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WO2002029000A2 - Procedes de reduction du rejet immunologique d'un foetus obtenu par la technique de transfert de noyau - Google Patents

Procedes de reduction du rejet immunologique d'un foetus obtenu par la technique de transfert de noyau Download PDF

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WO2002029000A2
WO2002029000A2 PCT/US2001/030925 US0130925W WO0229000A2 WO 2002029000 A2 WO2002029000 A2 WO 2002029000A2 US 0130925 W US0130925 W US 0130925W WO 0229000 A2 WO0229000 A2 WO 0229000A2
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embryo
mhc
nuclear
fetus
recipient
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WO2002029000A3 (fr
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Christopher J. Davies
Donald H. Schlafer
Jonathan R. Hill
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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Priority to US10/398,308 priority patent/US20040029825A1/en
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Publication of WO2002029000A3 publication Critical patent/WO2002029000A3/fr
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Priority to US12/468,208 priority patent/US20100016654A1/en
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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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates generally to animal cloning and, more specifically, to methods of minimizing immunological rejection of a nuclear fransfer ("NT") fetus.
  • NT nuclear fransfer
  • early fetal losses may be due to abnormalities of the embryo or its placenta, alterations in maternal uterine environment or feto-maternal interactions (Wilmut et al., 1986).
  • fetal abnormalities are known to be a major cause of pregnancy loss (Wilmut et al., 1986).
  • Fetal abnormalities, predominantly fetal oversize, have been observed as a result of in vitro embryo culture and this syndrome is believed to result from serum containing media (Thompson et al., 1995; Walker et al., 1996; Young et al., 1998).
  • NT fetuses that die during the first trimester are undersized, which probably represents the effects of "starvation” due to inadequate maternal-fetal contact and poor transfer of nutrients (Hill et al. 2000b).
  • the fetuses that die appear not to lose viability because of inherent fetal problems, but due to starvation from an inadequate placental nutrient transfer.
  • placentomes are visible using light microscopy with tenuous attachment of maternal and fetal epithelia and formation of micro villi. Contact with the maternal caruncle areas of the endometrium induces growth of villous processes that undergo hypertrophy and hype ⁇ lasia to form cotyledons (Noden, de Lahunta, 1990) and by Day 42 larger, more complex placentomes develop (King et al., 1979).
  • Placentomes are formed from extensive and complex branching of fetal villi and maternal crypts, serve as specialized areas for supplying nutrition to the developing conceptus. Villous projections assist in maintaining apposition and facilitate subsequent union. Binucleate cells form transient feto-maternal syncytia in the cow, which has been proposed to be central to villous expansion (Wooding, Flint, 1994). Chorioallantoic villous formation at the cotyledons is thought to be the primary site of transport of easily diffusible small molecules such as oxygen, carbon dioxide and also amino acids and glucose, whereas macromolecules are transported in the inte ⁇ lacentomal areas adjacent to uterine gland openings.
  • somatic cell NT For somatic cell NT to become a viable technique, its efficiency must be improved. Although the numbers of cloned calves born worldwide since 1998 has rapidly increased into the hundreds and press reports often detail the latest successful birth, these successes gloss over the huge amount of resources that must be devoted to producing each cloned calf. If the cloning technique can be improved so that pregnancy rates increase and fetal losses decrease to approximate those of in vitro produced embryos and fetuses, noted above, utilization of the technique would immediately increase. This would enable the use of cloning in commercial agriculture, facilitate production of transgenic animals, and dramatically reduce the costs to research institutions in maintaining recipient cows for cloned embryos. The present invention is directed to overcoming the above-noted deficiencies in art and otherwise minimizing the failure of NT pregnancies.
  • a first aspect of the present invention relates to a method of minimizing immunological rejection of a nuclear transfer fetus which includes transferring a nuclear transfer embryo into an embryo recipient under conditions effective for development of a nuclear fransfer fetus with minimal risk of immunological rejection of the fetus due to a maternal anti-fetal MHC class I ("MHC-I”) immune response.
