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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
  • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/805—Haemoglobins; Myoglobins
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2207/00—Modified animals
  • A01K2207/15—Humanized animals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/108—Swine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/01—Animal expressing industrially exogenous proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the use of transgenic pigs for the production of human
• the transgenic pigs of the invention may be used as an efficient and economical source of cell-free human hemoglobin that may be used for
• Oxygen absorbed through the lungs is carried by hemoglobin in red blood cells for delivery to tissues throughout the body. At high oxygen tensions, such as those found in the proximity of the lungs, oxygen binds to hemoglobin, but is released in areas of low oxygen tension, where it is needed.
• Each hemoglobin molecule consists of two alpha globin and two beta globin subunits. Each subunit, in turn, is noncovalently associated with an iron-containing heme group capable of carrying an oxygen molecule. Thus, each hemoglobin tetramer is capable of binding four molecules of oxygen.
• the subunits work together in switching between two conformational states to facilitate uptake and release of oxygen at the lungs and tissues, respectively.
• This effect is commonly referred to as heme-heme interaction or cooperativity.
• the hemoglobins of many animals are able to interact with biologic effector molecules that can further enhance oxygen binding and release. This enhancement is manifested in changes which affect the allosteric equilibrium between the two conformational states of hemoglobin.
• human and pig hemoglobin can bind 2, 3 diphosphoglycerate (2,3 DPG), which influences the equilibrium between the two conformational states of the tetramer and has the net effect of lowering the overall affinity for oxygen at the tissue level.
• 2,3-DPG increases the efficiency of oxygen delivery to the tissues.
• Hemoglobin protein is expressed in a tissue specific manner in red blood cells where it accounts for approximately ninety percent of total cellular protein.
• red blood cells which have lost their nucleus and all but a minimal number of organelles, are effectively membrane-enclosed packets of
• hemoglobin dedicated to oxygen transfer
• Human globin genes are found in clusters on chromosome 16 for alpha ( ⁇ ) globin and chromosome 11 for beta ( ⁇ ) globin.
• the human beta globin gene cluster consists of about 50 kb of DNA that includes one embryonic gene encoding epsilon ( ⁇ ) globin, two fetal genes encoding gamma ( ⁇ ) G and gamma A globin, and two adult genes encoding delta ( ⁇ ) and beta ( ⁇ ) globin, in that order (Fritschet al., 1980, Cell
• LCR locus control region
• transfused blood must be compatible with the blood type of the transfusion recipient; the donated blood supply may be unable to provide transfusions to individuals with rare blood types.
• hemoglobin produced by genetic engineering would not require blood type matching, would be virus-free, and would be available in potentially unlimited amounts.
• a transgenic animal is a non-human animal containing at least one foreign gene, called a transgene, in its genetic material.
• the transgene is contained in the animal's germ line such that it can be transmitted to the animal's offspring.
• a number of techniques may be used to introduce the transgene into an animal's genetic material,
• Transgenic animals may carry the transgene in all their cells or may be genetically mosaic.
• transgenic mice Although the majority of studies have involved transgenic mice, other species of transgenic animal have also been produced, such as rabbits, sheep, pigs (Hammer et al., 1985, Nature 315:680-683) and chickens (Salter et al., 1987, Virology 157:236-240). Transgenic animals are currently being
• phosphorylation, subunit assembly, etc. are critical for the activity of the molecule.
• transgenic livestock has proved problematic. Not only is it technically difficult to produce transgenic embryos, but mature transgenic animals that produce significant quantities of recombinant protein may prove inviable.
• the experience has been that pigs carrying a growth hormone encoding transgene (the only transgene introduced into pigs prior to the present invention) suffered from a number of health problems, including severe arthritis, lack of coordination in their rear legs, susceptibility to stress, anoestrus in gilts and lack of libido in boars (Wilmut et al., supra).
