genentech), a humanized anti-α4β7 antibody; OrthoClone OKT4A (Ortho Biotech), a humanized anti-CD4 IgG antibody; ANTOVA ™ (Biogen), a humanized anti-CD40L IgG antibody; ANTEGREN ™ (Elan), a humanized anti-VLA-4 IgG antibody; CAT-152 (Cambridge Ab Tech), a human anti-TGF-β ₂ antibody; Cetuximab (BMS), a monoclonal anti-EGF receptor (EGFr) antibody; Bevacizuma (Genentech), an anti-VEGF human monoclonal antibody; Infliximab (Centocore, JJ), a chimeric (mouse and human) monoclonal antibody used to treat autoimmune diseases; Gemtuzumab ozogamicin (Wyeth), a monoclonal antibody used in chemotherapy; And ranibizumab (Genentech), a chimeric (mouse and human) monoclonal antibody used to treat macular degeneration.

In one aspect, the invention includes G-CSF produced in poultry or algae. In one embodiment, the present invention includes G-CSF (TPD G-CSF) having a poultry derived glycosylation pattern, wherein the G-CSF is obtained from algal cells, eg, algal cells of chicken, quail or turkey. Human proteins comprising cytokines such as G-CSF produced in isolated or purified form of poultry, and cytokines such as G-CSF produced from poultry present in pharmaceutical compositions are also disclosed herein. Included in Isolation of proteins comprising G-CSF can be accomplished by methods readily apparent to those skilled in the art of protein purification. The construction of formulations useful for producing pharmaceutical compositions is also well known in the art.

The present invention includes transgenic poultry derived therapeutic or pharmaceutical proteins with poultry derived glycosylation patterns derived from algae. For example, the present invention includes interferon-α2 (TPD IFN-α2) derived from algae. TPD IFN-α2 (eg, species type b) exhibits a novel glycosylation pattern and contains novel protein glycoforms that are not generally observed in human peripheral blood leukocyte derived interferon-α2 (PBL IFN-α2b) (bands 4 and 5 are α-Gal extended disaccharides; see FIG. 9). TPD IFN-α2b also contains an O-linked carbohydrate structure similar to human PBL IFN-α2b, which is produced more efficiently in chickens than the human form.

The present invention contemplates an isolated polynucleotide comprising an optimized polynucleotide sequence of a protein produced as described herein. For example, the present invention includes an avian optimized coding sequence for human IFN-α2b, namely recombinant transgenic poultry derived interferon-α2b (TPD IFN-α2b) (SEQ ID NO: 1). The coding sequence for optimized human IFN-α2b comprises 498 nucleic acids and 165 amino acids (see SEQ ID NO: 1 and FIG. 11A). Similarly, the coding sequence for native human IFN-α2b comprises 498 nucleotides (NCBI Accession Number AF405539 and GI: 15487989) and 165 amino acids (NCBI Accession Number AAL01040 and GI: 15487990). The most frequently used codons for each specific amino acid found in the egg white proteins egg albumin, lysozyme, ovomucoid and ovotransferin are used in the design of optimized human IFN-α2b coding sequences inserted into the inventive vector. . More specifically, the DNA sequence for optimized human IFN-α2b is based on hen oval optimization codon usage and codons aggregated from chicken (Gallus gallus) egg albumin, lysozyme, ovomucoid and ovotransferrin proteins It is generated using the Wisconsin package, version 9.1 of the BACKTRANSLATE program (Genetics Computer Group Inc., Madison, Wis.) With a usage table. For example, the percent usage for the four codons of the amino acid alanine of the four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCU is used as the codon for most alanines in the optimized human IFN-α2b coding sequence. Vectors containing genes for optimized human IFN-α2b are used to generate transgenic birds expressing TPD IFN-α2b in the tissues and eggs of the birds.

As discussed in Example 13 (see below), TPD IFN-α2b is produced in chickens. However, TPD IFN-α2b can also be produced in other birds such as turkeys and quails. In one preferred embodiment of the invention, TPD IFN-α2b is expressed in chicken and turkey and their hard shell eggs. Carbohydrate assays, including monosaccharide assays and FACE assays (Example 14, see below), reveal sugar composition or novel glycosylation patterns of proteins. As such, TPD IFN-α2b represents the following monosaccharide residues: N-acetyl-galactosamine (NAcGal), galactose (Gal), N-acetyl-glucosamine (NAcGlu) and sialic acid (SA). However, there is no N-linked glycosylation in TPD IFN-α2b. Instead, TPD IFN-α2b is O-glycosylated at Thr-106. This type of glycosylation is similar to human IFN-α2, where the Thr residue at position 106 is unique in IFN-α2. Similar to native IFN-α, TPD IFN-α2b does not have mannose residues. FACE analysis shows six bands (FIG. 9) representing various sugar residues, where bands 1, 2 and 3 are unsialated sugar residues, mono-sialated sugar residues and di-sialated sugar residues, respectively. (FIG. 10). Sialic acid (SA) binding is alpha 2-3 for galactose (Gal) and alpha 2-6 for N-acetyl-galactosamine (NAcGal). Band 6 represents unsialated tetrasaccharide. Bands 4 and 5 are alpha-galactose (alpha-Gal) extending disaccharides that have not been observed in human PBL IFN-α2b or native human IFNs (relative hIFNs). 10 shows a comparison of TPD IFN-α2b (white egg hIFN) and human PBL IFN-α2b (natural hIFN). Small bands exist between bands 3 and 4 and between bands 4 and 5 in TPD IFN-α2b (see below).

The present invention contemplates an isolated polypeptide sequence of TPD IFN-α2b (SEQ ID NO: 2) (see also FIG. 11B) and pharmaceutical compositions thereof, wherein the protein is one or more of the carbohydrate structures described herein. O-glycosylated at Thr-106:

Where

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

In one embodiment of the invention, the percentages are as follows:

은 약 20%이고,(Ⅰ)

Is about 20%,

은 약 29%이고,(Ii)

Is about 29%,

은 약 9%이고,(Ⅲ)

Is about 9%,

은 약 6%이고,(Ⅳ)

Is about 6%,

은 약 7%이고,(Ⅴ)

Is about 7%,

은 약 12%이다.(Ⅵ)

Is about 12%.

Where

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

Small bands, considered about 17% in TPD IFN-α2b, exist between bands 3 and 4 and between bands 4 and 5.

In one embodiment, the present invention relates to a human protein having a poultry derived glycosylation pattern. In one embodiment, the poultry derived glycosylation pattern is obtained from avian fallopian cell, eg, coronary cell. For example, glycosylation patterns are described herein that have been demonstrated to be present in human proteins produced in the fallopian cells of chickens according to the invention.

In one embodiment, the invention relates to human G-CSF produced in algae (eg, algae fallopian cells), such as chickens, turkeys and quails, with a poultry derived glycosylation pattern. Mature hG-CSF amino acid sequence is shown in FIG. 18C. The nucleotide sequence used herein to generate G-CSF is shown in FIG. 18A and NCBI Accession NM 172219. Nucleotide sequences optimized for algal (eg chicken) codon usage are also contemplated for use to generate G-CSF, and other proteins such as human proteins produced according to the present invention.

The present invention includes eggs containing the G-CSF molecules of the present invention comprising one or more of the glycosylation structures shown below and algae that produce such eggs (eg, chickens, turkeys and quails):

Where

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

In one embodiment, the invention comprises a mixture of G-CSF molecules, the mixture having a glycosylated structure selected from at least one of structure A, structure B, structure C, structure D, structure E, structure F and structure G. It contains G-CSF molecules. The invention also includes mixtures of G-CSF molecules, which mixtures comprise G-CSF molecules having a glycosylated structure selected from one or more of structure A, structure B, structure C, structure D, structure E, structure F and structure G. And such mixtures are isolated or purified, for example from purified eggs or egg whites produced according to the invention. Also included are mixtures of G-CSF molecules, such mixtures comprising two, three, four, five, or six of the structures of structure A, structure B, structure C, structure D, structure E, structure F and / or structure G. Containing G-CSF molecules. Also included are mixtures of G-CSF molecules, such mixtures comprising two, three, four, five, or six of the structures of structure A, structure B, structure C, structure D, structure E, structure F and / or structure G. Containing G-CSF molecules, and such mixtures are isolated or purified, for example from eggs or egg whites produced according to the invention.

The present invention also encompasses individual G-CSF molecules comprising structure A. The invention also encompasses individual G-CSF molecules comprising structure B. The invention also includes individual G-CSF molecules comprising structure C. The present invention also encompasses individual G-CSF molecules comprising structure D. The invention also encompasses individual G-CSF molecules comprising the structure E. The invention also encompasses individual G-CSF molecules comprising the structure F. The invention also encompasses individual G-CSF molecules comprising the structure G. In one embodiment, the individual G-CSF molecules are present in a mixture of G-CSF molecules, which may be an isolated or purified mixture of G-CSF molecules, for example a mixture purified from eggs or egg whites produced according to the present invention. . In one embodiment, individual G-CSF molecules are isolated or purified, eg, purified as described herein (eg, purified by HPLC as described in Example 20).

Embodiments of the invention as specified herein with respect to G-CSF, for example mixtures of G-CSF molecules and individual G-CSF molecules (two paragraphs described above), are also generally produced in accordance with the invention. Applicable to each of the protein and its corresponding poultry derived glycosylation structure.

Transgenic chickens that lay eggs containing EPO were generated as described in Examples 22 and 23. Also, except that a second generation of EPO producing chickens used different producing cell lines as described in US Patent Application No. 2007/0077650, filed May 5, 2007, which is incorporated herein by reference in its entirety. Was essentially generated as described in Examples 22 and 23. This second line of EPO producing chickens has improved levels in the whites of transgenic chickens produced as described in US patent application Ser. No. 11 / 880,838, filed Jul. 24, 2007, which is incorporated herein by reference in its entirety. It appears to have a deletion in the CMV promoter and LTR, which provides EPO production. This higher EPO production line was used to obtain the EPO used for oligosaccharide analysis.

Proteins produced in transgenic algae according to the present invention may be purified from the whites by any useful process as would be apparent to those skilled in the art of protein purification. For example, EPO produced in transgenic birds in accordance with the present invention can be purified from whites by methods apparent to those skilled in the art of protein purification. Examples of purification protocols for EPO present in the whites are described in Example 24.

Human erythropoietin (hEPO) produced in chickens has been found to contain one O-linked carbohydrate chain and three N-linked carbohydrate chains. O-linked glycosylation was found in Ser-126 of EPO and N-linked glycosylation was found in Asn-24, Asn-38 and Asn-83. The mature erythropoietin amino acid sequence produced according to the present invention is shown in Figure 19B. The human nucleotide sequence encoding the EPO is shown in Figure 19A. Nucleotide sequences adapted for algal (eg chicken) codon usage are also contemplated for use in producing EPO and other proteins in accordance with the present invention.

Representative glycosylation structures have been determined for the erythropoietin of the invention, which is shown in Example 25 and FIGS. 20 and 21. In particular, it was confirmed that B-n, D-n, F-n, H-n, J-n, L-n, N-n, O-n, P-n and Q-n are present in the algae-derived EPO. In addition, the evidence indicates that one or more of the oligosaccharide structures A-n, C-n, E-n, G-n, I-n, K-n and M-n may also be present in the EPO. The data also showed that there may be a second form of Q-n where only four of the five terminal NAcGlu residues are present. This second form of Q-n may be in the form of a precursor of Q-n.

The present invention provides algae (eg chickens, turkeys and quails) that produce and lay egg whites containing eggs and egg whites, and the erythropoietin molecules of the invention comprising one or more of the glycosylation structures described herein. It includes.

In one embodiment, the invention provides that the mixture contains an erythropoietin molecule (eg, one or more erythropoietin molecules) having an O-linked glycosylation structure selected from structure Ao, structure Bo and structure Co. A mixture of erythropoietin molecules. O-linked glycosylation analysis to date confirmed the presence of A-o, B-o and C-o; Structure D-o, structure E-o, structure F-o and structure G-o are also believed to be present in poultry derived human EPO. The primary O-linked glycosylation present in algal derived EPO was determined to be C-o.

The invention also includes EPO having a glycosylated structure N-linked at three sites, wherein the structures at each of the three sites are An, Bn, Cn, Dn, En, Fn, Gn, Hn, In, Jn , Kn, Ln, Mn, Nn, On, Pn and Qn.