  • MHC-I maternal anti-fetal MHC class I
  • a second aspect of the present invention relates to a method of performing embryo transfer which includes: determining an MHC-I antigen type for a nuclear fransfer embryo and an MHC-I antigen type for embryo recipients and either (i) transferring the nuclear transfer embryo into a first embryo recipient having a compatible MHC-I antigen type under conditions effective for development of a nuclear transfer fetus from the nuclear fransfer embryo, or (ii) transferring the nuclear transfer embryo into a second embryo recipient having an incompatible MHC-I antigen type and (a) regulating MHC-I expression of the nuclear transfer embryo or (b) suppressing an immune response of the embryo recipient, under conditions effective for development of a nuclear transfer fetus from the nuclear fransfer embryo.
  • development of a healthy neonate from the nuclear transfer fetus/embryo is desired.
  • a third aspect of the present invention relates to an MHC-I microarray typing system which includes: a substrate and a plurality of oligonucleotide probes bound to the substrate, each of the plurality of oligonucleotides binding to at least one MHC-I allele, wherein each MHC-I allele binds to different oligonucleotide probes.
  • Trophoblast cells in NT embryos display abnormal expression of MHC-I antigen.
  • the abnormal MHC class I expression results in immunological rejection of these fetuses in a large proportion of NT pregnancies, particularly during the first trimester. This is in sha ⁇ contrast to normal pregnancies, where the rate of early embryonic loss is low and MHC incompatible pregnancies do not have a significantly increased amount of early embryonic loss.
  • the reason for this distinction is that in normal bovine pregnancy, there is no frophoblast MHC-I antigen expression in early pregnancy (Davies et al., 2000). Consequently, MHC-I antigen expression is not a target for immunologically mediated fetal rejection in normal pregnancies.
  • the present invention identifies two approaches for avoiding immunological rejection of MHC-I incompatible NT pregnancies.
  • the first approach involves matching NT donor cells and NT recipients for their MHC-I haplotype expression prior to transfer.
  • MHC-I antigen expression by NT frophoblasts is down-regulated, returning the NT frophoblasts to their normal MHC-I negative state. Both of these approaches minimize rejection of the NT fetus, particularly during the first trimester.
  • FIGS 1A-J illustrate the nucleotide sequence alignment of different
  • Figures 2A-D are photomicrographs comparing normal and cloned embryo development. Photomicrographs were originally photographed at 200X.
  • Figure 2 A the endometrium and attached chorioallantois from a normal bovine pregnancy are shown at 39 days gestation (H+E stain). Note trophoblast cells forming a pseudocolumnar layer of cells and the subjacent endometrium lined by an irregular layer of endometrial epithelial cells. Two endometrial glands and moderately cellular endometrial interstitium are evident in the endometrium.
  • FIG 2B the endometrium of a cow pregnant 35 days with a cloned embryo (fetal membranes are not shown; H+E stain) is shown containing a marked lymphoplasmacytic cellular infiltrate extending from just beneath the endometrial epithelium to deep within the endometrium. This is in marked confrast to the normal cellularity demonstrated in Figure 2 A.
  • Figures 2C-D illustrate sections of normal day 39 chorioallantois and endometrium (2C) and day 35 cloned embryonic placenta and opposing maternal endometrium (2D), respectively. Immunohistochemistry staining was performed with ILA19 antibody for bovine MHC-I antigen.
  • FIG. 2C is a graph illustrating the mild staining of the endometrial epithelial cells and complete absence of staining of trophoblast cells in Figure 2C. Confrast this to the intense class I staining of the trophoblast and endometrial cells in fetal and maternal tissues from a cow carrying a cloned fetus shown in Figure 2D. The trophoblast and endometrial cells show marked upregulation of MHC-I expression.
  • Figure 3 is a graph illustrating the interaggregate cd3 positive cells located in the endometrium of 3 cloned pregnancies (hatched bars) and 7 controls (clear bars). The counts are the number of cd3 positive cells per 0.584 mm 2 field at 1 Ox magnification.
  • the present invention is directed to new approaches for performing nuclear transfer ("NT") embryo fransfer into embryo recipients and, as a result, minimizing the immunological rejection of a developing NT fetus.
  • NT nuclear transfer
  • the incidence of NT fetus rejection when practicing an embodiment of the present invention is less than the historical incidence of NT fetus rejection, which is greater than about 80 percent for bovine during the first trimester (Hill et al., 2000a).
  • the NT embryo is prepared using donor and recipient cells from a non- human mammal, preferably a ruminant such as a cow, sheep, goat, buffalo, water buffalo, llama, alpaca, camel, giraffe, etc., or other mammals such as pig, horse, rabbit, mouse, or rat.