• transgenic mice carrying a growth hormone transgene which appeared to be healthy (Palmiter et al., 1982, Nature 300:611-615).
• healthy transgenic pigs which efficiently express their transgene(s) had not been produced.
• mice/human adult beta globin gene was described by Magram et al. in 1985 (Nature 315:338-340). Kollias et al. then reported regulated expression of human gamma-A, beta, and hybrid beta/gamma globin genes in transgenic mice (1986, Cell 46:89-94). Transgenic mice expressing human fetal gamma globin were studied by Enver et al. (1989, Proc. Natl. Acad. Sci. U.S.A. 86: 7033-7037) and Constantoulakis et al. (1991, Blood 77: 1326-1333). Autonomous developmental control of human embryonic globirr-gene switching in transgenic mice was observed by Raich et al. (1990, Science
• the present invention relates to the use of transgenic pigs for the production of human hemoglobin and/or human globin. It is based, at least in part, on the discovery that transgenic pigs may be generated that express human hemoglobin in their erythrocytes and are healthy, suffering no deleterious effects as a result of heterologous hemoglobin production.
• the present invention provides for transgenic pigs that express human globin genes.
• Such animals may be used as a particularly efficient and economical source of human hemoglobin, in light of (i) the relatively short periods of gestation and sexual maturation in pigs; (ii) the size and frequency of litters, (iii) the relatively large size of the pig which provides proportionately large yields of hemoglobin; and (iv) functional similarities between pig and human
• hemoglobins in the regulation of oxygen binding affinity which enables the transgenic pigs to remain healthy in the presence of high levels of human hemoglobin.
• the present invention also provides for recombinant nucleic acid constructs that may be used to generate transgenic pigs.
• recombinant nucleic acid constructs that may be used to generate transgenic pigs.
• such constructs place the human alpha and beta globin genes under the same promoter so as to avoid deleterious effects of gfobin chain imbalance and/or titration of transcription factors due to constitutive ⁇ -globin promoter activity in an
• inappropriate cell type e.g. a primitive
• the present invention provides for a hybrid hemoglobin that comprises human ⁇ globin and pig ⁇ globin.
• the whole blood from transgenic pigs expressing this hybrid hemoglobin appears to exhibit a P 50 that is
• the present invention also provides for a method of producing human hemoglobin comprising (i) introducing a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in at least some of its red blood cells; (ii) collecting red blood cells from the transgenic pig; (iii) releasing the contents of the collected red blood cells; and (iv) subjecting the released contents of the red blood cells to a purification procedure that substantially separates human hemoglobin from pig hemoglobin.
• a method of producing human hemoglobin comprising (i) introducing a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in at least some of its red blood cells; (ii) collecting red blood cells from
• human hemoglobin may be separated from pig hemoglobin by DEAE anion exchange column
• Construct ⁇ p ⁇ (the "185” construct); C. Construct ⁇ p ⁇ (the "290” construct); D. Construct ⁇ p ⁇ ; E. Construct ⁇ p ⁇ p ⁇ ; F. Construct ⁇ p ⁇ carrying a ⁇ 108 Asn -> Asp mutation (the "hemoglobin
• the present invention provides for a method of producing human hemoglobin that utilizes transgenic pigs, novel globin-encoding nucleic acid constructs, and transgenic pigs that express human hemoglobin.
• the present invention provides for a method of producing human globin and/or hemoglobin in
• Human hemoglobin is defined herein to refer to hemoglobin formed by globin chains encoded human globin genes (including alpha, beta, delta, gamma, epsilon and zeta genes) or variants thereof which are naturally occurring or the products of genetic engineering. Such variants are at least about ninety percent homologous in amino acid sequence to a naturally occurring human hemoglobin.
• the human hemoglobin of the invention comprises a human alpha globin and a human beta globin chain.