The invention also includes mixtures of erythropoietin molecules (eg, one or more erythropoitone molecules), wherein some or all of the erythropoietin molecules are structure Ao, structure Bo, structure Co, structure An , Structure Bn, structure Cn, structure Dn, structure E- n, structure Fn, structure Gn, structure Hn, structure In, structure Jn, structure Kn, structure Ln, structure Mn, structure Nn, structure On, structure Pn, structure Qn At least one glycosylation structure selected from In one embodiment, the mixture of erythropoietin molecules is purified or separated, for example from an egg or from whites produced in transgenic algae.

The present invention also includes individual erythropoietin molecules comprising structures A-o. The invention also encompasses individual erythropoietin molecules comprising structures B-o. The present invention also encompasses individual erythropoietin molecules comprising the structure C-o. The present invention also encompasses individual erythropoietin molecules comprising structures A-n. The invention also encompasses individual erythropoietin molecules comprising structures B-n. The present invention also encompasses individual erythropoietin molecules comprising the structure C-n. The present invention also includes individual erythropoietin molecules comprising the structure D-n. The present invention also encompasses individual erythropoietin molecules comprising the structure E-n. The present invention also encompasses individual erythropoietin molecules comprising the structure F-n. The present invention also encompasses individual erythropoietin molecules comprising the structure G-n. The present invention also encompasses individual erythropoietin molecules comprising the structure H-n. The invention also encompasses individual erythropoietin molecules comprising structures I-n. The present invention also encompasses individual erythropoietin molecules comprising the structure J-n. The present invention also encompasses individual erythropoietin molecules comprising the structure K-n. The present invention also encompasses individual erythropoietin molecules comprising the structure L-n. The present invention also encompasses individual erythropoietin molecules comprising the structure M-n. The present invention also encompasses individual erythropoietin molecules comprising the structure N-n. The present invention also encompasses individual erythropoietin molecules comprising structures O-n. The invention also encompasses individual erythropoietin molecules comprising the structure P-n. The invention also encompasses individual erythropoietin molecules comprising the structure Q-n. In one embodiment, the individual erythropoietin molecules are in a mixture of erythropoietin molecules produced in a transgenic bird, such as a transgenic chicken. In one embodiment, the individual erythropoietin molecules are present in a mixture of isolated or purified erythropoietin molecules, for example the mixture is isolated or purified from eggs or whites produced by transgenic algae. In one embodiment, the individual erythropoietin molecules are isolated or purified.

In the present invention, Asn-24, Asn-38 and Asn-83 glycosylation sites each include An, Bn, Cn, Dn, En, Fn, Gn, Hn, In, Jn, Kn, Ln, Mn, Nn, On, Pn and An exemplary EPO molecule is glycosylated with one of the Qn structures and Ser-126 is glycosylated with Ao, Bo or Co.

MALDI-TOF-MS analysis of the peptide product resulting from the proteolytic digestion of algal-derived EPO of the present invention revealed that essentially the same oligosaccharide structure exists at each of the three N-linked glycosylation sites. That is, it appears that each of the same ratios of N-linked oligosaccharides is present in each of the three N-linked glycosylation sites on the EPO molecule. This indicates that other N-glycosylated exogenous proteins produced according to the present invention may have similar N-linked glycosylation patterns. In addition, the data showed that each of the three N-linked sites was glycosylated extensively, and each site was always glycated more than 95%, possibly always more than 98%, for example always more than 99%. The erythropoietin analyzed was generated in transgenic chickens containing transgenes encoding the amino acid sequence of human 165 amino acid proteins after digestion of a single peptide. However, EPO generated in transgenic chickens using the nucleotide sequence encoding the 166 amino acid form of EPO results in the complement of oligosaccharides on the 166 amino acid protein that is identical to that found in 165 amino acid proteins.

N-linked oligosaccharides attached to human EPO produced in transgenic chickens have a small amount of terminal sialic acid moiety. That is, only a slight amount of N-linked oligosaccharide structure is terminal sialated. This is in contrast to EPO produced in human cells in which the N-linked oligosaccharide structure is extensively terminally sialated and human EPO produced in CHO cells. In addition, terminal N-acetyl glucosamine (NAcGlu) is widely present in the N-linked oligosaccharide structure of EPO produced in transgenic chickens, but not in the case of human EPO produced in human cells and CHO cells. not. In addition, there is no fucose in the N-linked oligosaccharide structure of EPO produced in transgenic chickens. However, it appears that fucose is present in all or most of the N-linked oligosaccharide structures of EPO produced in human cells and human EPO produced in CHO cells.

Combinations of glycosylated structures attached to erythropoietin are contemplated. For example, human erythropoietin molecules can be Ao, An, Bn and Cn, or Ao, Bn, Cn and Dn, or Ao, Dn, En and Fn, or Ao, En, Fn and Gn, or Bo, An , Dn and Hn, or Bo, En, Fn and Gn, or Bo, An, An and An, or Co, Dn, Dn and Cn, or Co, Fn, Gn and Hn, or Co, An, Bn and Cn, Or Co, An, Bn and Hn, or Co, An, Bn and En, or Co, An, Bn and Hn or other such combinations.

It is understood that the reported method of preparing the composition of the present invention is a method of preparing in algae, but the composition is not limited thereto. For example, certain molecules of the glycated protein molecules of the present invention may be used in other organisms, such as transgenic fish, transgenic mammals such as transgenic goats, or transgenic plants such as tobacco and moth frog rice (Lemna). It can be created in the minor (Lemna minor).

It is also contemplated that glycosylated structures demonstrated to be present in one protein of the invention may be present in another protein of the invention. For example, glycosylation structures found to be present in TPD G-CSF may also be present in TPD GM-CSF, TPD IFN and / or other TPD proteins. In another example, it is contemplated that glycosylation structures determined to be present in TPD IFN α2 may be present in TPD G-CSF, TPD GM-CSF and / or other transgenic poultry derived (TPD) proteins. The present invention also specifically contemplates human proteins generally having one or more of the TDP glycosylation structures described herein.

The present invention also discloses a PEGylated protein produced as described herein, for example, as described in US Patent Application No. 11 / 584,832, filed October 23, 2006, which is incorporated herein by reference in its entirety. It is contemplated that this may be advantageous as described.

For therapeutic use, the therapeutic protein produced according to the invention may be administered in raw form, but administration of the therapeutic protein as part of a pharmaceutical formulation is preferred.

Accordingly, the present invention provides pharmaceutical formulations comprising one or more pharmaceutically acceptable carriers and optionally other therapeutic and / or prophylactic ingredients, together with poultry derived glycosylation therapeutic proteins or pharmaceutically acceptable derivatives thereof, and such pharmaceutical formulations. Further provided are methods of administration. The carrier (s) is "acceptable" in the sense of being compatible with the other ingredients of the formulation and should not be harmful to the recipient of such carrier. Methods of treating a patient using the pharmaceutical compositions of the invention (eg, amount of pharmaceutical protein administered, frequency of administration and duration of treatment) can be determined using standard methods known to those of ordinary skill in the art. .

Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), intravaginal or parenteral administration. Pharmaceutical formulations include those suitable for administration by injection, including intramuscular, subcutaneous and intravenous administration. Pharmaceutical formulations also include those suitable for administration by inhalation or insufflation. Where appropriate, the formulations may conveniently be presented in separate dosage units and may be prepared by any of the methods well known in the art of pharmacy. The method of producing a pharmaceutical formulation typically comprises producing the therapeutic protein with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration may conveniently be presented in discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; Powder or granules; solution; Suspensions; Or as an emulsion. The active ingredient may also be provided as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binders, fillers, lubricants, disintegrants or wetting agents. Tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oil suspensions, solutions, emulsions, syrups or elixirs, or may be provided as a dry product consisting of water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifiers, non-aqueous vehicles (which may include edible oils) or preservatives.

The therapeutic proteins of the invention may also be formulated for parenteral administration (eg, injection, eg bolus injection or sustained infusion), and may be used in ampoules, prefilled syringes, single dosage forms of infusions, Or in a multidose container with preservatives added. The therapeutic protein can be injected, for example, by subcutaneous injection, intramuscular injection and intravenous infusion or injection.

The therapeutic protein may take the form of a suspension, solution or emulsion in an oil or aqueous vehicle and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. It is also contemplated that the therapeutic protein may be in powder form obtained by aseptic separation of sterile solid or lyophilization from solution, constructed using a suitable vehicle such as water that does not contain a sterile pyrogen before use.

For topical administration to the epidermis, the therapeutic proteins produced according to the invention may be formulated in ointments, creams or lotions, or transdermal patches. For example, ointments and creams may be formulated with an aqueous or oil base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oil base and will generally contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or coloring agents.

Formulations suitable for topical administration in the oral cavity include lozenges comprising the active ingredient in a flavoring base, usually sucrose and acacia or tragacanth; Pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; And mouthwashes comprising the active ingredient in a suitable liquid carrier.

Most preferably, pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are provided as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and suppositories are conveniently formed by cooling in a mold and mixing in the mold after mixing the active compound with the softening carrier (s) or dissolved carrier (s). Can be formed.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, carriers known to be suitable in the art.

For intranasal administration, the therapeutic proteins of the invention can be used in the form of liquid sprays or sprayable powders, or drops.

Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation, the therapeutic protein according to the invention may conveniently be delivered from a blower, nebulizer or other convenient means of delivering a pressurized pack or aerosol spray. The pressurized pack may comprise a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

For administration by inhalation or insufflation, the therapeutic protein according to the invention may take the form of a dry powder composition, eg a powder mixture of a compound and a suitable powder base, such as lactose or starch. The powder composition may be provided in unit dosage form, such as a capsule or cartridge, or in the form of a gelatin or blister pack into which the powder may be administered, for example with the aid of an inhaler or blower.

If desired, the formulations described above that are suitable for producing sustained release of the active ingredient can be used.

The pharmaceutical compositions according to the invention may also contain other active ingredients, for example antimicrobials or preservatives.

In one particular example, human EPO that can be produced and PEGylated as described herein is used in a pharmaceutical formulation in which 1 mL of each contains 0.05 mg of polysorbate 80, which is 2.12 mg in water for injection. Sodium monophosphate monohydrate, 0.66 mg sodium diphosphate anhydride, and 8.18 mg sodium chloride at pH 6.2 ± 0.2. In another specific example, human interferon alpha produced as described herein comprises 7.5 mg / ml sodium chloride, 1.8 mg / ml sodium diphosphate, 1.3 mg / ml sodium monophosphate, 0.1 mg / ml ede It is used in pharmaceutical formulations containing disodium tartrate dihydrate, 0.7 mg / ml of Tween® 80 and 1.5 mg / ml of m-cresol. In another specific example, the human G-CSF produced as described herein contains 0.82 mg / ml sodium acetate, 2.8 μl / ml glacial acetic acid, 50 mg / ml mannitol and 0.04 mg / ml Tween® 80 Used in pharmaceutical formulations.

It is also contemplated that the therapeutic proteins of the invention may be used with other therapeutic agents.

The compositions or compounds of the present invention can be used to treat a wide variety of diseases. For example, there are a number of diseases in which a therapeutic regimen is known to those skilled in the art, where therapeutic proteins obtained from cell cultures (eg, CHO cells) are used. The present invention contemplates that therapeutic proteins produced in algal systems containing poultry derived glycosylation patterns can be used to treat the disease. That is, the present invention contemplates the treatment of diseases known to be treatable by therapeutic proteins typically produced by using the therapeutic proteins produced according to the invention. For example, as will be understood in the art, erythropoietin produced according to the present invention may be used for anemia and kidney disease, eg, chronic renal failure (or other diseases that can be treated by administering the EPO of the present invention). It can be used to treat human diseases such as, and the G-CSF produced according to the present invention can be used to treat cancer patients.

In general, the dosage may vary depending on known factors such as the age, health and weight of the recipient, the type of concurrent treatment, the frequency of treatment, and the like. Usually, the dosage of active ingredient may be from about 0.0001 to about 10 milligrams per kilogram of body weight. The exact dosage, frequency of administration and duration of treatment can be determined by the skilled artisan in the administration of each therapeutic protein.

The following specific examples are intended to illustrate the invention and should not be considered as limiting the scope of the invention.

Example  One

Vector construction

The lacZ gene of pNLB, a replication-deficient avian leukemia virus (ALV) -based vector (Cosset et al., 1991), was replaced with an expression cassette consisting of the cytomegalovirus (CMV) promoter and the reporter gene β-lactamase. pNLB and pNLB-CMV-BL vector constructs are shown in FIGS. 3A and 3B, respectively.