  • a ruminant such as a cow, sheep, goat, buffalo, water buffalo, llama, alpaca, camel, giraffe, etc.
  • other mammals such as pig, horse, rabbit, mouse, or rat.
  • Suitable donor cells i.e., cells useful in the subject invention, may be obtained from any cell or organ of the body. This includes all somatic or germ cells. All cells of normal karyotype, including embryonic, fetal and adult somatic cells, may prove totipotent. Donor cells may be, but do not have to be, in culture.
  • TNT4 embryo-derived ovine cell line
  • OME ovine mammary epithelial cell derived cell line
  • SECL epithelial-like cell line derived from a 9-day old sheep embryo
  • Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression.
  • the reduction of serum levels in the culture medium has been used successfully to induce quiescence in both ovine and bovine cell lines.
  • the cells exit the growth cycle during the Gl phase and arrest in the so- called GO stage.
  • Such cells can remain in this state for several days (possibly longer depending upon the cell) until re-stimulated when they re-enter the growth cycle.
  • Quiescent cells arrested in the GO state are diploid.
  • the GO state is the point in the cell cycle from which cells are able to differentiate.
  • the recipient cell to which the nucleus from the donor cell is transferred may be an oocyte or another suitable cell.
  • Recipient cells at a variety of different stages of development can be used, from oocytes at metaphase I through metaphase II to zygotes and two-cell embryos. Methods for isolation of oocytes are well known in the art. Essentially, this includes isolating oocytes from the ovaries or reproductive tract of a mammal. A readily available source of bovine oocytes is slaughterhouse materials.
  • oocytes should be matured in vitro before these cells may be used as recipient cells for nuclear transfer. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries and maturing the oocytes in a maturation medium prior to enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post- aspiration (the "maturation period").
  • metaphase II stage oocytes which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes are collected surgically from either non-superovulated or superovulated cows or heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.
  • hCG human chorionic gonadotropin
  • the stage of maturation of the oocyte at enucleation and nuclear transfer has been reported to be significant to the success of NT methods (Prather et al. 1991).
  • successful mammalian embryo cloning practices use the metaphase II stage oocyte as the recipient oocyte, because at this stage it is believed that the oocyte can be or is sufficiently "activated" to treat the introduced nucleus as it does a fertilizing sperm.
  • the oocyte activation period generally ranges from about 10 to about 52 hours, preferably about 16 to about 42 hours post-aspiration.
  • Enucleation can be effected by known methods, such as described in U.S. Patent No. 4,994,384 to Prather et al. For example, enucleation may be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify those of which have been successfully enucleated. This screening may be effected by staining the oocytes with 1 ⁇ g/ml 33342 Hoechst dye in HECM, and then viewing the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, e.g., CRlaa plus 10% serum.
  • a suitable culture medium e.g., CRlaa plus 10% serum.
  • Suitable procedures for nuclear transfer include donor/recipient cell fusion (i.e., via PEG treatment, inactivated Sendai virus, or elecfrofusion) and microinjection.
  • the donor cell is first transferred into the perivitelline space of the enucleated oocyte. Thereafter, the cells can be fused by providing a pulse of electricity that is sufficient to cause a transient breakdown and subsequent reformation of the plasma membrane. If upon reformation the lipid bilayers intermingle, small channels will open between the two cells and, due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one (U.S. Patent No. 4,997,384 to Prather et al.).
  • a variety of elecfrofusion media can be used including e.g., sucrose, mannitol, sorbitol and phosphate buffered solution. Alternatively, fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham 1969).
  • the donor nuclei is simply removed from the donor cell and injected into the recipient cell (Collas & Barnes 1994).
  • parthenogenetic activation is typically required, at least if the cell is an oocyte, to stimulate the recipient cell into development.
  • Parthenogenic activation is typically achieved using electrical stimulation of the diploidized oocyte, which is believed to allow for increases in intracellular calcium concentration.
  • the interval between pulses for rabbit oocytes is approximately 4 minutes (Ozil 1990), and in the mouse 10 to 20 minutes (Cuthbertson & Cobbold 1985), while observations in the cow suggest that the interval is approximately 20 to 30 minutes (Robl et al. 1992).