• the human hemoglobin of the invention is defined herein to refer to hemoglobin formed by globin chains encoded human globin genes (including alpha, beta, delta, gamma, epsilon and zeta genes) or variants thereof which are naturally occurring or the products of genetic engineering. Such variants are at least about ninety percent homologous in amino acid sequence to a naturally occurring human hemoglobin.
• the human hemoglobin of the invention comprises a human alpha globin and a human beta
• human hemoglobin comprises at least two different globin chains, but may comprise more than two chains, to form, for example, a tetrameric molecule, octameric molecule, etc.
• human hemoglobin consists of two human alpha globin chains and two human beta globin chains.
• the present invention also provides for hybrid
• hemoglobin comprising human ⁇ globin and pig ⁇ globin.
• At least one human globin gene such as a human alpha and/or a human beta globin gene, under the control of a suitable promoter or promoters, is inserted into the genetic material of a pig so as to create a transgenic pig that carries human globin in at least some of its red blood cells.
• both human ⁇ and human ⁇ genes are expressed.
• only human ⁇ globin is expressed.
• human embryonic or fetal globin genes are expressed or are used as developmental expression regulators of adult genes.
• Human alpha and beta globin genes may be obtained from publicly available clones, e.g. as described in Swanson et al., 1992, Bio/Technol.
• Nucleic acid sequences encoding human alpha and beta globin proteins may be introduced into an animal via two different species of recombinant constructs, one which encodes human alpha globin, the other encoding human beta globin; alternatively, and preferably, both alpha and beta-encoding sequences may be comprised in the same recombinant construct.
• a suitable promoter is a promoter which can direct
• Such a promoter is preferably selectively active in erythroid cells. This would include, but is not limited to, a globin gene
• promoter such as the human alpha, beta, delta, epsilon or zeta promoters, or a globin promoter from another species. It may, for example, be useful to utilize pig globin promoter sequences.
• the human alpha and beta globin genes may be placed under the control of different promoters, but, since it has been inferred that vastly different levels of globin chain production may result in lethality, it may be
• a construct comprising the ⁇ construct (also termed the "116" construct; Swanson et al., 1992, Bio/Technol. 10:557-559; see Figure 2A) may be utilized.
• this construct when present as a transgene at high copy number, has resulted in deleterious effects in mice, it has been used to produce healthy transgenic pigs (see Example Section 6, infra).
• a construct comprising the ⁇ p/ ⁇ sequence (also termed the "185" construct), as depicted in Figure 1B may be used.
• Such a construct has the advantage of placing both alpha and beta globin-encoding sequences under the control of the same promoter (the alpha globin promoter).
• the present invention in further specific embodiments, provides for (i) the construct ⁇ p ⁇ , in which the human alpha and beta globin genes are driven by separate copies of the human beta globin promoter ( Figure 1C); (ii) the epf ⁇ p ⁇ construct, which
• ⁇ construct which comprises the human embryonic epsilon gene, the human adult alpha globin gene and the human adult beta globin gene linked in tandem from 5'- to 3' (Fig. 10);
• ⁇ construct which comprises the human adult alpha-globin gene, the human embryonic epsilon globin gene and the human adult beta globin gene linked in tandem from 5'- to 3' (Fig. 10);
• ⁇ construct which comprises the human adult alpha-globin gene, the human embryonic epsilon globin gene and the human adult beta globin gene linked in tandem from 5'- to 3' (Fig.
• the recombinant nucleic acid constructs described above may be inserted into any suitable plasmid, bacteriophage, or viral vector for
• Constructs may desirably be linearized for preparation of transgenic pigs.
• Vector sequence may desirably be removed.
• transgenic pig may be produced by any method known in the art, including but not limited to, microinjection, embryonic stem (ES) cell manipulation, electroporation, cell gun, trartsfection, transduction, retroviral infection, etc. Species of constructs may be introduced individually or in groups of two or more types of construct. According to a preferred specific embodiment of the invention, a transgenic pig may be produced by the methods as set forth in Example Section 6, infra.