To efficiently replace the lacZ gene of pNLB with a transgene, the intermediate adapter plasmid pNLB-adapter was first generated. Chewed back Apa I / Apa I fragments (in pNLB, 5 'Apa I and 3' LTR, present in the 289 bp upstream of lacZ of pNLB (Cosset et al., J. Virol. 65: 3388-94 (1991)) And Apa I) present at 3 ′ of the Gag segment were inserted into the chod-back Kpn I / Sac I site of pBluescriptKS (−) to generate the pNLB-adapter. The field-in MluI / XbaI fragment of pCMV-BL (Moore et al., Anal. Biochem. 247: 203-9 (1997)) was inserted into the chord-back KpnI / NdeI site of the pNLB-adapter , lacZ was replaced with the CMV promoter and BL gene (in pNLB, KpnI present 67 bp upstream of lacZ and NdeI present 100 bp upstream of lacZ stop codon) to generate pNLB-Adapter-CMV-BL. To generate pNLB-CMV-BL, the HindIII / BlpI insert of pNLB (containing lacZ) was replaced with the HindIII / BlpI insert of pNLB-Adapter-CMV-BL. The direct ligation of blunt-terminal fragments of pNLB to HindIII / BlpI produced mostly rearranged subclones for unknown reasons, so cloning of these two steps was necessary.

Example 2

Generation of pNLB-CMV-BL Progenitor Cluster

Sentas and Isoldes in F10 (Gibco), 5% neonatal serum (Gibco), 1% chicken serum (Gibco), 50 μg / ml Cayla Laboratories and 50 μg / ml hygromycin (Sigma) Incubated. Transduced particles were generated as described in Cosset et al., 1993, which is incorporated herein by reference, with the following exceptions. After 2 days of transfection of the retroviral vector pNLB-CMV-BL to 9 × 10 ⁵ Sentas (Example 1, supra), the virus was harvested in fresh medium and filtered for 6-16 hours. All of the above medium was used to transduce 3 × 10 ⁶ Isoldes in 3100 mm plates with polybrene added at a final concentration of 4 μg / ml. The next day, the medium was replaced with a medium containing 50 μg / ml pleomycin, 50 μg / ml hygromycin and 200 μg / ml G418 (Sigma). After 10-12 days, one G418 resistant colony was separated and transferred to a 24-well plate. After 7-10 days, titers from each colony were determined by G418 selection after transduction of Sentas. Typically, two of the 60 colonies generated titers of 1-3 × 10 ⁵ . These colonies are grown and incorporated by reference in Allioli et al., Dev. Biol. 165: 30-7 (1994), the virus was concentrated to 2-7 × 10 ⁶ . The integrity of the CMV-BL expression cassette was confirmed by analyzing for β-lactamase in the medium of cells transduced with NLB-CMV-BL transduced particles.

Transduction vector pNLB-CMV-BL was injected into the dorsum of 546 unincubated SPF White Leghorn embryos, of which 126 chicks hatched and β-lactamase to the blood (lactamase) ) Secretion was examined. To determine the concentration of active lactamase in unknown samples, a kinetic colorimetric analysis was used in which the purple substrate PADAC was specifically converted to a yellow compound by lactamase. Activity was quantified by monitoring the decrease in OD _5O nm over the standard reaction time and compared to a standard curve with varying levels of purified lactamase (referred to as "lactamase assay"). The presence or absence of lactase in the sample can also be determined by visually recording (“overnight lactase analysis”) the transition from purple to yellow in the test sample overnight or for several days. The overnight lactamase assay method is suitable for the detection of very low levels of lactamase or for the screening of large numbers of samples. At the age of 1-4 weeks, chick serum samples were tested for the presence of lactase. 27 chicks had very low levels of lactamase in the serum, which was detected only after overnight lactamase analysis, and as these chicks matured, no lactamase was detected anymore. As shown in Table 1 below and FIG. 4A, nine additional birds (three males and six females) had lactase serum levels ranging from 11.9 to 173.4 ng / ml after 6-7 months of incubation. .

Table 1: Expression of β-lactamase in NLB-CMV-BL-transduced chickens

¹ NA: not available.

² controls were obtained from untreated hens.

³ Average of 5 to 20 eggs.

Example 3

Β-lactamase Expression in the White of G0 Hens

57 young hens transduced with the pNLB-CMV-BL retroviral vector were sexually matured and the whites from each hen at 8 months of age were tested for β-lactamase (lactamase). Six of the 57 birds had significant levels of lactamase in the range of 56.3 to 250.0 ng / ml (Table 1, supra). Other hens in this group did not have detectable levels of lactamase in their whites after incubation of PADAC with samples for several days. No lactamase was detected in the whites from 42 hens transduced with NLB vectors without mock injected 24 hens and lactase transgenes. Stable lactamase expression was still detectable in the whites of 6 expressing hens after 6 months of the initial analysis (Table 1, see above).

Lactase was detected in the whites of all six hens by Western blot analysis using anti-β-lactamase antibody. White lactase was the same size as the purified lactase produced in the bacteria used as a standard. The amount detected in the whites by Western analysis was consistent with the amount determined by enzymatic analysis, indicating that a significant portion of the white lactase is biologically active. Hen-producing lactamase in whites stored at 4 ° C. did not lose activity after several months of storage and had no change in molecular weight. This observation allowed the storage of lactase-containing eggs for an extended period prior to analysis.

Example 4

Dot-line Delivery and Serum Expression of β-lactamase Transgenes in G1 and G2 Transgenic Chickens

DNA was extracted from sperm collected from 56 G0 roosters and three out of 56 birds with significant levels of transgenes on sperm DNA determined by quantitative PCR were selected for mating. These roosters were the same three with the highest levels of β-lactamase (lactamase) in their blood (cocks 2395, 2421 and 2428). Rooster 2395 developed three G1 transgene offspring (3 of 422 offspring), while the other two did not develop transgene offspring in a total of 630 offspring. Southern analysis of blood DNA from each of the three G1 transgenic chickens demonstrated that the transgene was intact and incorporated into their unique random locus. Serum of G1 transgenic chickens 5308, 5657 and 4133 after 6-11 weeks of hatching contained 0.03, 2.0 and 6.0 μg / ml of lactase, respectively. When the chickens were analyzed again at the age of 6-7 months, the levels of lactamase decreased to levels of 0.03, 1.1 and 5.0 μg / ml (FIG. 4A).

Hens 5657 and roosters 4133 were crossed with nontransgenic chickens to obtain progeny that were semiconjugated to the transgene. The family tree of transgenic chickens bred from rooster 4133 or hen 5657 and later generations is shown in FIG. 5. Transgenic rooster 5308 was also crossed, but its progeny exhibited very low or undetectable lactamase concentrations in serum and egg white. Active lactamase concentrations in the serum of randomly selected G2 transgenic chickens were measured 3 to 90 days after hatching. The five G2 transgenics crossed from hen 5657 all had active lactamase at a concentration of 1.9 to 2.3 μg / ml (compare with parental expression concentration of 1.1 μg / ml, FIG. 4B). All samples were collected for the same period, so the lactase concentration in the serum of descendants is expected to be higher than the lactase concentration in the serum of the parent because the concentration in hen 5657 decreases proportionally as the hen matures. It was. Similarly, five randomly selected transgenic chickens crossed from rooster 4133 all had similar but higher serum lactase concentrations than their parents (FIG. 4B).

Example 5

Β-lactamase Expression in the Whites of Transgenic Hens

Eggs from G1 hen 5657 contained 130 ng of active β-lactamase (lactamase) per ml of egg white (FIG. 6A). Lactamase concentrations were high in the first few eggs and then reached a stable plateau for more than nine months. Eggs from transgenic hens bred from hens 5657 and non-transgenic roosters had lactase concentrations similar to their parents (FIG. 6A). Hen 6978 was crossed from G2 hen 8617 and sibling G2 rooster 8839, which was homozygous for the transgene as determined by quantitative PCR and Southern analysis. As expected, the concentration of lactamase in the eggs of the new 6978 was nearly two times higher than its semiconjugated parent (FIG. 6B). Other G3 hens bred from hen 5657 were not analyzed because hen 6978 was the only female in its clutch. Eggs from hens 8867, 8868 and 8869 were harvested after 11 months, and they had similar concentrations of lactamase (Figures 6A and 6B), again indicating that expression levels in the egg white were consistent throughout the laying period.

Rooster 4133 was crossed with non-transgenic hens to obtain semiconjugate G2 hens. All 15 transgenic hens analyzed had lactamases at concentrations ranging from 0.47 to 1.34 μg / ml in the whites. Four representative hens are shown in FIG. 7A. After 6 months of analysis, the mean expression level decreased from about 1.0 μg / ml to 0.8 μg / ml (FIG. 7A). Expression levels were high in the first egg and were constant over the months. Thereafter, the concentration of lactase in the eggs was kept constant.

G2 hens 8150 and siblings G2 roosters 8191 were crossed to generate semiconjugate and homozygous G3 hens. All transgenic G3 hens expressed lactase at concentrations ranging from 0.52 to 1.65 μg / ml in their whites (FIG. 7B). The mean expression for homozygous G3 hens was 47% higher than the semiconjugate G2 hens and G3 hens. The amount of lactamase in eggs from G2 and G3 hens and their descendants crossed from rooster 4133 varied significantly (FIGS. 7A and 7B), but levels in eggs from any given hen in this group were relatively constant. . The mean expression of lactase was expected to double for the homozygous genotype. Western blot analysis demonstrated that the transgene accurately produced intact lactamase in the eggs of the G2 transgenic. The lactase levels detected in the western blot were also closely related to those determined by enzyme activity analysis, indicating that a significant portion of the white lactase is bioactive. Thus, retroviral vectors have been successfully used to meet stable and reliable expression of transgenes in chickens.

The deposition of lactase in egg yolk was detectable but lower than that in egg white. Seven G2 or G3 hens of the rooster 4133 strain were analyzed and the concentration in egg yolk ranged from 107 to 375 ng / ml, or about 20% of the concentration in egg white. There was no correlation between yolk yolk and egg white lactamase levels in hens provided (Harvey et al., "Expression of exogenous protein in egg white of transgenic chickens" (April 2002) Nat. Biotechnol. 20: 396-399).

Example 6

Generation of eponymous males

For pNLB-CMV-BL transduction, newly fertilized white leghorn eggs were used. Seven to ten microliters of concentrated particles were injected into the dorsal cavity of the windowed eggs and the chicks were hatched after the window was sealed. 546 eggs were injected. Blood DNA was extracted and analyzed for the presence of transgene using a probe-primer set designed to detect neo-resistant genes via Taqman analysis. As can be seen in Table 2 below, about 25% of all chicks had detectable levels of transgenes in their blood DNA.

Table 2: Summary of Gene Conversion Using NLB-CMV-BL Vectors

Example 7

Dotline Delivery of Transgene

Taqman detection of neo-resistant genes in sperm DNA was used to identify candidate G0 males for mating. Three G0 males were identified, each of which had an NLB-CMV-BL transgene in sperm DNA at levels above background. All G0 males positive for transgene in sperm were crossed with non-transgenic hens to fully identify transgenic G1 progeny.

For NLB-CMV-BL, 1026 chicks were crossed each and 3 G1 chicks were obtained for each transgene (Table 2, see above). Although equal numbers of chicks were crossed from each male, all G1 progeny occurred from males with the highest levels of transgenes in semen DNA.

Example 8

Southern analysis of G1 and G2

Southern blot analysis was performed on DNA from Gl and G2 transgenics to confirm the integrity and integrity of the inserted vector sequences. Blood DNA was digested with HindIII and hybridized to neo-resistant probes to detect junction fragments generated by the internal HindIII site (FIG. 3B) found in the pNLB-CMV-BL vector (FIG. 3B) and the genomic site adjacent to the integration site. It was. Each of the three G1 birds with NLB-CMV-BL had a conjoint fragment of unique size, indicating that the transgene was incorporated into three different genome sites. G1 was crossed with non-transgenic hens to obtain semiconjugate G2. As can be observed in Table 2 (see above), as expected for Mendelian segregation of a single integrated transgene, 50.8% of descendants from G1 roosters with NLB-CMV-BL were transgenic It was. Southern analysis of HindIII-digested DNA from G2 progeny detected contiguous fragments of similar size as those generated from their transgenic parents, indicating that the transgene was intact delivered.