  • activation can be effected by briefly exposing the fused NT embryo to a TL-HEPES medium containing 5 ⁇ M ionomycin and 1 mg/ml BSA, followed by washing in TL-HEPES containing 30 mg/ml BSA within about 24 hours after fusion, and preferably about 4 to 9 hours after fusion.
  • the reconstituted NT embryo may then give rise to one or more mammals, whether transgenic or non-transgenic.
  • the NT embryo will be cultured to a size of at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least about 50 cells.
  • Development to blastocyst stage can be carried out in vitro or in vivo (i.e., using a temporary pre-blastocyst recipient).
  • NT embryo and optionally developing the NT embryo to the blastocyst stage
  • the embryo recipient is preferably from the same species as the donor and recipient cells used to prepare the NT embryo, although dams from related species can, at least in some instances, be utilized to support gestation of the NT fetus. Synchronous transfers are desirable for success of the transfer, i.e., the stage of the NT embryo is in synchrony with the estrus cycle of the recipient female (Siedel 1981).
  • the method for minimizing immunological rejection of a NT fetus involves transferring, into an embryo recipient, an NT embryo having an MHC-I antigen type which is compatible with an MHC-I antigen type of the embryo recipient.
  • the MHC-I antigen type of the NT embryo and the embryo recipient Prior to transferring the NT embryo, the MHC-I antigen type of the NT embryo and the embryo recipient are determined. Matching of the NT embryo and the embryo recipient (into which transfer will subsequently occur) is based on the determined MHC-I antigen haplotypes thereof.
  • the determination of MHC-I antigen haplotype can be performed separately on individual NT embryos or it can be performed on a number of NT embryos in a single screening event.
  • MHC-I antigen haplotype for the embryo recipients.
  • a number of approaches can be utilized to perform the haplotyping, either alone or in combination. These include, without limitation, serological typing (Lewin 1996; Davies & Antczak 1991; Davies et al. 1994a); one dimensional-isoelectric focusing (Joosten et al. 1988; Davies et al. 1994a; Lewin 1996); DNA sequencing (Garber et al. 1993; Pichowski et al. 1996; Ellis et al. 1999); polymerase chain reaction amplification using allele specific primers (Ellis et al.
  • FIG. 1 A-J illustrate a nucleotide sequence alignment for a number of known MHC- I alleles. Probes can be selected based on the polymo ⁇ hism which exists among the various MHC-I alleles. Haplotype assignments for the NT embryo and the embryo recipient can be based on one or more of these methods.
  • an MHC-I microarray typing system can be used.
  • This typing system includes a substrate and a plurality of oligonucleotide probes bound to the substrate, each of the plurality of oligonucleotides binding to at least one MHC-I allele, wherein each MHC-I allele binds to different oligonucleotide probes (i.e., a different subset of oligonucleotide probes).
  • the method for minimizing immunological rejection of an NT fetus involves transferring, into an embryo recipient, an NT embryo having an MHC-I antigen type which is incompatible with an MHC-I antigen type of the embryo recipient.
  • a first approach to minimize immunological rejection in this situation involves down-regulation of MHC-I expression in the placenta of the NT embryo or fetus.
  • Down-regulation of MHC-I expression by placental trophoblast cells is preferred, although down-regulation of MHC-I expression by other placental cells is also beneficial.
  • Down-regulation of MHC-I expression (in placental cells of NT embryos) can be achieved by (i) modulating expression of an MHC-I transcription factor in the NT embryo or fetus; (ii) treating the NT embryo or fetus with a cytokine, a growth factor, or combinations thereof which is suitable to inhibit MHC-I expression; or (iii) both (i) and (ii).
  • MHC-I genes in bovine trophoblast cells may involve many of the same positive regulatory elements as human MHC-I genes (Harms & Splitter 1994; Harms et al. 1995; Barker et al. 1997).
  • down regulation of expression of "classical" MHC-I antigens on trophoblast cells involves both the absence of key transcription factors (CIITA and NF- ⁇ B/Rel family members p50 and p65) and the presence of specific negative regulatory factors (Gobin & van den Elsen 1999, 2000; Chiang & Main 1994; Coady et al. 1999; Peyman 1999).
  • TSU RNA suppressor element
  • the treatment can be carried out prior to transfer (i.e., in vitro), after fransfer (i.e., in utero), or both.