• estrus may be synchronized in sexually mature gilts (>7 months of age) by feeding an orally active progestogen (allyl tre ⁇ bolone, AT: 15 mg/gilt/day) for 12 to 14 days.
• an orally active progestogen allyl tre ⁇ bolone, AT: 15 mg/gilt/day
• AT allyl tre ⁇ bolone
• all gilts may be given an intramuscular injection (IM) of prostaglandin F 2a (Lutalyse: 10 mg/injection) at 0800 and 1600 hours.
• Twenty-four hours after the last day of AT consumption all donor gilts may be administered a single IM injection of pregnant mare serum
• gonadotropin (HCG: 750 IU) may be administered to all donors at 80 hours after PMSG.
• donor and recipient gilts may be checked twice daily for signs of estrus using a mature boar.
• Donors which exhibited estrus within 36 hours following HCG administration may be bred at 12 and 24 hours after the onset of estrus using artificial and natural (respectively)
• one- and two-cell ova may be surgically recovered from bred donors using the following procedure.
• General anesthesia may be induced by administering 0.5 mg of acepromazine/kg of bodyweight and 1.3 mg ketamine/kg of bodyweight via a peripheral ear vein.
• the reproductive tract may be exteriorized following a mid-ventral laparotomy.
• a drawn glass cannula (O.D. 5 mm, length 8 cm) may be inserted into the ostium of the oviduct and anchored to the infundibulum using a single silk (2-0) suture.
• Ova may be flushed in retrograde fashion by inserting a 20 g needle into the lumen of the oviduct 2 cm anterior to the uterotubal junction.
• Sterile Dulbecco's phosphate buffered saline (PBS) supplemented with 0.4% bovine serum albumin (BSA) may be infused into the oviduct and flushed toward the glass cannula.
• the medium may be collected into sterile 17 ⁇ 100 mm polystyrene tubes. Flushings may be transferred to 10 ⁇ 60 mm petri dishes and searched at lower power (50 x) using a Wild M3 stereomicroscope. All one- and two-cell ova may be washed twice in Brinster's Modified Ova Culture-3 medium (BMOC-3) supplemented with 1.5% BSA and
• Ova may be stored at 38°C under a 90% N 2 , 5% O 2 , 5% CO 2 atmosphere until microinjection is performed.
• One- and two-cell ova may be placed in a Eppendorf tube (15 ova per tube) containing 1 ml HEPES Medium supplemented with 1.5% BSA and centrifuged for 6 minutes at 14000 ⁇ g in order to visualize pronuclei in one-cell and nuclei in two-cell ova. Ova may then be transferred to a 5 - 10 ⁇ l drop of HEPES medium under oil on a depression slide. Microinjection may be performed using a Laborlux microscope with
• concentration of about lng/ ⁇ l of Tris-EDTA buffer may be injected into one pronuclei in one-cell ova or both nuclei in two-cell ova.
• Microinjected ova may be returned to microdrops of BMOC-3 medium under oil and maintained at 38°C under a 90% N 2 , 5% CO 2 , 5% O 2 atmosphere prior to their transfer to suitable-recipients. Ova may preferably be transferred within 10 hours of recovery.
• ova preferably be utilized for embryo transfer. Recipients may be anesthetized as described earlier. Following exteriorization of one oviduct, at least 30 injected one-and/or two-cell ova and 4-6 control ova may be transferred in the following manner.
• the tubing from a 21 g ⁇ 3/4 butterfly infusion set may be connected to a 1 cc syringe.
• the ova and one to two mis of BMOC-3 medium may be aspirated into the tubing.
• the tubing may then be fed through the ostium of the oviduct until the tip reaches the lower third or isthmus of the oviduct. The ova may be subsequently expelled as the tubing is slowly withdrawn.
• the exposed portion of the reproductive tract may be bathed in a sterile 10% glycerol-0.9% saline solution and returned to the body cavity.
