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WO1999005255A1 - Methodes d'augmentation ou de reduction des taux de survie d'embryons de preimplantation - Google Patents

Methodes d'augmentation ou de reduction des taux de survie d'embryons de preimplantation Download PDF

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WO1999005255A1
WO1999005255A1 PCT/US1998/015124 US9815124W WO9905255A1 WO 1999005255 A1 WO1999005255 A1 WO 1999005255A1 US 9815124 W US9815124 W US 9815124W WO 9905255 A1 WO9905255 A1 WO 9905255A1
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embryo
protein
modified
cells
development
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Carol M. Warner
Abigail S. Mcelhinny
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Northeastern University China
Northeastern University Boston
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Northeastern University China
Northeastern University Boston
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8775—Murine embryos
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • 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 fast Ped allele is the presence of the Ped gene product, the Qa-2 antigen encoded by the Q7 and Q9 genes, whereas the slow Ped allele is the absence of the Qa-2 antigen.
  • the expressed Qa-2 protein is attached to the embryonic cell surface by a posttranslationally added glycosylphosphatidylinositol (GPI) anchor .
  • GPI glycosylphosphatidylinositol
  • GPI -anchored proteins have recently assumed importance in protein engineering of surfaces of antigen-presenting cells without gene transfer (Tykocinski et al . , PCT International Application WO 96/12009) .
  • GPIs serve as membrane anchors for numerous cell -surface proteins, and isolated GPI-anchored proteins have an unusual capacity to reintegrate with cell-surface membranes.
  • cell surfaces can be coated, or "painted, " with an externally added protein.
  • GPI moiety can be recombinantly engineered into the extracellular domain of a conventionally anchored protein (also see Caras et al . , U.S. Patent No. 5,109,113, 1992) and that recombinant GPI-reanchored chimeric proteins can be produced in microgram-to-milligram quantities. Therefore, it was suggested that cell-surface engineering using GPI-anchored proteins provides a general means for modifying antigen- presenting cell surfaces exogenously.
  • GPI-linked proteins incorporate within minutes into recipient plasma membranes, even into cells that are normally difficult to transfect. Painting can be performed on a small number of cells, with nearly 100% efficiency. Additionally, unlike in gene transfer, the amount of protein incorporated into the cell surface can be easily controlled. Finally, multiple GPI- linked proteins can be painted onto the cell surface simultaneously, and many of the GPI-linked proteins that have been painted onto cells have been shown to retain their function, particularly in ligand binding (Table I below) .
  • FcRIII binds IgG Jurkat cells Nagaraj an for et al . , endocytosis 1995
  • Thy-1 T-cell T-cells Zhang et activation al., 1992 a
  • the Qa-2 protein or other appropriate protein, can be "painted" on the surface of oocytes or preimplantation embryos.
  • the proteins enter the cell membrane because they contain a lipid, preferably a GPI, "tail" (Fig. 1) .
  • Fig. 1 shows a schematic of protein painting of exogenous Qa-2 protein onto recipient cells.
  • the lipid molecules in the GPI tail spontaneously, but stably, incorporate into the outer leaflet of the lipid bilayer of the recipient cell membranes.
  • preimplantation embryonic survival could either be enhanced or reduced. It is advantageous to use protein painting to alter oocytes or embryos because, unlike genetic engineering with DNA, engineering with proteins is transient and should have no long-lasting effects on the offspring. Paint e . g. , Qa-2 protein, onto the surface of Ped slow preimplantation embryos would be a practical alternative to the difficult and tedious procedure of microinjection of the Ped gene(s) involved in embryonic cleavage and survival . Examples are given that show that partially purified Qa-
  • the Ped gene product (the Ped gene product) can be painted onto the surface of T cells and preimplantation embryos in vi tro, and that the exogenous Qa-2 protein can increase embryonic cleavage rate after equilibration on the cell surface enhancing the likelihood of embryonic survival.