Example 9

Screening of G3 Offspring Homozygous for Transgene

To obtain transgenic chickens homozygous for the transgene, G2 semiconjugate birds (eg, progeny of the same G1 male) with NLB-CMV-BL integrated at the same site were crossed. Two groups were crossed: the first group was hens and roosters from G1 4133 males, and the second group was hens and roosters from G1 5657 hens. Taqman analysis was used to quantitatively detect neo-resistant transgenes in G3 progeny using standard curves. Standard curves were prepared using known amounts of genomic DNA from G1 transgenic 4133 males, which are semi-conjugated to the transgene as determined by Southern analysis. The standard curve ranged from 10 ³ to 1.6 × 10 ⁴ full copies of transgene or 0.2 to 3.1 transgene copies per diploid genome. As the reaction components were not limited during the exponential growth phase, amplification was very efficient and produced reproducible values for the number of copies provided. There was a reproducible 1-cycle difference between each standard curve that was twice as different in copy number.

To determine the number of transgene alleles in the G3 progeny, DNA was amplified and compared to standards. DNA from non-transgenics was not amplified. Birds homozygous for the transgenic allele generated a plot that initiated amplification one cycle earlier than birds that were semiconjugate for the allele. The sequence detection program was able to calculate the number of alleles in unknown DNA samples based on the periodic curve (Ct), where the standard curve and amplification plots of the samples showed significant elevations. The data is shown in Table 3 below.

To confirm Taqman copy number analysis, the DNA of selected birds was analyzed by Southern blotting using probes complementary to the PstI-digested DNA and neo-resistant genes to detect 0.9 kb fragments. Detection of small fragments was chosen because of the greater amount of transfer of smaller DNA from the gel to the membrane. The signal strength of the 0.9 kb band was in good agreement with the copy number of the G3 transgenic bird as determined by Taqman analysis. The copy number of an additional 18 G3 transgenic birds analyzed by Southern blotting was also consistent with that determined by Taqman. A total of 33 offspring were analyzed for 4133 strains, of which 9 (27.3%) were non-transgenic, 16 (48.5%) were semiconjugates and 8 (24.2%) were homozygotes. A total of 10 offspring were analyzed for the 5657 lineage, of which 5 (50.0%) were non-transgenic, 1 (10.0%) was a semiconjugate and 4 (40.0%) were homozygotes. The observed ratios of non-transgenic, semiconjugate and homozygote for the G3 progeny of the 4133 strains were not statistically different from the expected 1: 2: 1 ratio as determined by the χ2 test (P is 0.05 or less). ). The offspring of the 5657 lineage did not have the expected distribution, but this may be due to the small number of tested offspring (Harvey et al., "Consistent production of transgenic chickens using replication deficient retroviral vectors and high-throughput screening procedures" (February 2002). ) Poultry Science 81: 202-212.

Table 3: Determination of transgene copy number in G3 offspring crossed from G2 transgenic

¹ Std. No .: standard number; NTC: No template control.

² Ct: periodic threshold; Period in which the fluorescence of the sample shows a significant increase above the background.

Copies per ^three diploid genomes were determined by dividing the mean by 5100 and rounding the nearest first decimal point.

⁴ NA: Not applicable.

Example 10

Vector Construction for pNLB-MDOT-EPO Vectors

In accordance with the teachings of Example 1 of the specification (vector construction), a pNLB-MDOT-EPO vector was generated and the EPO encoding sequence was replaced with a BL encoding sequence (FIG. 8B). Instead of using a CMV promoter, MDOT was used (FIG. 13). MDOT is a synthetic promoter containing elements from both the ovomucoid (MD) and ovotransferrin (TO) promoters (pNLB-MDOT-EPO vector, a.k.a. pAVIJCR-A145.27.2.2).

DNA sequence for human EPO based on hen oval optimization codon usage was determined using the Wisconsin package, version 9.1, with codon usage tables compiled from chicken (Gallus gallus) egg albumin, lysozyme, ovomucoid, and ovotransferrin proteins. (Genetics Computer Group, Inc., Madison, Wis.) Was created using the BACKTRANSLATE program. DNA sequences were synthesized and cloned into 3 'overhang T of pCRII-TOPO (Invitrogen) by Integrated DNA Technologies, Coralville, Iowa, under contract. The EPO coding sequence was then separated from pEpoMM using HindIII and FseI, purified from 0.8% agarose-TAE gel and ligated to HindIII and FseI digested alkaline phosphatase-treated pCMV-IFNMM. The resulting plasmid was pAVIJCR-A137.43.2.2 containing cytomegalovirus immediate early promoter / enhancer and an EPO coding sequence regulated at the SV40 polyA site. Plasmid pAVIJCR-A137.43.2.2 was digested with NcoI and FseI and the appropriate fragment was ligated to NcoI and FseI-digested fragments of pMDOTIFN to contain pAVIJCR-A137.87.2 containing EPO driven by the MDOT promoter. 1 was obtained. To clone the EPO coding sequence regulated by the MDOT promoter into the NLB retroviral plasmid, the plasmids pALVMDOTIFN and pAVIJCR-A137.87.2.1 were digested with KpnI and FseI. Appropriate DNA fragments were purified on 0.8% agarose-TAE gels and then ligated and transformed into DH5 α cells. The resulting plasmid was pNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2).

Example 11

Generation of Transgenic Chickens and Complete Transgenic G1 Chickens Expressing EPO

Generation of NLB-MDOT-EPO transduced particles was performed as described for NLB-CMV-BL (see Example 2). About 300 white leghorn eggs were observed according to the Speksnijder method (US Pat. No. 5,897,998), and then about 7 × 10 ⁴ transduced particles were injected per egg. Eggs were hatched 21 days after injection and human EPO levels were measured by EPO ELISA from serum samples collected from chicks 1 week after hatching.

For screening for G0 roosters containing EPO transgene in sperm, DNA was extracted from rooster sperm samples by Chelex-100 extraction (Walsh et al., 1991). Subsequently, the DNA samples were prepared with primers "neo for-1" (5'-TGGATTGCACGCAGGTTCT-3 '; SEQ ID NO: 5) and "neo rev-1" (5'-TGCCCAGTCATAGCCGAAT-3'; SEQ ID NO: 6) Transgenes were detected by application to Taqman® analysis in a 7700 sequence detector (Perkin Elmer) using FAM labeled NEO-PROBE1 (5'-CCTCTCCACCCAAGCGGCCG-3 '; SEQ ID NO: 7). Eight G0 roosters with the highest levels of transgenes in sperm samples were crossed with non-transgenic White Leghorn (SPAFAS) hens by artificial insemination. Blood DNA samples were screened for the presence of transgenes by Taqman® analysis as described above.

Of 1,054 offspring, 16 chicks were found to be transgenic (G1 birds). Chick serum was tested for the presence of human EPO by EPO ELISA and EPO was present at about 70 nanograms / ml (ng / ml). Egg whites of eggs from G1 hens were also tested for the presence of human EPO by EPO ELISA and found to contain about 70 ng / ml of human EPO. EPO present in eggs (ie, derived from optimized coding sequences of human EPO) has been shown to be biologically active when tested in human EPO reactive cell lines (HCD57 murine erythrocyte cells) in cell culture assays.

Example 12

Vector Construction for pNLB-CMV-IFN

In accordance with the teachings of Example 1, the IFN encoding sequence was replaced with the BL encoding sequence of Example 1 to generate the pNLB-CMV-IFN vector (FIG. 8A).

An optimized human IFN-α2b coding sequence was generated in which the most frequently used codons for each specific amino acid found in the egg white proteins egg albumin, lysozyme, ovomucoid and ovotransferin were inserted into the vector of the present invention. It was used in the design of the sequence. More particularly, the DNA sequence for optimized human IFN-α2b was based on hen oval optimization codon usage and codons aggregated from chicken (Gallus gallus) ovalbumin, lysozyme, ovomucoid and ovotransferrin proteins Along with the usage table, the Wisconsin package, version 9.1 (Genetics Computer Group Inc., Madison, Wis.), Was created using the BACKTRANSLATE program. For example, the percent frequency of use for the four codons of amino acid alanine in the four egg white proteins was 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCU was used as the codon for most alanines in the optimized human IFN-α2b coding sequence. Vectors containing genes for optimized human IFN-α2b were used to generate transgenic birds expressing transgenic poultry derived IFN-α2b (TPD IFN-α2b) in tissues and eggs of poultry.

The templates and primer oligonucleotides listed in Table 4 below were 94 ° C. for 1 minute; 50 ° C. for 30 seconds; And PCR using Pfu polymerase (Stratagene, La Jolla, Calif.) Using 20 cycles of 72 ° C. for 1 minute 10 seconds. Purification of PCR products from 12% polyacrylamide-TBE gels by the "crush and soak" method (Maniatis et al. 1982), followed by amplification reactions using only IFN-1 and IFN-8 as primers. Combined into template (see Table 4). The resulting PCR product was digested using HindIII and XbaI, gel purified from 2% agarose-TAE gel and then ligated with HindIII and XbaI digested alkaline phosphatase-treated pBluescript KS (Stratagene), The plasmid pBluKSP-IFNMagMax was generated. Both strands were sequenced by periodic sequencing in an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Foster City, Calif.) Using universal T7 or T3 primers. Mutations in pBluKSP-IFN derived from the original oligonucleotide template were corrected by site-specific mutagenesis using a Transformer site-specific mutagenesis kit (Clontech, Palo Alto, Calif.). The IFN coding sequence was then separated from the calibrated pBluKSP-IFN using HindIII and XbaI, purified from 0.8% agarose-TAE gel, and the HindIII and XbaI digested alkaline phosphatase-treated pCMV-BetaLa-3B ligated to -dH. The resulting plasmid was pCMV-IFN containing cytomegalovirus immediate early promoter / enhancer and IFN coding sequence regulated by the SV40 polyA site. To clone the IFN coding sequence regulated by the CMV promoter / enhancer into the NLB retroviral plasmid, pCMV-IFN was first digested with ClaI and XbaI, and then both ends of the Klenow fragment of DNA polymerase ( New England BioLabs, Beverly, Mass.). The pNLB-adapter was digested with NdeI and KpnI, and both ends were blunted by T4 DNA polymerase (New England BioLabs). Appropriate DNA fragments were purified on 0.8% agarose-TAE gels and then ligated and transformed into DH5 α cells. The resulting plasmid was pNLB-Adapter-CMV-IFN. This plasmid was then digested with MluI, partially digested with BlpI, and the appropriate fragments gel purified. pNLB-CMV-EGFP was digested with MluI and BlpI, then treated with alkaline phosphatase and gel purified. MluI / BlpI partial fragments of pNLB-Adapter-CMV-IFN were ligated to large fragments derived from MluI / BlpI digests of pNLB-CMV-EGFP to generate pNLB-CMV-IFN.

Table 4

Example 13

Generation of Transgenic Chickens and Complete Transgenic G1 Chickens Expressing IFN

Transduced particles of pNLB-CMV-IFN were generated following the procedure of Example 2. About 300 white leghorn (line 0) eggs were observed according to the Speksnijder method (US Pat. No. 5,897,998) and about 7 × 10 ⁴ transduced particles were injected per egg. The eggs hatched 21 days after infusion and human IFN levels were measured by IFN ELISA from serum samples collected from chicks 1 week after hatching.

To screen G0 roosters containing IFN transgene in sperm, DNA was extracted from rooster sperm samples by Chelex-100 extraction (Walsh et al., 1991). Subsequently, DNA samples were prepared using the "neo for-1" (5'-TGGATTGCACGCAGGTTCT-3 '; SEQ ID NO: 5) and "neo rev-1" (5'-GTGCCCAGTCATAGCCGAAT-3'; SEQ ID NO: 6) primers and Transgenes were detected by application to Taqman® analysis in a 7700 sequence detector (Perkin Elmer) using FAM labeled NEO-PROBE1 (5'-CCTCTCCACCCAAGCGGCCG-3 '; SEQ ID NO: 7). Three G0 roosters with the highest levels of transgenes in sperm samples were mated with non-transgenic White Leghorn (SPAFAS) hens by artificial insemination.

Blood DNA samples were screened for the presence of transgenes by Taqman® analysis as described above. Of 1,597 offspring, one rooster was found to be transgenic (a.k.a. "Alphie"). Alfie's serum was tested by hIFN ELISA for the presence of hIFN and hIFN was present at 200 ng / ml.