  • a suitable cytokine or growth factor is introduced into the growth medium in which the NT embryo resides following nuclear transfer, such as the above-described medium utilized for activation.
  • a suitable cytokine or growth factor can be administered via intrauterine delivery or intravenous injection.
  • Suitable cytokines that can be employed to down-regulate MHC-I expression levels include, without limitation, several interleukins such as IL-4, IL-10 and IL-13, leukemia inhibitory factor ("LIF”) and transforming growth factor- ⁇ ("TGF- ⁇ ”), which has both cytokine and growth factor activities, or combinations thereof (Mitchell et al. 1993 ; Robertson et al. 1994; Moreau et al. 1999). While IL- 10 can directly down-regulate MHC-I expression (see Moreau et al. 1999), it is believed that the other cytokines act indirectly by inhibiting the production of inflammatory cytokines (particularly INF-gamma) that induce MHC-I expression.
  • interleukins such as IL-4, IL-10 and IL-13, leukemia inhibitory factor (“LIF”) and transforming growth factor- ⁇ (“TGF- ⁇ ”), which has both cytokine and growth factor activities, or combinations thereof (Mitchell et al. 1993
  • Suitable growth factors that can also be employed to down-regulate MHC-I expression levels include, without limitation, insulin, epidermal growth factor (“EGF”), granulocyte/macrophage colony-stimulating factor (“GM-CSF”), TGF- ⁇ , insulin-like growth factor(s) (“IGFs”), interleukin-3 (“IL-3”), or combinations thereof (Mitchell el al. 1993; Robertson et al. 1994).
  • a second approach to minimize immunological rejection in this situation involves suppressing an immune response of the embryo recipient.
  • Suppression of the embryo recipient's immune response to the MHC-I incompatible embryo or fetus is effected by administering an amount of an immunosuppresive drug to the embryo recipient under conditions effective to suppress the anti-MHC-I immune response.
  • Suitable immunosuppressive drugs include, without limitation, cyclosporin A, tacrolimus, and sirolimus. These exemplary immunosuppressive drugs are believed to cause immunosuppression by blocking signaling pathways in lymphocytes, thereby blocking immunological rejection.
  • These immunosuppressive drugs can be administered systemically (i.e., intravenous) to the embryo recipient.
  • Antigen positive cells in the placentomal and inte ⁇ lacentomal endometrium are enumerated by digital image processing with NIH Image software (Gr ⁇ nig et al. 1995).
  • Cytokine immunohistochemistry can be used to compare cytokine production between groups and to identify cytokine producing cells at the uterine/placental interface. For each pregnancy, sections from at least two placentomal and two inte ⁇ lacentomal blocks would be assessed. Staining can be done using the three- stage avidin-biotin system described above. Antibodies against the following cytokines can be used: IL-2 (mAb 14.1, VMRD), IL-4 (mAb CC303, Serotec; Weynants et al. 1998), IL-10 (goat anti-human IL-10, R & D Systems; Brown et al.
  • IL-12 mAb CC301, Serotec
  • IFN- ⁇ mAb CC302, Serotec
  • TNF- ⁇ mAb 2C4-1D3 and polyclonal rabbit anti-bovine TNF- ⁇ , generously provided by Dr. Ted Elsasser; Palmer et al. 1998; Kenison et al. 1990; Sileghem et al. 1992
  • TGF ⁇ l and TGF ⁇ 2 rabbit anti-human TGF ⁇ l and TGF ⁇ 2 from R & D Systems; Munson et al. 1996)
  • GM-CSF mAb CC305, Serotec
  • cytokine positive cells can be based on cell location and mo ⁇ hologic features.
  • the leukocyte differentiation antigen immunohistochemistry described above would be invaluable in the inte ⁇ retation of the cytokine immunohistochemistry.
  • the number of positive cells and the intensity of staining would be assessed using digital image analysis with NIH Image software (Gr ⁇ nig et al. 1995).
  • Example 1 Microarray MHC-I Typing
  • a bovine MHC-I microarray typing system was prepared by providing
  • MHC-I typing array is based on 118 known cDNA or genomic sequences from the BoLA Nomenclature Web Site and GenBank. As shown in Tables 1-4 below, two series of exon 2 probes and two series of exon 3 probes are provided.