  • a GPI-linked "tail" could be attached to an appropriate apoptosis-related gene product and be used either to prevent or increase fragmentation, depending on whether the desired outcome was increased embryo survival or embryo death.
  • gene products that suppress apoptosis such as anti-apoptotic members of the Bcl-2 family.
  • the surface- modified oocyte would then be fertilized and allowed to develop.
  • the surface-modified embryo would be cultured in a growth-promoting environment having conditions that promote the implantation, the rate of development, and the probability of survival of the embryo after its transfer to a recipient mother.
  • a GPI-linked "tail” could be engineered onto a protein that would recognize and bind only to an embryo (as opposed to maternal tissue) , to cause embryonic demise.
  • a composition containing such a construct could be incorporated into a vaginal suppository to provide a new method of contraception.
  • Fig. 1 depicts a schematic of protein painting of exogenous Qa-2 protein onto recipient cells.
  • Fig. 2 depicts a FACScan analysis of various titrations of B6.K2 lymphocyte lysate on B6. Kl (Qa-2 " ) T cells for incorporation of Qa-2 protein;
  • Fig. 3 depicts a FACScan analysis of various culture conditions used to incorporate exogenous Qa-2 protein onto B6.K1 T cells;
  • Fig. 4 depicts bar graphs showing the effect of Qa-2 crosslinking on the proliferative responses of B6.K1 T cells incubated with various concentrations of B6.K2 lymphocyte lysate (Fig. 4A) and, as a control, of B6.K2 lymphocytes incubated with various concentrations B6. Kl lysate (Fig. 4B) ;
  • Fig. 5 depicts a bar graph showing the effect of Qa-2 cross-linking on the proliferative responses of B6.K1 T cells incubated with various concentrations of B6.K2 lymphocyte lysate for 0, 2, or 8 hours or with PHA as a control;
  • Fig. 6 depicts I-PCR analysis of B6.K1 preimplantation embryos painted with exogenous Qa-2 protein containing lysate .
  • GPI-linked Qa-2 antigen can be purified, e.g., from T lymphocytes from C57BL/6 mice or from the murine thymoma cell line, Rl .1 , which overexpresses class I molecules.
  • the purified Qa-2 can then be painted onto Qa-2 negative (Qa-2 " ) T lymphocytes from B6.K1 mice (as test cells) .
  • the painted protein is checked for stability, the GPI -anchor is confirmed, and the functionality of the anchored protein is determined by Qa-2 -mediated crosslinking .
  • the painted Qa-2 is established as being functional, it is painted onto B6.K1 (Qa-2 " ) preimplantation embryos at different stages of development . The embryos are checked for a change in phenotype from Ped slow to Ped fast by quantitating the number of cells per embryo.
  • a similar protocol can be used to paint a Qa-2 homolog onto embryos in another mammalian system.
  • the GPI "tail" is transferred from the mouse protein onto a human or domestic animal equivalent to Qa-2 (e.g., HLA-G would be a suitable human protein) , and the synthetic construct is used for protein painting.
  • This method would be useful therapeutically to achieve pregnancies in women who formerly could not become pregnant because their embryos were developing too slowly.
  • heterologous construct e.g., human HLA-G with the mouse Qa-2 GPI "tail"-- is advantageous compared to using the intact mouse Qa-2 protein, in terms of preventing or minimizing the risk of unwanted immunological reactions of the human maternal immune system against the treated embryo.
  • Cells are cultured in medium comprising: RPMI 1640 + 10% fetal calf serum (FCS) + antibiotics + 5 mM glutamine.
  • Nylon wool- enriched T cells are obtained from C57BL/6 mouse spleens in the same medium.
  • Cells (approximately 1x10 s total) are lysed in lysis buffer (comprising 50 mM Tris-HCl pH 8.0 , 1% Triton X-100, 5 mM iodoacetamide, 1 mM PMSF, 1% aprotinin, and 2 mM diisopropyl fluorophosphate) for 1 hour at 4° C.