Alfie's sperm was used for artificial insemination of non-transgenic SPAFAS (White Leghorn) hens. As detected by Taqman® analysis, 106 of the 202 offspring (about 52%) contained transgenes. This crossing result followed the Mendel genetic pattern, indicating that Alfie is transgenic.

Example 14

Carbohydrate Analysis of Transgenic Poultry-Derived Interferon-α2b (TPD IFN-α2b)

Experimental results showed a new glycosylation pattern in alpine-derived interferon-α2b (ie, TPD IFN-α2b). TPD IFN-α2b was found to contain two new protein glycoforms not commonly observed in human peripheral blood leukocyte derived interferon-α2b (PBL IFN-α2b) or natural human interferon-α2b (natural hIFN) (band 4 and 5 is α-Gal extended disaccharide; see FIG. 9). TPD IFN-α2b was also found to contain an O-linked carbohydrate structure similar to human PBL IFN-α2b and was produced more efficiently in chickens than the human form.

Coding sequences for human IFN-α2b were optimized (Example 12, supra) to generate recombinant IFN-α2b coding sequences. TPD IFN-α2b was then generated in chickens (Example 13, supra). Carbohydrate assays, including monosaccharide assays and FACE assays, have revealed sugar composition or novel glycosylation patterns of proteins. As such, TPD IFN-α2b exhibited the following monosaccharide residues: N-acetyl-galactosamine (NAcGal), galactose (Gal), N-acetyl-glucosamine (NAcGlu) and sialic acid (SA). N-linked glycosylation was not found in TPD IFN-α2b. Instead, TPD IFN-α2b was found to be O-glycosylated at Thr-106. This type of glycosylation is similar to human IFN-α2 where the Thr residue at position 106 is unique for IFN-α2. In addition, TPD IFN-α2b was found to have no mannose residues. FACE analysis showed six bands representing various sugar residues (FIG. 9), where bands 1, 2 and 3 are each unsialized sugar residues, mono-sialated sugar residues and di-sialized sugar residues. (FIG. 10). Sialic acid (SA) binding is alpha 2-3 for galactose (Gal) and alpha 2-6 for N-acetyl-galactosamine (NAcGal). Band 6 represents unsialated tetrasaccharide. Bands 4 and 5 were found to be alpha-galactose (alpha-Gal) extended disaccharides not observed in human PBL IFN-α2b. 10 shows a comparison of TPD IFN-α2b (white egg hIFN) and human PBL IFN-α2b (natural hIFN). Small bands were present between bands 3 and 4 and bands 4 and 5 in TPD IFN-α2b (see below).

The protein was found to be O-glycosylated at Thr-106 with the following specific residues:

Where

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

The percentages are as follows:

은 약 20%이고,(Ⅰ)

Is about 20%,

은 약 29%이고,(Ii)

Is about 29%,

은 약 9%이고,(Ⅲ)

Is about 9%,

은 약 6%이고,(Ⅳ)

Is about 6%,

은 약 7%이고,(Ⅴ)

Is about 7%,

은 약 12%이다.(Ⅵ)

Is about 12%.

Small bands, considered about 17% in TPD IFN-α2b, existed between bands 3 and 4 and between bands 4 and 5.

Example 15

Expression of MAb from Plasmid Transfection and Retrovirus Transduction with EMCV IRES in Algal Cells

Light chain of human monoclonal antibody from the single vector pCMV-LC-emcvIRES-HC by placement of IRES from the cardiomyocardial virus (EMCV) between the light (LC) and heavy (HC) coding sequences of the human monoclonal antibody. (LC) and heavy chain (HC) (Jang et al. (1988) "A segment of the 5 'nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation" J. Virol. 62: 2636-2643). Transcription was driven by the CMV promoter.

To test the expression of monoclonal antibodies from two separate vectors, LC or HC linked to the CMV promoter was cotransfected into LMH / 2a cells, an estrogen-reactive chicken liver cell line (Binder et al. 1990) "Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line" Mol. Endocrinol. 4: 201-208). Cotransfection of pCMV-LC and pCMV-HC yielded 392 ng / ml MAb as determined by MAb ELISA, whereas transfection of pCMV-LC-emcvIRES-HC resulted in 185 ng / ml MAb. Generated.

The CMV-LC-emcv-HC cassette was inserted into a retroviral vector based on Moroni murine leukemia virus (MLV) to generate pL-CMV-LC-emcvIRES-HC-RN-BG. LMH cells, the parent cell line of LMH / 2a (see Kawaguchi et al. (1987) "Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH" Cancer Res. 47: 4460-4464) This is because they are not neomycin resistant. LMH cells were transduced with L-CMV-LC-emcvIRES-HC-RN-BG retroviral vector, selected as neomycin and passaged for several weeks. LMH cells were transduced individually and neomycin selected using LXRN, the parent MLV vector. Medium from LXRN cells was negative for MAb, whereas medium from L-CMV-LC-emcvIRES-HC-RN-BG-transduced cells contained 22 ng / ml of MAb.

Example 16

Generation of Transgenic Chickens and Complete Transgenic G1 Chickens Expressing MAb

The pNLB-CMV-LC-emcv-HC vector was generated by replacing the CMV-BL cassette of pNLB-CMV-BL of Example 1 with the CMV-LC-emcv-HC cassette of Example 15.

According to the procedure of Example 2, transduced particles of pNLB-CMV-LC-emcv-HC were generated. About 300 white leghorn (line 0) eggs were observed according to the Speksnijder method (US Pat. No. 5,897,998), followed by injection of about 7 × 10 ⁴ transduced particles per egg. Eggs were hatched 21 days after injection and human MAb levels were measured by ELISA from serum samples collected from chicks 1 week after hatching.

G0 roosters containing transgene in sperm were identified by Taqman® analysis. Three G0 roosters with the highest levels of transgenes in sperm samples were crossed with Non-Transgenic White Leghorn by artificial insemination.

Over 1000 offspring were screened and more than 10 chicks were found to be transgenic (G1 birds). Chick serum was tested with ELlSA for the presence of MAb. It was found that MAb is present in serum at an amount of at least 10 μg / ml. Egg whites of eggs from G1 hens were also tested by EKISA for the presence of MAb and found to be present in whites in an amount of at least 10 μg / ml.

Example 17

Construction of pNLB-CMV-hG-CSF

This vector construct effectively replaces the IFN coding region of the pNLB-CMV-IFN vector of Example 12 with the coding sequence of G-CSF. From primers 5'GCSF (ggggggaagctttcaccatggctggacctgcca; SEQ ID NO: 32) and 3'GCSF (actagacttttcagggctgggcaaggtggcg; SEQ ID NO: 33) from pORF9-hG-CSFb (cat. No. Porf-hgcsfb, Invivogen, San Diego, CA) hG-CSF ORF (human granulocyte colony stimulating factor open reading frame) was amplified to generate 642 base pairs (bp) of PCR product. present adjacent to the 3 'end of the INF coding sequence to provide a pNLB-CMV-hG-CSF construct having the sequence 3' of the same G-CSF coding sequence as found in pNLB-CMV-IFN alpha-2b 86 bp fragment of pNLB-CMV-IFN alpha-2b was amplified by PCR using primers 5'GCSF-NLB (ccagccctgaaaagtctagtatggggattggtg; SEQ ID NO: 34) and 3'GCSF-NLB (gggggggctcagctggaattccgcc; SEQ ID NO: 35) . Two PCR products (642 bp and 86 bp) were mixed and fused by PCR amplification using primers 5'GCSF and 3'GCSF-NLB. PCR products were cloned into pCR®4Blunt-TOPO® plasmid vector (Invitrogen) according to the manufacturer's instructions and electroporated into DH5α-E cells to generate pFusion-hG-CSF-NLB. pFusion-hG-CSF-NLB was digested with HindIII and BlpI, and 690 bp G-CSF fragment was gel purified. The IFN alpha-2b coding sequence was separated from pNLB-CMV-IFN alpha-2b by digestion with BlpI. The vector was then ligated again and clones lacking the IFN coding insert were selected to generate pNLB-CMV-delta MFN alpha-2b. pNLB-CMV-delta IFN alpha-2b was digested with BlpI, partially digested with HindIII, and 8732 bp BlpI- HindIII vector fragments were gel purified. An 8732 bp fragment was ligated into a 690 bp HindIII / BlpI G-CSF fragment to generate pNLB-CMV-G-CSF. G-CSF ORP was confirmed by sequencing.

Example 18

Generation of Transgenic Chickens Expressing Human Granulocyte Colony Stimulating Factor (hG-CSF)

Generation of NLB-CMV-hG-CSF transduced particles was performed as described for NLB-CMV-BL in Example 2. The embryos of 277 stage X eggs were injected with 7 μl of NLB-CMV-hG-CSF transduced particles (titers 2.1 × ^{10 7} -6.9 × 10 ⁷ ). 86 chicks hatched and were sexually mature. Sixty chicks, equally gendered, divided into 30 males and 30 females, were tested for G-CSF. White eggs from 21 hens were analyzed by ELISA for the presence of hG-CSF. Five hens were found to have significant levels of hG-CSF protein in egg white at levels ranging from 0.05 ug / ml to 0.5 μg / ml.

DNA was extracted from rooster sperm samples by Chelex-100 extraction (Walsh et al., 1991). The DNA samples were then subjected to primers SJ-G-CSF for (cagagcttcctgctcaagtgctta: SEQ ID NO: 36) and SJ-G-CSF rev (ttgtaggtggcacacagcttct: SEQ ID NO: 37) and probe SJ-G-CSF probe (agcaagtgaggaagatccagggcg: sequence The transgene was detected by application to Taqman® analysis on a 7700 sequence detector (Perkin Elmer) using Listing No. 38). Cocks with the highest levels of transgenes in sperm samples were crossed with non-transgenic White Leghorn (SPAFAS) hens by artificial insemination.

Blood DNA samples were screened for the presence of transgenes by Taqman ™ analysis as described above. Of 2,264 offspring, 13 G1s were found to be transgenic, each was seropositive for the presence of G-CSF, and one hen (XGF498) was approximately 136.5 ng / s in serum, as measured by ELISA. with ml G-CSF and 5.6 μg / ml G-CSF in whites.

Two roosters (QGF910 and DD9027), the same strain as XGF498 (and therefore have the same transgene inserted at the same position in the genome), were crossed with non-transgenic hens to produce eggs containing poultry derived G-CSF. Female offspring were generated. Milligrams of G-CSF were purified from whites collected from eggs of QGF910 and DD9027 progeny. The pattern of representative glycosylation structures of poultry derived G-CSF was determined from G-CSF obtained as described in Example 20.

Example 19

Generation of Transgenic Chickens Expressing Human Cytotoxic Lymphocyte Antigen 4-Fc Fusion Protein (CTLA4-Pc)

PNLB-1.8OM-CTLA4Fc and pNLB-3.9OM-CTLA4Fc were constructed as described in US patent application Ser. No. 11 / 047,184, filed Jan. 31, 2005, which is incorporated herein by reference in its entirety. Generation of pNLB-1.8OM-CTLA4Fc and pNLB-3.9OM-CTLA4Fc transduced particles was performed as described for pNLB-CMV-BL in Example 2. 193 white leghorn eggs were injected with 7 μl of pNLB-1.8OM-CTLA4Fc transduced particles (titer of ˜4 × 10 ⁶ ) and 72 chicks hatched. 199 white leghorn eggs were injected with 7 μl of pNLB-3.9OM-CTLA4Fc transduced particles (titer of ˜4 × 10 ⁶ ) and 20 chicks hatched.

White eggs from 30 hens produced with pNLB-1.8OM-CTLA4Fc particles were analyzed by ELISA for the presence of CTLA4-Fc. One hen was found to have significant levels of CTLA4-Fc protein at an average level of 0.132 μg / ml (5 eggs analyzed) in the whites.

White eggs from seven hens produced with pNLB-3.9OM-CTLA4Fc particles were analyzed by ELISA for the presence of CTLA4-Fc. Two hens have significant levels of CTLA4-Fc protein in the egg white, of which one averages 0.164 μg / ml (5 eggs analyzed) and the second positive hen averages 0.123 μg / ml ( 5 eggs analyzed) level.