  • the exon 2 probes include 25 series A probes for codons 61-68 and 30 series B probes for codons 71-78 (see also Figures 1A-J).
  • the exon 3 probes include 27 series A probes for codons 111-118 and 31 series B probes for codons 151-158 (see also Figures 1 A-J). Together, these probes (and the corresponding polymo ⁇ hisms) define an undetermined number of MHC-I haplotypes.
  • BoLA-ClEx2B27 CAGAGATTGCGAACGGGC 18 53 61
  • a hemi-nested PCR protocol was used to amply exons 2 and 3 together from genomic DNA (primers BoClFP-E2A/E2B and BoClRP-E3C) followed by amplification of each exon independently.
  • primer sequences are as follows:
  • Class I exon 2 mixture of BoClFP-E2A/E2B (SEQ ID Nos: 28, 29) acgtggacga cacg (c/g) agttc 20
  • BoClFP-E3D (SEQ ID No: 31) tggtcggggc gggtcagggt ctcac 25
  • Cryopreserved aliquots of cell suspensions from a Nellore fetus removed by hysterotomy at Day 45 of gestation were used to provide donor cells.
  • the donor cells were derived from cells frozen at passage 2 (Day 10 of culture), then thawed and cultured in 4 well Nunc plates containing Dulbecco's Modified Eagles medium (DMEM-F12) + 10% v:v fetal bovine serum (FBS) + 1% v:v penicillin/streptomycin at 37°C in air containing 5% CO 2 . At 50% confluence they were serum starved (0.5% FBS) for 5 days prior to NT.
  • DMEM-F12 Dulbecco's Modified Eagles medium
  • FBS v:v fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • Recipient oocytes were slaughterhouse derived and matured for 17 hours in Medium 199 (M 199; Gibco Laboratories Inc.; Grand Island, NY) supplemented with 10% v:v fetal calf serum (FCS; Gibco), FSH 0.1 units/ml (Sioux Biochem; Sioux City, IA), LH 0.1 units/ml (Sioux Biochem), estradiol 1 ⁇ g/ml (Sigma; St Louis, MO), 0.1 mM Cysteamine (Sigma M 9768), and 1% penicillin- streptomycin.
  • FCS v:v fetal calf serum
  • FSH FSH 0.1 units/ml
  • Sioux Biochem Sioux City, IA
  • LH 0.1 units/ml estradiol 1 ⁇ g/ml
  • Sigma St Louis, MO
  • 0.1 mM Cysteamine Sigma M 9768
  • penicillin- streptomycin The cumulus-oocyte complexes
  • Oocytes were enucleated beginning at 19 h post maturation. Prior to enucleation, oocytes were placed for 15 min in Hepes-buffered Ml 99 containing Hanks salts (H-M199; Gibco) with 4 mg/ml fatty acid free BSA (Sigma) plus 7.5 ⁇ g/ml cytochalasin B (Sigma) and 5 ⁇ g/ml Hoechst 33342 (Sigma). Oocytes were selected for the presence of a polar body and homogeneous cytoplasm. Suitable oocytes were enucleated in H-M199 with 7.5 ⁇ g/ml cytochalasin B using a beveled 25 ⁇ m outside diameter glass pipette.
  • Fusion parameters were 1x40 ⁇ sec 2.25 kV/c DC fusion pulses delivered by a BTX Elecfrocell Manipulator 830 (BTX; San Diego, CA). Oocyte-fibroblast fusion was assessed 20 - 30 minutes later by light microscopy and unfused couplets were refused. Oocyte activation were performed 3-5 h after fusion at 27 h post maturation, by a 4 min incubation in Hepes buffered Ml 99 + 5 ⁇ M ionomycin (Calbiochem; San Diego, CA), then 4 minutes in 30 mg/ml H199 + BSA followed by washing in 4 mg/ml BSA in H-M199.
  • the fused oocytes were transferred into 2 mM DMAP in Ml 99 + 3 mg/ml BSA for 4 h followed by transfer to the embryo culture medium for 7 days.