  • the lysate is then centrifuged at 10,000 x g for 1 hour.
  • the supernatant, containing Qa-2 is passed over a CNBr-activated Sepharose (Pierce) affinity column coupled to anti-Qa-2 monoclonal antibodies.
  • lysis buffer comprising 50 mM Tris-HCl pH 8.0 , 1% Triton X-100, 5 mM iodoacetamide, 1 mM PMSF, 1% aprotinin, and 2 mM diisopropyl fluorophosphate
  • Anti-Qa-2 monoclonal antibodies are commercially available (e.g., 1-1-2 (mouse IgGa) , Cat. No. 06321D and 1-9-9 (mouse IgG2a) , Cat. No. 06331D, Pharmingen (San Diego, CA) ; 1416.6 (mouse IgG2b) , Cat. No. MCA1012, Serotec (Washington, D.C.)).
  • Qa-2 is eluted with 50 mM glycine-HCl pH 3.0/1% Triton X-100.
  • One-ml fractions are collected and neutralized with 1 M Tris-HCl pH 10.
  • Qa-2 can be subjected to SDS-PAGE and Western blot analysis to determine purity and specificity.
  • B6.K2 Qa-2 + mice or B6.Kl(Qa-2 " ) mice were rubbed over a wire mesh screen and rinsed with phosphate-buffered saline (PBS) to create a cell suspension, as described previously.
  • PBS phosphate-buffered saline
  • the cell suspension was enriched for lymphocytes by Ficoll-Hypaque density gradient centrifugation, and the lymphocyte layer was rinsed twice in PBS.
  • the density was adjusted to about 5xl0 7 cells/ml in lysis buffer (comprised of 50 mM NaCl + 2% NP-40) , and 200 ⁇ l of a mammalian protease-inhibitor cocktail (Sigma) were added.
  • the cells were vortexed vigorously for several seconds, and were placed on ice for one to two hours, with intermittent vortexing to ensure lysis. At this point, the suspension was centrifuged at 1500xg for 10 minutes at 4°C, and the pellet was discarded. The supernatant was centrifuged at 10,000xg for an additional 15 minutes, and was frozen at -20°C until it could be concentrated. For concentration of the lysate with simultaneous detergent removal, 2 ml of the lysate were placed into a Centricon-10 and centrifuged at 5000xg for one hour (Millipore) .
  • the centrifugation step was repeated 5 times, and the retentate was diluted with PBS to a final volume of 2 ml with each spin, in order to ensure complete removal of the detergent.
  • the final concentrated retentate was aliquoted and kept at - 20°C until use, at which point it was diluted in PBS and titered on Qa-2 negative (Qa-2 " ) T-cells by FACScan analysis, as discussed in Example I below.
  • the cells are washed three times in PBS and subjected to FACScan analysis to confirm attachment of the Qa-2 to the recipient cell membrane.
  • Crosslinking studies using a protocol previously established for Qa-2 -mediated T cell activation (Robinson et al . , 1989), are then carried out to establish that the Qa-2 antigen not only has been incorporated into the cell membrane, but also is functional.
  • the optimized conditions determined for painting Qa-2 protein onto T cells are then used as a starting point to determine the optimum conditions for painting Qa-2 onto preimplantation mouse embryos.
  • Example I Painting of B6.K1 T-cells with Qa-2 protein from partially purified B6.K2 lysate
  • Qa-2 protein was first painted onto the surface of T lymphocytes, to determine optimal conditions for painting Qa-2 onto embryos of the Ped slow phenotype, e . g. , B6.K1 mice lacking Qa-2 protein expression.
  • Nylon wool-enriched T-cells were obtained from B6.K1 mice and B6.K2 mice (as the Qa-2 + control) and washed three times in PBS in order to remove residual fetal calf serum (FCS) .
  • the cells were resuspended at a density of 5xl0 6 cells/ml in PBS, and 100 ⁇ l of the cell suspension were added to FACScan tubes (Falcon) for a final concentration of 5xl0 5 cells/tube.