Example 20

Carbohydrate Analysis of G-CSF from Transgenic Poultry

TPD G-CSF oligonucleotide structures were determined using the following analytical techniques well known to those skilled in the art. MALDI-TOF-MS (Matrix assisted laser desorption ionization time-of-flight mass spectrometry) analysis and ESI MS / MS (electrospray ionization) for oligosaccharides after release from the peptide backbone Electrospray ionization tandem mass spectrometry was performed. O-linked oligosaccharides were chemically released from the protein, permethylated using a NaOH method involving reaction with methyl iodide under anhydrous DMSO and extracted with chloroform before analysis. Direct mass spectrometry was performed on intact glycated G-CSF. Analysis of polysaccharide structures was also performed using HPLC analysis. Briefly, after release from the protein backbone, the structures were separated using HPLC. Samples of the individual polysaccharide species were digested with specific enzymes and the degradation products were analyzed by HPLC to provide structural determinations as understood in the art.

The determined structure is shown below. Interestingly, structures C and D may be in the form of precursors of structure E shown in the figures. Structure A is always present at about 20% to about 40% in poultry derived glycosylated G-CSF, and structure B is always present at about 5% to about 25% in poultry derived glycated G-CSF, and poultry derived glycated G-CSF Structure C is always present at about 10% to about 20%, and structure D is always present at about 5% to about 15% in poultry derived glycosylated G-CSF, and structure E is always about Present in 1% to about 5%, and structure F in poultry-derived glycated G-CSF is always present from about 10% to about 25%, and structure G in poultry-derived glycated G-CSF is always about 20% to about 30% It was evaluated to exist as, but is not limited thereto.

GC / MS (gas chromatography) on poultry derived G-CSF that has been hydrolyzed, N-acetylated and spiked with TMS derived arabitol (internal standard) using methods readily available to those skilled in the art Monosaccharide analysis by mass spectrometry). The derived sample was compared to a standard mixture of similarly derived sugars. Sialic acid analysis of poultry-derived G-CSF was performed after spiking with ketodeoxynonulosonic acid followed by lyophilization followed by hydrolysis, desalting and refreeze drying. Analysis of the samples was performed on a Dionex BioLC system using appropriate standards. This analysis indicated the presence of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid (N-acetylneuraminic acid) as can be observed in Table 5. The data in Table 5 supersedes preliminary data generated by HPAEC-PAD analysis in which it is determined that a higher percentage of N-acetylglucosamine is present.

Table 5

Binding assays were performed on polar methylated glycan samples of poultry derived G-CSF hydrolyzed in TFA and reduced in sodium borodeuteride. The borate was removed by three additions of methanol: ice cold acetic acid (9: 1), followed by lyophilization and acetylation with acetic anhydride. After purification by extraction with chloroform, the samples were tested by GC / MS. Mixtures of standards were also performed under the same conditions. The binding was determined as follows:

Iii. Sialic acid bonds were 2-3 for galactose and 2-6 for N-acetylgalactosamine.

Ii. Galactose binding was 2-3 for N-acetylgalactosamine and 2-4 for N-acetylglucosamine.

Iii. N-acetylglucosamine binding was 2-6 for N-acetylgalactosamine.

Example 21

In Vitro Cell Proliferation Activity of Poultry-Derived G-CSF (TPD G-CSF)

NFS-60 cell proliferation assays were used to demonstrate in vitro biological activity of TPD G-CSF. Briefly, NFS-60 cells were maintained in growth medium containing GM-CSF. Confluence cultures were harvested, washed and plated at a cell density of 10 ⁵ cells per well with growth medium only. TPD G-CSF and bacterially derived human G-CSF (ie Neupogen®) were serially diluted in growth medium and added to separate wells in triplicate. Cell proliferation was determined by metabolic reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and quantified by spectrophotometry. The specific activity of algae-derived G-CSF was determined by comparing ED ₅₀ of Neupogen® with ED ₅₀ of purified algae-derived G-CSF. Over a period of 14 days it was determined that the specific activity of TPD G-CSF exceeds the specific activity of bacterial derived G-CSF Neuupogen® (non-glycosylated G-CSF). See FIG. 17.

Example 22

pNLB-CMV-Des-Arg166-EPO Construction

PNLB-CMV-IFN described in Example 12 was digested with HindIII and EcoRI to replace the hIFN α2 coding sequence and signal peptide coding sequence with the EPO coding sequence + signal peptide (SEQ ID NO: 42) shown below. Since a number of EcoRI and HindIII sites exist in the vector, a RecA-assisted restriction endonuclease (RARE) digestion method was used to cleave the desired site. The following oligonucleotides were used in the RARE process: pnlbEcoRI3805rare (5'-GAC TCC TGG AGC CCG TCA GTA TCG GCG GAA TTC CAG CTG AGC GCC GGT CGC TAC CAT TAC-3 ') (SEQ ID NO: 43) and pnlbHinDIII3172rare (5) '-TAA TAC GAC TCA CTA TAG GGA GAC CGG AAG CTT TCA CCA TGG CTT TGA CCT TTG CCT TAC-3') (SEQ ID NO: 44). A linearization vector of 8740 bp was obtained and gel purified.

EPO inserts were prepared by overlapping PCR as follows. A first PCR product was generated by amplification of a synthetic EPO sequence cloned into a standard cloning vector using Pfu polymerase and the following primers: 5'pNLB / Epo (5'-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3 ') (SEQ ID NO: 45 ) And pNLB / 3'Epo (5'-TCCCCATACTAGACTTTTTACCTATCGCCGGTC-3 ') (SEQ ID NO: 46). A second PCR product was generated by amplification of the region of pNLB-CMV-hIFN alpha-2b with Pfu polymerase and the following primers. 3'Epo / pNLB (5'-ACCGGCGATAGGTAAAAAGTCTAGTATGGG-3 ') (SEQ ID NO: 47) and pNLB / SapI (5'-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3') (SEQ ID NO: 48). The two PCR products were mixed and reamplified using the following primers: 5'pNLB / Epo (5'-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3 ') (SEQ ID NO: 45) and pNLB / SapI (5'-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3' ) (SEQ ID NO: 48).

The fusion PCR product was cleaved with HindIII and EcoRI, and 633 bp fragments were gel purified. 8740 bp and 633 bp fragments were ligated to generate pNLB-CMV-EPO.

EPO  1-synthesis EPO  Sequence (610 nt )

An EPO coding sequence was generated that encodes the 165 amino acid form of EPO from which the terminal codons (encoding arginine at position 166) have been removed. The 179 bp region of pNLB-CMV-EPO corresponding to the sequence from the Eco 47III site present in the EPO coding sequence within pNLB-CMV-EPO to the EcoRI site downstream of the EPO stop codon is located at the terminal arginine codon (position 166). ) Was removed to synthesize aspartic acid (amino acid 165) to the terminal amino acid codon, resulting in 176 bp Eco 47III / EcoRI fragment. The fragment was synthesized by Integrated DNA Technologies (Coralville, Iowa 52241) and cloned into pDRIVE vector (Qiagen, Inc) to generate pDRIVE-des-Arg166-EPO. 176 bp Eco 47III / EcoRI fragment was subcloned into the Eco 47III / EcoRI site of pNLB-CMV-EPO to generate pNLB-CMV-Des-Arg166-EPO.

Transduced particles were prepared essentially from pNLB-CMV-Des-Arg166-EPO as described in Example 2.

Example  23

human Erythropoietin  Expressive Transgenic  Generation of chickens

Eggs of 1234 white leghorn chickens were observed and transduced particles were injected as essentially described in Example 2. 334 eggs hatched. DNA was extracted from rooster sperm samples by Chelex-100 extraction (Walsh et al., 1991). The DNA samples were then subjected to Taqman ™ analysis on a 7700 sequence detector (Perkin Elmer) to detect the transgene. Seven hatched G0 roosters were tested positive for the NLB-CMV-EPO transgene. Three of the chimeric dotline transgenic roosters tested positive for the NLB-CMV-EPO transgene were crossed with nontransgenic hens by artificial insemination, producing 1190 offspring, 14 of which were transgenes. Positive pointline transgenic G1. The egg whites of the eggs produced by the G1 pointline transgenic hens or their descendants contained about 0.4 to 1.9 μg / ml EPO as determined by ELISA.

Example  24

Transgenic  Poultry origin EPO Tablets

The egg whites from the eggs of transgenic chickens producing EPO in the fallopian tubes were diluted with 3 volumes of 50 mM sodium acetate (pH 4.6), mixed, filtered and loaded onto a Sepharose cation exchange column. The column was washed with 50 mM sodium acetate (pH 5.0) containing 100 mM NaCl, then EPO was eluted with the same acetic acid buffer containing 500 mM NaCl with 0.05% Tween 20. EPO eluted from the Sepharose column was loaded onto a phenyl Sepharose hydrophobic interaction chromatography column. The column was equilibrated with 2 M NaCl, 50 mM Tris-HCl, pH 7.2, 0.05% Tween 20. The same buffer was used to wash the column after loading of the preparation. Then washed with water. EPO was then eluted with 30% IPA. The EPO preparation was then applied to a reversed phase HPLC column and the EPO was eluted with increasing concentrations of ethanol in 0.1% trifluoroacetic acid. A peak of EPO elution occurred at an ethanol concentration of about 53%. The final EPO preparation was concentrated and diafiltration was used to replace the solvent with 0.1 M sodium phosphate buffer (pH 7.0).

Example  25

Transgenic  Poultry origin Erythropoietin  Carbohydrate Analysis

Oligosaccharide structures for algal derived human EPO were determined by using the following analytical techniques well known to those skilled in the art.

O-linked oligosaccharides were chemically released from the protein and N-linked oligosaccharides were enzymatically released from the protein. After release, O-linked oligosaccharides and N-linked oligosaccharides were polar methylated using a NaOH method involving reaction with methyl iodide under anhydrous DMSO and then extracted with chloroform before analysis. The structure was separated using HPLC.

MALDI-TOF-MS (Matrix assisted laser desorption ionization time-of-flight mass spectrometry) for oligosaccharides after release and purification from the peptide backbone as understood in the art Analysis and ESI MS / MS (electrospray ionization tandem mass spectrometry) were performed. Samples of the individual polysaccharide species were also cleaved with specific enzymes and the cleaved products were analyzed by HPLC as understood in the art.

The O-linked oligosaccharide structure and N-linked oligosaccharide structure shown below were identified. Binding analysis of the structures showed the binding shown in FIGS. 20 and 21.

N-linked EPO structures are shown below.

The O-linked EPO structure is shown below.

Where

Gal is galactose,

NAcGal is N-acetyl-galactosamine,

NAcGlu is N-acetyl-glucosamine,

SA is sialic acid.

Example  26

Transgenic  Poultry origin EPO Carbohydrate Analysis

Monosaccharide analysis of EPO obtained from transgenic chickens by GC / MS (gas chromatography-mass spectrometry) was performed. Samples were spiked with arabitol (internal standard) using methods readily available to those skilled in the art, hydrolyzed, N-acetylated and TMS derivatized. Derivatized samples were compared to a standard mixture of similarly derivatized sugars. After spiking with ketodeoxynonulosonic acid, lyophilized, hydrolyzed, desalted and refreeze dried, sialic acid analysis of EPO was performed. Analysis of the samples was performed on a Dionex BioLC system using appropriate standards. Table 6 shows the quantification of the monosaccharides detected for EPO. Small amounts of contaminating xylose, fucose and glucose were also detected in monosaccharide analysis. The data in Table 6 replaces preliminary data generated by HPAEC-PAD analysis.

Table 6

Example  27

TPD  human EPO of In vitro  Cell proliferation activity

In vitro biological activity of poultry derived human EPO was demonstrated using TF-1 cell proliferation assay. Two separate samples representing two fractions recovered from the initial ion exchange purification step (SP1 column: 130 mM NaCl and 250 mM NaCl) were tested. Each of the two fractions exhibited essentially the same cell proliferative activity, which later also indicated that the glycated erythropoietin contained in the two fractions was essentially identical. Briefly, TF-1 cells were maintained in growth medium containing GM-CSF (2 ng / ml). Confluence cultures are harvested, washed, and stored in wells of standard 96 well plates (each well is 0.32 cm 2) at a cell density of 10 ⁴ cells per well in growth medium containing no GM-CSF. Plated. Algal derived EPO was diluted in growth medium and added in triplicates to separate wells. After 5 days cell proliferation was determined by metabolic reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and quantified spectrophotometrically. In vitro activity of purified EPO is shown in FIG. 22.