  • Embryos were cultured in 50 :1 drops of a derivative of synthetic oviductal fluid serum-free medium (BARC-1; Wells and Powell, 2000) under mineral oil (Sage Biopharma, Bedminster, NJ) in a 5% CO 2 ,
  • a fibroblast cell line was derived from an in vivo produced Day 45 Nellore fetus. To produce the fetus, three embryos recovered non-surgically from a donor cow were transferred the same day into three recipient cows, all of which were pregnant at Day 45. The Nellore cell line was selected with a goal of amplifying any differences that may arise between tissue types of the donor tissue (Bos indicus) and recipient cows (Bos taurus - Angus). Fetal fibroblasts were derived from passage 2 cells (10-15 days in culture) and serum starved for 5 days prior to NT. NT was performed as previously described (Hill et al. 2000a) except that embryos were cultured for 7 days in a defined serum- free medium (BARC-1; Wells & Powell 2000).
  • Day 7 embryos were shipped in a temperature-controlled 39°C incubator to a commercial embryo fransfer center (Trans Ova, Iowa) for transfer into synchronous recipient cows.
  • the per embryo survival rate to Day 35 was 23% when transferred in pairs and the recipient cow pregnancy rate was 50%.
  • Six cloned fetuses were recovered from 5 recipient cows between Day 35-50 of gestation.
  • Tissue samples were collected within 30 minutes of slaughter. If feasible, separate placentomal and inte ⁇ lacentomal samples were collected. However, in the Day 35 placentas, distinction between cotyledonary and intercotyledonary areas by visual inspection is difficult. Tissues were be fixed in 4% paraformaldehyde for histology and for immunohistochemistry by freezing in OCT freezing compound. Fetal heart, liver, lung, kidney, gut, and flank muscle were also processed for histology. For immunohistochemistry, 2 x 2.5 cm rectangular sections of apposed placenta and uterus would be excised, anchored in plastic boats with OCT, and immediately frozen in isopentane chilled in liquid nitrogen.
  • Frozen tissues were held on dry ice and then transferred to a -80°C freezer for storage. For sectioning, blocks were warmed to -30°C and cryostat sectioned at 8 ⁇ m. Sections were transferred to slides, dried at room temperature for 30 minutes, fixed in cold acetone for 15 minutes, air dried for 30 minutes, and returned to the freezer for storage. If "normal" placentomes, with villus crypt interdigitation, and "failing" placentomes, where attachment is not occurring, were present, at least two tissue blocks containing each type of placentome were collected.
  • Tissues were collected and processed on site as described above. Pregnant tracts were initially selected for gestational age by palpation of amniotic vesicle. After opening the uterus, the crown rump length was measured and the fetal age determined using a formula developed for purebred Holsteins by Rexroad et al. (1974). MHC-I immunocytochemistry was performed on frozen sections from
  • cryostat sections were blocked with normal goat serum and incubated with a 1 :6000 dilution of IL-A19 anti-bovine MHC-I mAb (Bensaid et al. 1989; generously provided by Jan Naessens, ILRI, Kenya) or control antibody for two hours at 37°C. Detection of antigen/antibody complexes were achieved using a three stage avidin-biotin system and the AEC chromogen.
  • 5a and 5b were twins.
  • Each of the 3 positive placentas was at 35 days of gestation while the 3 negative placentas were at 40 or 50 days. Based on these results, fetuses that do not express MHC-I are able to develop more normal placentation and have a higher probability of reaching the 2 nd trimester of pregnancy.
  • Non- viable fetuses were present in the cloned group. Two of 6 cloned fetuses were non- viable (as determined by lack of heartbeat on ultrasonographic scan on the previous day and confirmed by presence of amniotic hemorrhage at slaughter). One of these non- viable fetuses was MHC-I positive (Day 35 single) whereas the other was negative (a Day 50 twin).
  • lymphocyte cd3 positive
  • aggregates in the stratum compactum of the intercotyledonary areas of endometrium. Interspersed between these aggregates were increased numbers of lymphocytes and plasma cells mainly distributed immediately beneath the epithelium and adjacent to the endometrial glands. Aggregates were defined as areas of cd3 positive cells where more than 20 cells were in contact with each other. Objective counts of numbers of aggregates and interaggregate cd3 positive lymphocytes were determined by visual estimation using a 0.292mm 2 reticle to delineate linear boundaries per field. Mean counts per field were totaled per section, and means per case were calculated. A minimum of 5 fields per section, 4 sections from inte ⁇ lacentomal tissues, per case, was scored.