  • Qa-2 was partially purified from a B6.K2 lymphocyte lysate, and the lysate was incubated at various dilutions with 5xl0 5 B6.K1 T-cells suspended in PBS.
  • Fig. 2 is a
  • B6.K1 T-cells suspended in PBS were incubated with: (i) 50 ⁇ l of PBS only (peak 1) , to detect autofluorescence; (ii) 50 ⁇ l of various dilutions of B6.K1 lymphocyte lysate (peak ), to detect non-specific binding; or (iii) 50 ⁇ l of various dilutions of B6.K2 lymphocyte lysate (peak 3), to detect incorporation of exogenous Qa-2 protein.
  • FIG. 2 Panel A shows incubation with undiluted lysate; Panel B shows incubation with 1:2 dilution of lysate; Panel C shows incubation with 1:10 dilution of lysate; Panel D shows incubation with 1:100 dilution of lysate; and Panel E shows incubation with 1:1000 dilution of lysate.
  • T-lymphocytes in a concentration-dependent manner, with optimal Qa-2 incorporation occurring when using B6.K2 lysate dilutions of approximately 1:2, 1:10, and 1:100.
  • EXAMPLE II Optimal conditions for painting Qa-2 protein onto B6. Kl T-cells It has previously been shown that the incorporation of GPI-linked proteins is inhibited by bovine serum albumin (BSA) and by low temperatures (reviewed in Medof , 1996) . Therefore, the optimal conditions for incorporation of exogenous Qa-2 protein onto the surface of B6.K1 T-cells were determined (as a starting point for determining the optimal conditions for painting Qa-2 onto preimplantation embryos) . It was determined that painting of the Qa-2 protein onto the cell surface was inhibited by BSA and by low temperature (as shown in Fig. 3) , two hallmark characteristics of GPI-linked protein incorporation (reviewed in Ilangumanran et al .
  • BSA bovine serum albumin
  • FIG. 3 shows the FACScan analysis of optimal conditions for incorporation of exogenous Qa-2 protein on B6.K1 T-cells.
  • B6.K1 T-cells (5xl0 5 cells/FACScan tube) were incubated for 1 hour with: (i) 50 ⁇ l of a 1:100 dilution, or otherwise noted dilutions, of B6.K2 lymphocyte lysate (peak 2); or (ii) PBS only (peak 1) .
  • Panel A shows the results of a
  • Example III Crosslinking of the Qa-2 protein painted onto B6.K1 cells
  • the exogenously added Qa-2 protein was crosslinked to determine if those cells would undergo T-cell activation.
  • the same conditions were used as for cell isolation, in terms of media, solution concentrations, incubation times, and lysate concentrations (discussed later with reference to Fig. 4) , except that the cells were washed in RPMI 1640 medium after incubation with the partially purified Qa-2 protein.
  • B6.K1 T-cells After incubating the B6.K1 T-cells in either a 1:10 or 1:100 dilution of the partially purified B6.K2 lymphocyte lysate, the cells were then incubated with 1-12-1 anti-Qa-2 monoclonal antibody (mAb) and the exogenous Qa-2 protein cross-linked in the presence of PMA, to determine if the B6.K1 T-cells would undergo activation.
  • mAb monoclonal antibody
  • B6.K2 T-cells alone, or B6.K2 T-cells incubated with a 1:10 or 1:100 dilution of B6.K1 lymphocyte lysate were included as positive controls.
  • Fig. 4 depicts the results of crosslinking the exogenous Qa-2 protein painted onto B6.K1 T-cells. Specifically, B6.K1 T-cells (5xl0 5 cells/well) were incubated with either
  • B6.K2 T-cells were incubated under the same conditions in PBS only, or in a 1:10 or 1:00 dilution of B6.K1 lymphocyte lysate (lacking Qa-2 protein) .
