WO1993025071A9 - Utilisation de porcs transgeniques pour la production d'hemoglobine humaine - Google Patents

Utilisation de porcs transgeniques pour la production d'hemoglobine humaine

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Publication number
WO1993025071A9
WO1993025071A9 PCT/US1993/005629 US9305629W WO9325071A9 WO 1993025071 A9 WO1993025071 A9 WO 1993025071A9 US 9305629 W US9305629 W US 9305629W WO 9325071 A9 WO9325071 A9 WO 9325071A9
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WO
WIPO (PCT)
Prior art keywords
human
pig
hemoglobin
globin
transgenic
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PCT/US1993/005629
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English (en)
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WO1993025071A1 (fr
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Priority to EP93915317A priority Critical patent/EP0655888A4/fr
Priority to AU45343/93A priority patent/AU687743B2/en
Priority to JP5512779A priority patent/JPH07507921A/ja
Publication of WO1993025071A1 publication Critical patent/WO1993025071A1/fr
Publication of WO1993025071A9 publication Critical patent/WO1993025071A9/fr
Priority to FI945829A priority patent/FI945829A/fi
Priority to NO944811A priority patent/NO944811L/no

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  • 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 (e) globin, two fetal genes encoding gamma ( ⁇ ) G and gamma A globin, and two adult genes encoding delta ( ⁇ ) and beta ( ⁇ ) globin, in that order (Fritsch et al., 1980, Cell
  • 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
  • Methods of expressing recombinant protein via transgenic livestock have an important theoretical advantage over protein production in recombinant bacteria and yeast; namely, the ability to produce large, complex proteins in which post-translational modifications, including glycosylation,
  • 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 globin 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 globin chain imbalance and/or titration of transcription factors due to constitutive ⁇ -globin promoter activity in an
  • inappropriate cell type e.g. a primitive
  • the constructs comprise the pig adult beta globin gene regulatory region, comprising the promoter or the 3' region of the pig beta globin gene.
  • 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 j6l08 Asn -> Asp mutation (the "hemoglobin
  • hemoglobin by DEAE chromatography A. Hemolyzed mixture of human and pig red blood cells; B.
  • Figure 7. Oxygen affinity of transgenic hemoglobin.
  • Figure 8. DNA sequence of the pig adult beta globin gene regulatory region, including the promoter region. Sequence extending to 869 base pairs upstream of the ATG initiator codon (boxed) of the pig beta globin gene is shown. The position of the initiation of mRNA, the cap site, is indicated by an arrow. The sequences corresponding to GATA transcription factor binding sites are underlined.
  • Figure 9 Comparison of pig (top) and human (bottom) beta globin regulatory sequences. Differences in the two sequences are marked by asterisks.
  • cassettes for the production of human hemoglobin in transgenic pigs are provided.
  • FIG. 13 Map of plasmid pSaf/Pige (k), containing the pig ⁇ gene.
  • hemoglobin is run as a standard.
  • Figure 20 Molecular modeling of hybrid human ⁇ /pig ⁇ and human ⁇ /human ⁇ hemoglobin molecules. ⁇ subunits are in blue, ⁇ subunits in red. Above the middle helix of the ⁇ human (blue) one can see a gap in the green contour (see arrow). In the hybrid this gap is filed in. This difference is due to a change at 0112 Cys- - - >Val where
  • Valine contributes to greater hydrophobic
  • transgenic pig hemosylate transgenic pig hemosylate
  • FIG. 24 Oxygen binding curve for Hb Presbyterian.
  • Figure 25 Purification of Hb Yoshizuka from
  • 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 by 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.
  • 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
  • hemoglobins 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 or 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.
  • the pig epsilon globin gene is contained in plasmid psaf/pig ⁇ (k) ( Figure 13), deposited with the ATCC and assigned accession number 75373.
  • 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. For example, as discussed in Section 10, infra, the use of the endogenous pig ⁇ globin gene control region, as contained in plasmid Pgem5/Pig ⁇ pr (K), deposited with the ATCC and assigned accession number 75371 and having the sequence set forth in Figure 8, has been shown to operate particularly efficiently.
  • 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 1A) 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; see 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).