All documents mentioned in the above specification (eg, US patents, US patent applications, publications) are incorporated herein by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not overly limited to such specific embodiments. In addition, various modifications of the described methods for carrying out the invention which are apparent to those skilled in the art are considered to be within the scope of the following claims.

  <110> Ivarie, Robert D.       Liu, Guodong       Rapp, Jeffrey C.       Morris, Julie A.       Harvey, Alex J. <120> AVIAN DERIVED ERYTHROPOIETIN   <130> AVI-000CIP5PCT   <160> 50 <170> PatentIn version 3.1 <210> 1 <211> 498 <212> DNA <213> Artificial Sequence   <220> <223> Transgenic Poultry Derived IFN alpha 2b CDS <400> 1 tgcgatctgc ctcagaccca cagcctgggc agcaggagga ccctgatgct gctggctcag 60 atgaggagaa tcagcctgtt tagctgcctg aaggataggc acgattttgg ctttcctcaa 120 gaggagtttg gcaaccagtt tcagaaggct gagaccatcc ctgtgctgca cgagatgatc 180 cagcagatct ttaacctgtt tagcaccaag gatagcagcg ctgcttggga tgagaccctg 240 ctggataagt tttacaccga gctgtaccag cagctgaacg atctggaggc ttgcgtgatc 300 cagggcgtgg gcgtgaccga gacccctctg atgaaggagg atagcatcct ggctgtgagg 360 aagtactttc agaggatcac cctgtacctg aaggagaaga agtacagccc ctgcgcttgg 420 gaagtcgtga gggctgagat catgaggagc tttagcctga gcaccaacct gcaagagagc 480 ttgaggtcta aggagtaa 498 <210> 2 <211> 165 <212> PRT <213> Artificial Sequence   <220> <223> Transgenic Poultry Derived IFN alpha 2b <400> 2 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp             20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln         35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe     50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu                 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys             100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu         115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg     130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu                 165 <210> 3 <211> 579 <212> DNA <213> Artificial Sequence   <220> <223> Transgenic Poultry Derived EPO CDS <400> 3 atgggcgtgc acgagtgccc tgcttggctg tggctgctct tgagcctgct cagcctgcct 60 ctgggcctgc ctgtgctggg cgctcctcca aggctgatct gcgatagcag ggtgctggag 120 aggtacctgc tggaggctaa ggaggctgag aacatcacca ccggctgcgc tgagcactgc 180 agcctgaacg agaacatcac cgtgcctgat accaaggtga acttttacgc ttggaagagg 240 atggaggtgg gccagcaggc tgtggaggtg tggcagggcc tggctctgct gagcgaggct 300 gtgctgaggg gccaggctct gctggtgaac agctctcagc cttgggagcc tctgcagctg 360 cacgtggata aggctgtgag cggcctgaga agcctgacca ccctgctgag ggctctgggc 420 gctcagaagg aggctatcag ccctccagat gctgcaagcg ctgcccctct gaggaccatc 480 accgctgata cctttaggaa gctgtttagg gtgtacagca actttctgag gggcaagctg 540 aagctgtaca ccggcgaggc ttgcaggacc ggcgatagg 579 <210> 4 <211> 193 <212> PRT <213> Artificial Sequence   <220> <223> Transgenic Poultry Derived EPO <400> 4 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu             20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu         35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu     50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu                 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser             100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly         115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu     130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu                 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp             180 185 190 Arg      <210> 5 <211> 19 <212> DNA <213> Artificial Sequence   <220> <223> neo for-1 primer <400> 5 tggattgcac gcaggttct 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence   <220> <223> neo rev-1 primer <400> 6 gtgcccagtc atagccgaat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence   <220> <223> NEO-PROBE1 <400> 7 cctctccacc caagcggccg 20 <210> 8 <211> 83 <212> DNA <213> Artificial Sequence   <220> IFN-A primer <400> 8 atggctttga cctttgcctt actggtggct ctcctggtgc tgagctgcaa gagcagctgc 60 tctgtgggct gcgatctgcc tca 83 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence   <220> IFN-1 primer <400> 9 cccaagcttt caccatggct ttgacctttg cctt 34 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence   <220> IFN-2 primer <400> 10 ctgtgggtct gaggcagat 19 <210> 11 <211> 100 <212> DNA <213> Artificial Sequence   <220> IFN-B primer <400> 11 gacccacagc ctgggcagca ggaggaccct gatgctgctg gctcagatga ggagaatcag 60 cctgtttagc tgcctgaagg ataggcacga ttttggcttt 100 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence   <220> <223> IFN-2b primer <400> 12 atctgcctca gacccacag 19 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence   <220> IFN-3b primer <400> 13 aactcctctt gaggaaagcc aaaatc 26 <210> 14 <211> 62 <212> DNA <213> Artificial Sequence   <220> IFN-C primer <400> 14 ctcaagagga gtttggcaac cagtttcaga aggctgagac catccctgtg ctgcacgaga 60 tg 62 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> IFN-3c primer <400> 15 gattttggct ttcctcaaga ggagtt 26 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence   <220> IFN-4 primer <400> 16 atctgctgga tcatctcgtg c 21 <210> 17 <211> 95 <212> DNA <213> Artificial Sequence   <220> IFN-D primer <400> 17 atccagcaga tctttaacct gtttagcacc aaggatagca gcgctgcttg ggatgagacc 60 ctgctggata agttttacac cgagctgtac cagca 95 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence   <220> IFN-4b primer <400> 18 gcacgagatg atccagcaga t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence   <220> IFN-5 primer <400> 19 atcgttcagc tgctggtaca 20 <210> 20 <211> 78 <212> DNA <213> Artificial Sequence   <220> IFN-E primer <400> 20 gctgaacgat ctggaggctt gcgtgatcca gggcgtgggc gtgaccgaga cccctctgat 60 gaaggaggat agcatcct 78 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence   <220> IFN-5b primer <400> 21 tgtaccagca gctgaacgat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence   <220> IFN-6 primer <400> 22 cctcacagcc aggatgctat 20 <210> 23 <211> 83 <212> DNA <213> Artificial Sequence   <220> IFN-F primer <400> 23 ggctgtgagg aagtactttc agaggatcac cctgtacctg aaggagaaga agtacagccc 60 ttgcgcttgg gaagtcgtga ggg 83 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence   <220> IFN-6b primer <400> 24 atagcatcct ggctgtgagg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence   <220> IFN-7 primer <400> 25 atgatctcag ccctcacgac 20 <210> 26 <211> 65 <212> DNA <213> Artificial Sequence   <220> IFN-G primer <400> 26 ctgagatcat gaggagcttt agcctgagca ccaacctgca agagagcttg aggtctaagg 60 agtaa 65 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence   <220> <223> IFN-7b primer <400> 27 gtcgtgaggg ctgagatcat 20 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence   <220> <223> IFN-8 primer <400> 28 tgctctagac tttttactcc ttagacctca agctct 36 <210> 29 <211> 69 <212> DNA <213> Artificial Sequence   <220> <223> Lysozyme signal sequence <400> 29 ccaccatggg gtctttgcta atcttggtgc tttgcttcct gccgctagct gccttagggc 60 cctctagag 69   <210> 30 <211> 671 <212> DNA <213> Artificial Sequence   <220> <223> MDOT promoter linked to IFN-MM CDS <400> 30 atcgataggt accgggcccc ccctcgaggt gaatatccaa gaatgcagaa ctgcatggaa 60 agcagagctg caggcacgat ggtgctgagc cttagctgct tcctgctggg agatgtggat 120 gcagagacga atgaaggacc tgtcccttac tcccctcagc attctgtgct atttagggtt 180 ctaccagagt ccttaagagg tttttttttt ttttggtcca aaagtctgtt tgtttggttt 240 tgaccactga gagcatgtga cacttgtctc aagctattaa ccaagtgtcc agccaaaatc 300 gatgtcacaa cttgggaatt ttccatttga agccccttgc aaaaacaaag agcaccttgc 360 ctgctccagc tcctggctgt gaagggtttt ggtgccaaag agtgaaaggc ttcctaaaaa 420 tgggctgagc cggggaaggg gggcaacttg ggggctattg agaaacaagg aaggacaaac 480 agcgttaggt cattgcttct gcaaacacag ccagggctgc tcctctataa aaggggaaga 540 aagaggctcc gcagccatca cagacccaga ggggacggtc tgtgaatcaa gctttcacca 600 tggctttgac ctttgcctta ctggtggctc tcctggtgct gagctgcaag agcagctgct 660 cgtgggttgc g 671 <210> 31 <211> 24 <212> PRT <213> Artificial Sequence   <220> <223> MDOT promoter linked to IFN-MM CDS <400> 31 Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser Trp Val Ala             20 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence   <220> <223> 5 'GCSF Primer <400> 32 ggggggaagc tttcaccatg gctggacctg cca 33 <210> 33 <211> 31 <212> DNA <213> Artificial Sequence   <220> <223> 3 'GCSF Primer <400> 33 actagacttt tcagggctgg gcaaggtggc g 31 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence   <220> <223> 5 'GCSF-NLB Primer <400> 34 ccagccctga aaagtctagt atggggattg gtg 33 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence   <220> <223> 3 'GCSF-NLB Primer <400> 35 gggggggctc agctggaatt ccgcc 25 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence   <220> <223> SJ-G-CSF Primer <400> 36 cagagcttcc tgctcaagtg ctta 24 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SJ-G-CSF rev Primer <400> 37 ttgtaggtgg cacacagctt ct 22 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence   <220> <223> SJ-G-CSF probe <400> 38 agcaagtgag gaagatccag ggcg 24 <210> 39 <211> 612 <212> DNA <213> Artificial Sequence   <220> <223> G-CSF Nucleotide Coding Sequence <400> 39 atggctggac ctgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg 60 cacagtgcac tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc 120 cagagcttcc tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg 180 ctccaggaga agctgtgtgc cacctacaag ctgtgccacc ccgaggagct ggtgctgctc 240 ggacactctc tgggcatccc ctgggctccc ctgagcagct gccccagcca ggccctgcag 300 ctggcaggct gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag 360 gccctggaag ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc 420 gccgactttg ccaccaccat ctggcagcag atggaagaac tgggaatggc ccctgccctg 480 cagcccaccc agggtgccat gccggccttc gcctctgctt tccagcgccg ggcaggaggg 540 gtcctagttg cctcccatct gcagagcttc ctggaggtgt cgtaccgcgt tctacgccac 600 cttgcccagc cc 612 <210> 40 <211> 204 <212> PRT <213> Artificial Sequence   <220> <223> G-CSF Plus Signal Peptide <400> 40 Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro             20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu         35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys     50 55 60 Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80 Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser                 85 90 95 Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu             100 105 110 Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu         115 120 125 Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala     130 135 140 Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160 Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg                 165 170 175 Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu             180 185 190 Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro         195 200 <210> 41 <211> 174 <212> PRT <213> Artificial Sequence   <220> <223> G-CSF Mature Peptide <400> 41 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln             20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val         35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys     50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser                 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp             100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro         115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe     130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro                 165 170 <210> 42 <211> 610 <212> DNA <213> Artificial Sequence <220> <223> Synthetic EPO equence <400> 42 aagctttcac catgggcgtg cacgagtgcc ctgcttggct gtggctgctc ttgagcctgc 60 tcagcctgcc tctgggcctg cctgtgctgg gcgctcctcc aaggctgatc tgcgatagca 120 gggtgctgga gaggtacctg ctggaggcta aggaggctga gaacatcacc accggctgcg 180 ctgagcactg cagcctgaac gagaacatca ccgtgcctga taccaaggtg aacttttacg 240 cttggaagag gatggaggtg ggccagcagg ctgtggaggt gtggcagggc ctggctctgc 300 tgagcgaggc tgtgctgagg ggccaggctc tgctggtgaa cagctctcag ccttgggagc 360 ctctgcagct gcacgtggat aaggctgtga gcggcctgag aagcctgacc accctgctga 420 gggctctgag ggctcagaag gaggctatca gccctccaga tgctgcaagc gctgcccctc 480 tgaggaccat caccgctgat acctttagga agctgtttag ggtgtacagc aactttctga 540 ggggcaagct gaagctgtac accggcgagg cttgcaggac cggcgatagg taaaaaggcc 600 ggccgagctc 610 <210> 43 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pnlbEcoRI3805rare <400> 43 gactcctgga gcccgtcagt atcggcggaa ttccagctga gcgccggtcg ctaccattac 60 <210> 44 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pnlbHinD III3172rare <400> 44 taatacgact cactataggg agaccggaag ctttcaccat ggctttgacc tttgccttac 60 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> 5 'pNLB / Epo <400> 45 ggggggaagc tttcaccatg ggcgtgcacg ag 32 <210> 46 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pNLB / 3'Epo <400> 46 tccccatact agacttttta cctatcgccg gtc 33 <210> 47 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 3'Epo / pNLB <400> 47 accggcgata ggtaaaaagt ctagtatggg 30 <210> 48 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> pNLB / SapI <400> 48 gggggggctc ttctcagctg gaattccgcc gatac 35 <210> 49 <211> 495 <212> DNA <213> Artificial Sequence <220> <223> Mature EPO Nucleotide Coding Sequence <400> 49 gctcctccaa ggctgatctg cgatagcagg gtgctggaga ggtacctgct ggaggctaag 60 gaggctgaga acatcaccac cggctgcgct gagcactgca gcctgaacga gaacatcacc 120 gtgcctgata ccaaggtgaa cttttacgct tggaagagga tggaggtggg ccagcaggct 180 gtggaggtgt ggcagggcct ggctctgctg agcgaggctg tgctgagggg ccaggctctg 240 ctggtgaaca gctctcagcc ttgggagcct ctgcagctgc acgtggataa ggctgtgagc 300 ggcctgagaa gcctgaccac cctgctgagg gctctgggcg ctcagaagga ggctatcagc 360 cctccagatg ctgcaagcgc tgcccctctg aggaccatca ccgctgatac ctttaggaag 420 ctgtttaggg tgtacagcaa ctttctgagg ggcaagctga agctgtacac cggcgaggct 480 tgcaggaccg gcgat 495 <210> 50 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Mature EPO Amino Acid Sequence <400> 50 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His             20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe         35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp     50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp                 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu             100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala         115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val     130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp                 165  