• the connective tissue encompassing the linea alba, the fat and the skin may be sutured as three separate layers.
• An uninterrupted Halstead stitch may be used to close the Una alba.
• the fat and skin may be closed using a simple continuous and mattress stitch, respectively.
• a topical antibacterial agent e.g. Furazolidone
• Furazolidone may then be administered to the incision area.
• piglets may be processed, i.e. ears notched, needle teeth clipped, 1 cc of iron dextran administered, etc.
• a tail biopsy and blood may also be obtained from each pig.
• Pigs producer! according to this method are described in Example Section 6, infra, and are depicted in Figure 2. Such pigs are healthy, do not appear to be anemic, and appear to grow at a rate comparable to that of their non-transgenic
• Such pigs may transmit the transgene to their offspring.
• hemoglobin such pigs, examples of which follow, represent preferred, non-limiting, specific
• a transgenic pig contains at least twenty copies of a globin transgene.
• the P 50 of whole blood of a transgenic pig according to the invention is increased by at least ten percent over the P 50 of the whole blood of a comparable non-transgenic pig,taking into
• the present invention provides for a non-pregnant transgenic pig that carries and expresses a human globin transgene in which the P 50 of whole blood of the transgenic pig is at least ten percent greater than the P 5 ⁇ of whole blood of a comparable non-pregnant non-transgenic pig at the same altitude.
• the present invention provides for a transgenic pig in which the amount of human globin produced relative to total hemoglobin is at least two percent, more
• Section 6 infra describes transgenic pigs which serve as working examples of preferred, non- limiting, specific examples of the invention.
• the present invention provides for a method for producing human hemoglobin comprising introducing a transgene or transgenes encoding human hemoglobin, such as a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human
• hemoglobin in at least some of its blood cells.
• the present invention also provides for a method of producing human hemoglobin comprising (i) introducing a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in at least some of its red blood cells; (ii) collecting red blood cells from the transgenic pig; (iii) releasing the contents of the collected red blood cells to form a lysate; (iv) subjecting the lysate of the red blood cells to a purification procedure that substantially separates human
• hemoglobin from pig hemoglobin and (v) collecting the fractions that contain purified human hemoglobin.
• hemoglobin may be separated from pig hemoglobin by DEAE anion exchange column chromatography.
• red blood cells are obtained from the pig using any method known in the art.
• the red blood cells are then lysed using any method, including hemolysis in a hypotonic solution such as distilled water, or using techniques as described in 1981, Methods in Enzymology Vol. 76, and/or tangential flow filtration.
• transgenic pig it may be useful to perform a small- scale electrophoretic analysis of the hemolysate, such as, for example, isoelectric focusing using standard techniques.
• human hemoglobin may be separated from pig hemoglobin using ion exchange chromatography.
• Any ion exchange resin known in the art or to be developed may be utilized, including, but not limited to, resins comprising diethylaminoethyl, Q-Sepharose, QCPI (I.B.F.) Zephyr, Spherodex, ectiola, carboxymethylcellulose, etc.
• the resin results in a separation of human and pig hemoglobin comparable to that achieved using DEAE resin.
• a hemolysate of transgenic pig red blood cells prepared as above may be applied to a DEAE anion exchange column equilibrated with 0.2 M glycine buffer at pH 7.8 and washed with 0.2 M glycine pH 7.8/5 mM NaCl, and may then be eluted with a 5-30 mM NaCl gradient, or its equivalent (see, for example, Section 9 infra).
• human and pig hemoglobin separates readily upon such treatment, with human hemoglobin eluting earlier than pig hemoglobin. Elution may be monitored by optical density at 405 nm and/or electrophoresis of aliquots taken from serial fractions. Pig hemoglobin, as well as tetrameric hemoglobin composed of heterodimers formed between pig and human globin chains, may be separated from human hemoglobin by this method. Human hemoglobin produced in a transgenic pig and separated from pig hemoglobin by this method has an oxygen binding capability similar to that of native human hemoglobin.