  • a construct coding for di-alpha globin like polypeptides may be introduced to form transgenic pigs that produce human hemoglobins with decreased dimerization and an increased half-life (WO Patent 9013645).
  • a construct comprising the human adult alpha globin and epsilon globin gene, the pig beta globin gene control region and the human beta globin gene (the "339 construct, see Figure 1R) may be used.
  • the pig epsilon globin gene may permit correct
  • the pig epsilon globin gene as contained in plasmid pSaf/Pig ⁇ , deposited with the ATCC and assigned accession number 75373, is shown in Figure 13.
  • 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'
  • ⁇ e ⁇ 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'
  • the LCR ⁇ construct which comprises the human embryonic epsilon globin gene, the human adult beta globin gene and the human adult alpha-globin gene linked in tandem from 5'- to 3' (Fig. 1V);
  • the ⁇ p ⁇ construct carrying a mutation that results in a lysine residue (rather than a tyrosine residue) at amino acid number 42 of the ⁇ -globin protein (Fig.
  • transgenic pigs expressing human hemoglobin three types of hemoglobin dimers are detectable: pig ⁇ /pig ⁇ , human ⁇ /human ⁇ , and hybrid human ⁇ /pig ⁇ .
  • three types of hemoglobin dimers are detectable: pig ⁇ /pig ⁇ , human ⁇ /human ⁇ , and hybrid human ⁇ /pig ⁇ .
  • hybrid hemoglobin it may be desirable to decrease the amount of hybrid hemoglobin. Accordingly, the molecular basis for the formation of hybrid hemoglobin has been investigated using molecular modeling studies. Based on the information derived from these studies, the human alpha and beta globin structures can be modified to increase the level of human ⁇ /human ⁇ dimers (See Section 11.), so that in further embodiments of the invention, constructs comprising the ⁇ p ⁇ sequence may be modified to code for ⁇ or ⁇ globin proteins
  • the present invention provides for constructs which encode human ⁇ globin and human ⁇ globin carrying one or more of the
  • the construct carrying such mutation (s) is the ⁇ p ⁇ construct.
  • the present invention provides for constructs which encode human ⁇ globin and human ⁇ globin carrying one or more of the following mutations in the ⁇ globin molecule: (1) a Leu instead of Val at position 33; (ii) a Val or lie instead of Cys at position 112; (iii) a Val or Leu instead of Ala at position at position 115; (iv) a His instead of Gly at position 119; (v) a Met instead of Pro at position 125; (vi) an lie instead of Ala at position 128;
  • the construct carrying the mutation (s) is the ⁇ p ⁇ construct.
  • constructs the untranslated 3' end of the pig beta globin gene as contained in plasmid pPig3' ⁇ ( Figure 16) as deposited with the ATCC and assigned accession number 75372. (see, for example, construct 354 in Figure 12 and Figures 426 and 427 in Figure 14). Such constructs may also be useful in the expression of non-globin protein in pig erythrocytes.
  • the pig beta globin control region depicted in Figures 8 and 9 may be used in constructs that encode non-globin proteins for the expression of said proteins in transgenic pig or other non-human erythrocytes.
  • the recombinant nucleic acid constructs described above may be inserted into any suitable plasmid, bacteriophage, or viral vector for
  • the present invention further provides for isolated and purified nucleic acids comprising the pig adult beta globin promoter regulatory region, the pig 3' beta globin region, and the pig epsilon globin gene as comprised, respectively, in plasmids
  • Constructs may desirably be linearized for preparation of transgenic pigs.
  • Vector sequence may desirably be removed.
  • the recombinant constructs described above may be used to produce a transgenic pig by any method known in the art, including but not limited to, microinjection, embryonic stem (ES) cell manipulation, electroporation, cell gun, transfection, transduction, retroviral infection, etc.
  • Species of constructs may be introduced individually or in groups of two or more types of construct.
  • a transgenic pig may be produced by the methods as set forth in Example Section 6, infra. Briefly, estrus may be synchronized in sexually mature gilts (>7 months of age) by feeding an orally active progestogen (allyl trenbolone, AT: 15 mg/gilt/day) for 12 to 14 days. On the last day of AT feeding 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
  • IM intramuscular injection
  • 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.
  • 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 lina 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.
  • all 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 produced according to this method are described in Example Section 6, infra, and are