  • the cd3 positive aggregates were rare in the seven controls (4/158; 0.03% of fields), but found in over half the fields examined in the three clones (39/62; 62.9% of fields, p ⁇ 0.001, Chi-square test). The mean number of aggregates per field was thus significantly higher in clones than controls (0.639 V 0.09 vs 0.025 V 0.012; p ⁇ 0.001, Mann- Whitney rank sum test). These aggregates contained hundreds of cd3 positive lymphocytes in cross section. As illustrated in Figure 3, cd3 positive lymphocytes located away from these aggregates (interaggregate cd3 positive cells) were also found to be significantly higher in the cloned pregnancies (p ⁇ 0.001).
  • the dead clone had the highest number of cd3 positive aggregates (0.8 aggregates per field) and interaggregate cd3 cells (133 V 38 cells per field; bar 1 in Figure 3) whereas the dead confrol fetus had no aggregates and a nonnal number of interaggregate cells (28 V 7 cells per field; bar 10 in Figure 3).
  • the crown rump length for the dead clone was less than half that expected for a Day 35 fetus (0.7 cm vs expected of 1.9 cm). This indicated either failure of fetal development or a hostile uterine environment. While lymphocytic infiltration in the uterus of the non- viable fetus may logically be explained by release of fetal antigens to the endometrium, no signs of inflammation were present in endometrium of the other non- viable fetus - the Day 50 MHC-I negative clone. Thus, trophoblast MHC-I expression correlated with endometrial lymphocytic accumulations. This small group of clones provides compelling evidence that a substantial proportion of the high early embryonic mortality observed in cloned pregnancies is due to inappropriate trophoblast MHC-I expression and immunologically mediated placental rejection.
  • Alexander BM Johnson MS, Guardia RO, Graaf WLvd, Senger PL, Sasser RG, 1995. Embryonic loss from 30 to 60 days post breeding and the effect of palpation per rectum on pregnancy. Theriogenology 43:551-556. Allen WR, 1982. Immunological aspects of the endometrial cup reaction and the effect of xenogeneic pregnancy in horses and donkeys. J. Reprod. Fertil. Suppl. 31:57-94:57-94.
  • Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner. Infection and Immunity 62:4697-4708.
  • Cibelli JB Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM, 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258.
  • Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca 2+ . Nature 316:541-542.
  • Graham CF 1969. The fusion of cells with one and two cell mouse embryos. Wister Inot. Symp. Monogr., 9:19.

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Abstract

La présente invention concerne un procédé permettant de minimiser le rejet immunologique d'un foetus obtenu par la technique de transfert de noyau. Ce procédé consiste à transférer un embryon obtenu par transfert de noyaux dans un récepteur d'embryon dans des conditions permettant le développement de cet embryon avec un risque minime de rejet immunologique du foetus en raison de la réponse immunitaire MHC-I anti-foetale maternelle. Après avoir déterminé un type d'antigène MHC-I pour les récepteurs d'embryons, cet embryon est (i) transféré dans un premier récepteur d'embryon présentant un type d'antigène MHC-I compatible dans des conditions permettant le développement du foetus à partir de l'embryon de transfert de noyau, ou (ii) transféré dans un second récepteur d'embryon présentant un type d'antigène MHC-I incompatible. Ce procédé consiste ensuite à réguler l'expression du MHC-I de l'embryon de transfert ou à supprimer une réponse immunitaire du récepteur d'embryon dans des conditions permettant de développer le foetus de transfert.
PCT/US2001/030925 2000-10-03 2001-10-03 Procedes de reduction du rejet immunologique d'un foetus obtenu par la technique de transfert de noyau Ceased WO2002029000A2 (fr)

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US12/468,208 US20100016654A1 (en) 2000-10-03 2009-05-19 Methods of Minimizing Immunological Rejection of A Nuclear Transfer Fetus

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PINTO-CORREIA ET AL.: 'Factors involved in nuclear reprogramming during early development in the rabbit' MOLECULAR REPRODUCTION AND DEVELOPMENT vol. 40, 1995, pages 292 - 304, XP002956444 *
TROUNSON A.: 'Nuclear transfer in human medicine and animal breeding' REPROD. FERTIL. DEV. vol. 13, 2001, pages 31 - 39, XP002956443 *

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