• human hemoglobin may be separated from pig hemoglobin (including human/pig hemoglobin hybrids) using QCPI ion exchange resin as follows:
• Buffer A 10mM Tris, 20mM Glycine pH 7.5
• the column may then be washed with 2 volumes of Buffer A, and then with 20 column volumes of a 0-50mM NaCl gradient (10 column volumes of Buffer A + 10 column volumes of 10mM Tris, 20mM Glycine, 50mM NaCl Ph 7.5) or, alteratively, 6 column volumes of 10mM Tris, 20mM Glycine, 15mM NaCl, pH 7.5, and the O.D. 280 absorbing material may be collected in fractions to yield the separated
• hemoglobin human hemoglobin being identified, for example, by isoelectric focusing using appropriate standards.
• the QCPI column may be cleaned by elution with 2 column volumes of 10mM Tris, 20mM Glycine, 1M NaCl, pH 7.5.
• the present invention also provides for essentially purified and isolated human/pig hybrid hemoglobin, in particular human ⁇ /pig ⁇ hybrid hemoglobin.
• Pig ⁇ /human ⁇ hybrid has not been observed to form either in vitro in reassociation experiments or in vitro in transgenic pigs.
• the present invention provides for hybrid hemoglobin and its use as a blood substitute, and for a pharmaceutical composition comprising the
• Hybrid hemoglobin may be prepared from transgenic pigs, as described herein, and then
• hybrid hemoglobin may be prepared using nucleic acid constructs that comprise both human and pig globin sequences which may then be expressed in any suitable microorganism, cell, or transgenic animal.
• a nucleic acid construct that comprises the human ⁇ and pig ⁇ globin genes under the control of a suitable promoter may be expressed to result in hybrid hemoglobin.
• human ⁇ globin and pig ⁇ globin genes, under the control of cytomegalovirus promoter may be transfected into a mammalian cell such as a COS cell, and hybrid hemoglobin may be harvested from such cells.
• such constructs may be
• hemoglobin hybrid it may be desirable to modify the hemoglobin hybrid so as to render it non-immunogenic, for
• NUCLEIC ACID CONSTRUCTS Constructs 116 (the ⁇ construct), 185 (the ⁇ p ⁇ construct), or 263 (the ⁇ p ⁇ construct) were microinjected into pig ova as set forth below in order to produce transgenic pigs.
• donor and recipient gilts were checked twice daily for signs of estrus using a mature boar.
• Donors which exhibited estrus within 36 hours following HCG administration were bred at 12 and 24 hours after the onset of estrus using artificial and natural (respectively) insemination.
• Eppendorf tube (15 ova per tube) containing 1 ml HEPES Medium supplemented with 1.5% BSA and centrifuged for 6 minutes at 14000 ⁇ g in order to visualize pronuclei in one-cell and nuclei in two-cell ova. Ova were then transferred to a 5 -10 ⁇ l drop of HEPES medium under oil on a depression slide. Microinjection was
• construct DNA lng/ ⁇ l of Tris-EDTA buffer
• microdrops of BMOC-3 medium under oil and maintained at 38°C under a 90% N 2 , 5% CO 2 , 5% O 2 atmosphere prior to their transfer to suitable recipients. Ova were transferred within 10 hours of recovery.
• the tubing from a 21 g ⁇ 3/4 butterfly infusion set was connected to a 1 cc syringe.
• the ova and one to two mis of BMOC-3 medium were aspirated into the tubing.
• the tubing was then fed through the ostium of the oviduct until the tip reached the lower third or isthmus of the oviduct.
• the ova were subsequently expelled as the tubing was slowly withdrawn.
• connective tissue encompassing the linea alba, the fat and the skin were sutured as three separate layers.
• a topical antibacterial agent (Furazolidone) was then administered to the incision area.