  • Such pigs are healthy, do not appear to be anemic, and appear to grow at a rate comparable to that of their non-transgenic littermates. 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 50 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.
  • hemoglobin For purposes of ascertaining whether human hemoglobin is being produced by a particular 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 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), 263 (the ⁇ p ⁇ construct) 339, 293 and 294 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.
  • One- and two-cell ova were placed in an Eppendorf tube (15 ova per tube) containing 1 ml HEPES Medium supplemented with 1.5% BSA and centrifuged for 6 minutes at 14000 x 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
  • Microinjected ova were 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 were transferred within 10 hours of recovery.
  • the exposed portion of the reproductive tract was bathed in a sterile 10% glycerol-0.9% saline solution and returned to the body cavity.
  • prostaglandin F 2a (10 mg/ injection) at 0800 and 1400 hours on day 112 of gestation. In all cases, recipients farrowed within 34 hours
  • Hybrid human hemoglobin (“HB") produced are set forth in Table II, infra. Total hemoglobin was
  • Figure 3 presents the results of isoelectric focussing and triton acid urea gels of hemoglobin produced by three of these pigs (numbers 12-1, 9-3, and 6-3) which demonstrate the expression of human alpha and beta globin in these animals.
  • 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
  • Table IV presents hemoglobin expression data of offspring of pig 38-4 carrying the "185" construct (the " ⁇ p ⁇ ” construct; see Figure 1B).
  • Table V presents hemoglobin expression data of offspring of pig 38-4 carrying the "185" construct (the " ⁇ p ⁇ ” construct; see Figure 1B).
  • isoelectric focussing which demonstrates the levels of hemoglobin expression in representative transgene positive 38-4 offspring.
  • 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
  • 25 lambda of pig blood, 25 lambda of human blood, or a 25 lambda mixture of 12.5 lambda human blood and 12.5 lambda pig blood were treated as follows.
  • the blood was pelleted at a setting of 5 on microfuge for 2 minutes, then washed three times with 100 lambda 0.9 percent NaCl.
  • the cells were lysed with 50 lambda H 2 O, then spun at high speed to confirm lysis.
  • 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.
  • Equal proportions of human and of pig blood were mixed and lysed, and the resulting hemolysate was subjected to DEAE chromatography as described supra.
  • 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.
  • 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
  • FIG. 15 depicts an isoelectric focusing gel analysis of hemoglobin produced by pig 70-3; equal amounts of hemoglobin from transgenic pig 6-3,
  • the amount of human hemoglobin produced by pig 70-3 is greater than the amount produced by pig 6-3. It has been calculated that 38 percent of the total
  • hemoglobin produced by pig 70-3 is human hemoglobin
  • 10 percent of total hemoglobin produced by pig 6-3 is human hemoglobin (see Table II and Section 6.2. supra, for data and calculations). This suggests that the pig beta globin promoter region is more efficient than the human beta globin promoter in transgenic pigs.
  • transgenic pigs was determined by running isoelectric focussing gels and densitometric scanning of the individual bands (FIG. 18). As indicated in Figure 17, both pig 70-3 and pig 80-4 expressed high levels of authentic human hemoglobin. To obtain the copy number of transgenes, genomic DNA (isolated from the tail) was digested with EcoR I and a Southern Blot was performed. The probe used was a 427 bp Ncol/Bam HI fragment of human beta globin gene containing the first exon, first intron and part of the second exon.
  • the pig and hybrid hemoglobin structures were modeled using the following four steps: (1) hydrogen atoms were added to the X-ray model and their positions modified using energy minimization; (2) amino acid residue
  • HINT is software from Virginia Commonwealth
  • TABLE VI represents the differences between the unmodified dimer and the one with the specified replacement.
  • TABLE VII has the same format as TABLE VI with the following two
  • Hb Presbyterian The amino acid substitution generated in Hb Presbyterian results in the comigration of Hb Presbyterian with the hybrid (h ⁇ p ⁇ ) hemoglobin on isoelectric focussing gels. Based on previous results with the purification of human hemoglobin from hybrid and porcine hemoglobins and the more positive nature of the Hb Presbyterian it should be easier to purify this variant hemoglobin on an anion exchange resin.