Claims (106)

A composition comprising isolated human erythropoietin (EPO) comprising an algae derived oligosaccharide structure. The composition of claim 1, wherein at least about 50% of the N-linked oligosaccharides present on the EPO do not contain sialic acid. The composition of claim 1, wherein at least about 60% of the N-linked oligosaccharides present on the EPO do not contain sialic acid. The composition of claim 1, wherein at least about 70% of the N-linked oligosaccharides present on the EPO do not contain sialic acid. The composition of claim 1, wherein at least about 80% of the N-linked oligosaccharides present on the EPO do not contain sialic acid. The composition of claim 1, wherein at least about 90% of the N-linked oligosaccharides present on the EPO do not contain sialic acid. The composition of claim 1, wherein at least about 50% of the N-linked oligosaccharide structure types do not contain sialic acid. The composition of claim 1, wherein at least about 50% of the N-linked oligosaccharides present on the EPO contain terminal N-acetylglucosamine. The composition of claim 1, wherein at least about 60% of the N-linked oligosaccharides present on the EPO contain terminal N-acetylglucosamine. The composition of claim 1, wherein at least about 70% of the N-linked oligosaccharides present on the EPO contain terminal N-acetylglucosamine. The composition of claim 1, wherein at least about 80% of the N-linked oligosaccharides present on the EPO contain terminal N-acetylglucosamine. The composition of claim 1, wherein at least about 90% of the N-linked oligosaccharides present on the EPO contain terminal N-acetylglucosamine. The composition of claim 1, wherein at least about 50% of the N-linked oligosaccharide structure types present on the EPO contain terminal N-acetylglucosamine. A composition comprising glycosylated EPO, wherein at least about 80% of the N-linked oligosaccharides present on the EPO do not contain fucose. 15. The composition of claim 14, wherein at least about 90% of the N-linked oligosaccharides present on the EPO do not contain fucose. 15. The composition of claim 14, wherein said EPO is obtained from a transgenic alga containing a transgene encoding said EPO. The composition of claim 14, wherein said EPO is obtained from a transgenic chicken containing a transgene that encodes said EPO. The composition of claim 14, wherein said EPO is produced in avian fallopian cell. The composition of claim 14, wherein said EPO is present in the egg of the hard shell. The composition of claim 14, wherein said EPO is human EPO. A composition comprising isolated EPO molecules, wherein the EPO is produced in a transgenic chicken fallopian cell and the EPO is separated from the egg white of a transgenic chicken containing a transgene that encodes the EPO. The composition of claim 21, wherein said EPO is human EPO. The composition of claim 21, wherein said fallopian cells are tubular gland cells. The composition of claim 21 wherein said EPO is contained in a hard shell egg. The composition of claim 21, wherein said EPO is N-glycosylated. The composition of claim 21, wherein the EPO is O-glycosylated. The composition of claim 21, wherein said composition is a pharmaceutical formulation. The composition of claim 21, wherein the EPO has an amino acid sequence of SEQ ID NO: 50. A composition comprising isolated EPO having a chicken derived glycosylation pattern. 30. The composition of claim 29, wherein said EPO is human EPO. The composition of claim 29, wherein said EPO is produced in fallopian cells. 30. The composition of claim 29, wherein said EPO is contained in a hard shell egg. 30. The composition of claim 29, wherein said EPO is glycosylated in fallopian cells of a chicken. 30. The composition of claim 29, wherein said fallopian tube cells are coronary gland cells. The composition of claim 29, wherein the composition is a pharmaceutical formulation. The composition of claim 29, wherein said glycosylation pattern is different from the glycosylation pattern of EPO produced in CHO cells and human cells. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

38. The separation mixture of claim 37, wherein said EPO is obtained from a hard shell egg. 38. The separation mixture of claim 37, wherein said EPO is N-glycosylated. 38. The isolation mixture of claim 37, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. 38. The separation mixture of claim 37 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

43. The separation mixture of claim 42 wherein said EPO is present in an egg of a hard shell. 43. The separation mixture of claim 42 wherein said EPO is N-glycosylated. 43. The isolation mixture of claim 42, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. The separation mixture of claim 42, wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

48. The separation mixture of claim 47 wherein said EPO is present in the egg of a hard shell. 48. The separation mixture of claim 47 wherein said EPO is N-glycosylated. 48. The separation mixture of claim 47 wherein said EPO has an amino acid sequence of SEQ ID NO: 50. 48. The separation mixture of claim 47 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

53. The separation mixture of claim 52 wherein said EPO is present in the egg of a hard shell. The separation mixture of claim 52, wherein said EPO is N-glycosylated. The isolation mixture of claim 52, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. The separation mixture of claim 52 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

58. The separation mixture of claim 57 wherein said EPO is present in the egg of a hard shell. 59. The separation mixture of claim 57 wherein said EPO is N-glycosylated. 58. The isolation mixture of claim 57, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. 59. The separation mixture of claim 57 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

63. The separation mixture of claim 62 wherein said EPO is present in an egg of a hard shell. 63. The separation mixture of claim 62 wherein said EPO is N-glycosylated. 63. The isolation mixture of claim 62, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. 63. The separation mixture of claim 62 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

68. The separation mixture of claim 67 wherein said EPO is present in an egg of a hard shell. 68. The separation mixture of claim 67 wherein said EPO is N-glycosylated. 68. The isolation mixture of claim 67, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. The separation mixture of claim 67 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

73. The separation mixture of claim 72 wherein said EPO is present in an egg of a hard shell. 73. The separation mixture of claim 72 wherein said EPO is N-glycosylated. 73. The isolation mixture of claim 72, wherein said EPO has the amino acid sequence of SEQ ID NO: 50. The separation mixture of claim 72 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

78. The separation mixture of claim 77 wherein said EPO is present in an egg of a hard shell. 78. The separation mixture of claim 77 wherein said EPO is N-glycosylated. 78. The separation mixture of claim 77 wherein said EPO has an amino acid sequence of SEQ ID NO: 50. 78. The separation mixture of claim 77 wherein said EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

83. The separation mixture of claim 82 wherein said EPO is present in the egg of a hard shell. 83. The separation mixture of claim 82 wherein said EPO is N-glycosylated. 83. The separation mixture of claim 82, wherein said EPO has an amino acid sequence of SEQ ID NO: 50. The separation mixture of claim 82 wherein the EPO is present in the pharmaceutical formulation. Separation mixture of EPO molecules comprising EPO molecules glycosylated with the structure:

88. The separation mixture of claim 87 wherein said EPO is present in an egg of a hard shell. 88. The separation mixture of claim 87 wherein said EPO is O-glycosylated. 88. The separation mixture of claim 87 wherein said EPO has an amino acid sequence of SEQ ID NO: 50. 88. The separation mixture of claim 87 wherein said EPO is present in the pharmaceutical formulation. A composition comprising an EPO containing a glycosylation pattern, wherein the EPO is produced in alveolar fall cells. A composition comprising an EPO containing a glycosylation pattern, wherein said glycosylation pattern is different from the glycosylation pattern of EPO produced in human cells or CHO cells, wherein said EPO is produced in chicken fallopian cells. 94. The composition of claim 93, wherein said EPO is isolated. 94. The composition of claim 93, wherein said fallopian tube cells are coronary gland cells. A method of treating a patient comprising administering to the patient an EPO obtained from a therapeutically effective amount of a transgenic bird. 97. The method of claim 96, wherein said therapeutically effective amount is an amount that increases a patient's erythrocyte cell number to a desired amount. A composition comprising isolated glycosylated human protein molecules produced in the fallopian tube of a transgenic chicken, wherein said transgenic chicken contains a transgene that encodes a human protein molecule, wherein said protein molecule is not generally present in a human protein. A composition containing chicken-derived oligosaccharides. A composition comprising isolated protein molecules produced in the fallopian tubes of a transgenic chicken, wherein said transgenic chicken contains a transgene encoding a protein molecule, said protein molecule containing a chicken derived oligosaccharide, and said protein molecule EPO, G-CSF, GM-CSF, interferon β, fusion protein, CTLA4-Fc fusion protein, growth hormone, cytokines, structural interferon, lysozyme, β-casein, albumin, α-1 antitrypsin, anti Thrombin III, collagen, factor VII, VII, VII (etc.), fibrinogen, lactoferrin, protein C, tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin, immunoglobulins, antibodies, immunotoxins , Factor VII, b-domain deletion factor VII, factor VIIa, factor VII, anticoagulant; Hirudin, alteplase, tpa, reteplase, tpa, tpa lacking three of the five domains, insulin, insulin lispro, insulin aspart, insulin glargine, Persistent insulin analogs, glucagon, tsh, polytropin-beta, fsh, pdgh, inf-beta 1b, ifn-alpha 1, ifn-alpha, ifn-alpha 1a, ifn-alpha 1b, ifn-alpha 2, ifn-alpha 2a, ifn-alpha 2b, ifn-beta 1a, ifn-gamma 1b, il-2, H-11, hbsag, ospa, dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diphtheria toxin fusion protein, tnfr-Igg fragment fusion protein laronidase, dnaases, alefacept, tositumomab, murine mab, alemtuzumab, ras Burasease, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (Igg1), anakinra (anakin) ra), biological modulators, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factor, pegvisomant, human growth hormone receptor antagonist, recombinant activating protein c, O'Malley Umalizumab, immunoglobulin e (Ige) blocker, Ibritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigment hormone, somatomedin, corpus luteum formation Hormone, chorionic gonadotropin, hypothalamic hormone, etanercept, antidiuretic hormone, prolactin and thyroid stimulating hormone, immunoglobulin polypeptide, immunoglobulin polypeptide D region, immunoglobulin polypeptide J region, immunoglobulin polypeptide C region, immune Globulin light chain, immunoglobulin heavy chain, immunoglobulin heavy chain variable region, immunoglobulin light chain variable region and A composition selected from the group consisting of large peptides. 107. The composition of claim 99, wherein said composition is in a pharmaceutical formulation. 107. The composition of claim 99, wherein said protein is N-glycosylated. 107. The composition of claim 99, wherein said protein is O-glycosylated. A method of preparing glycosylated erythropoietin comprising generating a transgenic algae containing a transgene encoding erythropoietin, wherein the erythropoietin is packaged in an egg of a hard shell produced by the algae. How). Eggs laid by algae containing EPO or G-CSF. An egg produced by an alga containing the protein of claim 99. Whites containing at least 0.5 micrograms of exogenous protein per ml.

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