• prostaglandin F 2a (10 mg/injection) at 0800 and 1400 hours on day 112 of gestation. In all cases, recipients farrowed within 34 hours
• Table III presents the profiles of offspring of pig number 9-3, which shows that the F1 generation of transgenic pigs are capable of expressing
• the birth weights of the transgenic pigs have been approximately equivalent to the birth weights of their non-transgenic littermates. As the transgenic pigs matured, their weights remained comparable to the weights of control animals.
• red blood cells were collected by centrifugation of 5000 rpm for 3 minutes in an eppendorf microcentrifuge and washed three times with an equal volume (original blood) of 0.9% NaCl. Red cells were lysed with 1.5 volumes deionized H 2 O, centrifuged at 15,000 rpm, and the supernatant was fractionated by anion exchange chromatography.
• DEAE cellulose chromatography DE-SE manufactured by
• 50 lambda of the lysed cells was then combined with 50 lambda 0.2 M Na Acetate, pH 4.5, put on ice and then incubated in a cold room overnight. After adding 1.9 ml 0.1 M NaH 2 PO 4 4, pH 7.4 each sample was spun in centricon tubes at 4°C and 5K until about 0.5 ml remained. Then 1 ml of 0.1 M NaH 2 PO 4 pH 7.4 was added and spun through at about 5K until about 0.2 ml volume was left. The hemoglobin was then washed from the walls of the centricon tube, an eppendorf adaptor was attached, and a table top microfuge was used to remove each sample from its centricon tube. The samples were then analyzed by isoelectric focusing.
• the order of elution of the proteins from the anion exchange column was not as expected. Based on the relative pi's of the proteins as deduced from the IEF gels, the predicted order of elution would be first the hybrid (human ⁇ /pig ⁇ ) followed by the authentic human ⁇ /human ⁇ . The last protein to elute from the anion exchange column then would be the endogenous pig ⁇ /pig ⁇ protein. However, under all the conditions currently attempted the order of elution was altered such that the human hemoglobin was the first to elute. The second peak was an enriched fraction of the hybrid followed very closely by the pig hemoglobin.
• human hemoglobin was separated from pig hemoglobin and from human ⁇ globin/pig beta globin heterologous hemoglobin. As shown in Figure 4D, human hemoglobin was substantially purified by this method. 7.2.3. PIG ALPHA GLOBIN/HUMAN BETA GLOBIN HETEROLOGOUS HEMOGLOBIN DOES NOT APPEAR TO FORM BASED ON REASSOCIATION DATA
• hemoglobin-expressing transgenic pigs It was possible, however, that this observation could be explained by relatively low levels of human beta globin expression. Alternatively, association between pig alpha globin and human beta globin may be
• Buffer A 10mM Tris, 20mM Glycine pH 7.5;
• Buffer B 10mM Tris, 20mM Glycine, 15 mM NaCl pH 7.5;
• Buffer C 10mM Tris, 20mM Glycine, 1M NaCl pH 7.5;
• Buffer D 10mM Tris, 20mM Glycine, 50 mM NaCl pH 7.5; QCPI column 10ml Equilibrated in Buffer A; Trio purification system. 10mg of hemoglobin prepared from transgenic pig 6-3 was diluted in 20ml Buffer A. 20ml of sample was loaded at a flow rate of 5ml/min onto the QCPI column, and washed with 2 column volumes of Buffer A. The column was then washed with 20 column volumes of a 0-50mM NaCl gradient. (10 column volumes Buffer A + 10 column volumes of Buffer D) and the O.D. 280 absorbing material was collected. The column was then cleaned with 2 column volumes of Buffer C, and then re-equilibrated with 2 column volumes of Buffer A.
• transgenic pigs of the invention were all found to produce significant amounts of human ⁇ /pig ⁇ globin hybrid hemoglobin (the pig ⁇ /human ⁇ hybrid was not observed). Significantly, pigs that expressed higher percentages of hybrid also appeared to exhibit

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