Abstract

La présente invention concerne l'utilisation de porcs transgéniques pour produire de l'hémoglobine humaine dans lesquels, dans certains modes de réalisation, le promoteur de globine bêta du porc est utilisé pour faciliter l'expression de l'hémoglobine humaine. Les porcs transgéniques de l'invention peuvent être utilisés comme une source efficace et économique d'hémoglobine humaine sans cellule pouvant servir dans les transfusions et autres applications médicales chez l'homme.
PCT/US1993/005629 1992-06-12 1993-06-11 Utilisation de porcs transgeniques pour la production d'hemoglobine humaine WO1993025071A1 (fr)

Priority Applications (5)

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EP93915317A EP0655888A4 (fr) 1992-06-12 1993-06-11 Utilisation de porcs transgeniques pour la production d'hemoglobine humaine.
AU45343/93A AU687743B2 (en) 1992-06-12 1993-06-11 Production of human hemoglobin in transgenic pigs
JP5512779A JPH07507921A (ja) 1992-06-12 1993-06-11 トランスジェニックブタにおけるヒトヘモグロビンの生産
FI945829A FI945829A (fi) 1992-06-12 1994-12-12 Ihmisen hemoglobiinin valmistaminen transgeenisissa sioissa
NO944811A NO944811L (no) 1992-06-12 1994-12-12 Produksjon av humant hemoglobin i transgene griser

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US89764892A 1992-06-12 1992-06-12
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US98789092A 1992-12-08 1992-12-08
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US5922854A (en) * 1991-06-14 1999-07-13 Kumar; Ramesh Purifying Human Hemogloblin from transgenic pig red cells and plasmids containing pig globin nucleic acids
US5821351A (en) * 1994-06-10 1998-10-13 Dnx Biotherapeutics Production of hemoglobin having a delta-like globin
AU698317B2 (en) 1995-02-25 1998-10-29 Cancer Research Technology Limited Transgenic animals as model of psoriasis
US6022738A (en) * 1995-03-03 2000-02-08 Mount Sinai School Of Medicine Of The City University Of New York Vectors for expression of globin genes
US5741894A (en) * 1995-09-22 1998-04-21 Baxter International, Inc. Preparation of pharmaceutical grade hemoglobins by heat treatment in partially oxygenated form
US5861483A (en) 1996-04-03 1999-01-19 Pro-Neuron, Inc. Inhibitor of stem cell proliferation and uses thereof
JP4360915B2 (ja) 2002-01-07 2009-11-11 ユーロスクリーン・ソシエテ・アノニム Ｇタンパク質共役受容体ｇｐｒ４３のリガンドおよびその使用
US20050227251A1 (en) 2003-10-23 2005-10-13 Robert Darnell Method of purifying RNA binding protein-RNA complexes
ES2440953T3 (es) 2005-03-31 2014-01-31 The General Hospital Corporation Modulación de la actividad de HGF/HGFR para tratar un linfedema
EP1943265B1 (fr) 2005-10-01 2012-09-12 Charles Stout Promoteurs de fusion regulables
CN101886075B (zh) * 2010-07-02 2011-12-21 东北农业大学 猪rosa26启动子及其应用
GB201417042D0 (en) * 2014-09-29 2014-11-12 Fkd Therapies Oy Method
EP3746485B1 (fr) 2018-02-02 2023-11-22 Bloodworks Criblage d'anticorps utilisant un(des) antigène(s) transgénique(s)

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AU6505690A (en) * 1989-09-26 1991-04-28 Richard R Behringer Erythroid-specific gene expression system
CA2111348A1 (fr) * 1991-06-14 1992-12-23 John S. Logan Production d'hemoglobine humaine dans des porcs transgeniques

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