  • the T-cells were then incubated with 50 ⁇ g/ml of 1-12-1 anti-Qa-2 mAb, for 30 minutes at room temperature, and then cultured for 48 hours in second antibody and PMA.
  • One ⁇ Ci of [ 3 H-TdR] was added for the last 6 hours of culture, and the cells were harvested. Results are from one experiment, performed in triplicate, and presented as mean cpm ⁇ SEM.
  • Fig. 4A shows that B6.K1 T-cells incubated with the B6.K2 lysate for 1 hour could not be activated by crosslinking with an Ab and PMA.
  • Example IV Effect of allowing "equilibration" of the Qa-2 protein painted onto B6.K1 cells It has been proposed that other factors, namely, the association of GPI-linked proteins with accessory molecules, is required for the exogenous GPI-linked protein's full signaling function (Morgan et al . , 1993; van de Berg et al . , 1995) . To address this issue, another crosslinking study was performed where the exogenously added Qa-2 protein was allowed to "equilibrate" in the plasma membrane before being crosslinked.
  • B6.K1 T-cells were first incubated for either 0, 2, or 8 hours after incorporation of Qa-2 protein, and then treated with 1-2-1 mAb and crosslinking solution, or with PHA.
  • Fig. 5 shows the effect on T cell activation, via crosslinking of exogenous Qa-2 protein painted onto B6.K1 T- cells, after allowing equilibration of the Qa-2 in the plasma membrane.
  • B6.K1 T-cells were incubated with a 1:10 or 1:100 dilution of B6.K2 lymphocyte lysate for Qa-2 protein painting.
  • the cells were washed and resuspended in RPMI 1640 medium, and placed into the wells of a 96-well microtiter plate (time 0) . After 0, 2, or 8 hours in culture to allow Qa-2 equilibration, the cells were incubated with a 1:100 dilution of PHA, or were incubated with 1-12-1 mAb for 30 minutes and cultured in crosslinking solution, as previously described. After 42 hours in culture, 1 ⁇ Ci of [ 3 H-TdR] was added to each well, and the cells were cultured for an additional 6 hours before harvesting. The results are from one experiment and are presented as mean cpm from duplicate wells ⁇ SEM.
  • Fig. 5 shows that the Qa-2 -painted T-cells were activated by PHA at all the timepoints assayed.
  • a mouse embryo test system is used to develop appropriate conditions for use in the desired end-product species. For example, one may paint Qa-2 onto B6.K1 mouse embryos, ranging from the two-cell to the blastocyst (32- cell) stage of development, and assess their conversion from the Ped slow to the Ped fast phenotype .
  • protein painting maybe performed on oocytes (unfertilized eggs) .
  • concentrations of Qa-2 protein ranging from the attomolar to the millimolar range, may be tested and the optimal concentration chosen that converts the phenotype of the mouse oocytes or embryos from Ped slow to Ped fast .
  • Ped slow embryos were also painted. It was found that Qa2 protein incorporated into the membranes of Ped slow 8-cell embryos, as determined by Immuno-PCR (I- PCR) analysis (Fig. 6, discussed later in Example V) . To determine functionality, the exogenous Qa-2 protein was added to Ped slow embryos and allowed to equilibrate in the plasma membrane for 24 or 48 hours, at which point the number of cells per embryo was quantified. It was found that the addition of exogenous Qa-2 protein to embryos had no effect on their rate of cleavage after 24 hours in culture, compared to control embryos that were not painted with Qa-2 protein
  • CBA/Ca (Qa-2-) or B6.Kl(Qa-2-) 8 -cell embryos were collected into Whitten-Biggers (WB) medium, pooled, and washed three times in WB without BSA. The embryos were then incubated with the partially purified lysate from B6.K1 splenocytes (Qa-2 " control) or B6.K2 (Qa-2 + ) splenocytes, at dilutions ranging from 1:100 to 1:200 (in WB without BSA), for 45 minutes at room temperature to minimize damage from any residual detergent .
  • WB Whitten-Biggers
  • the embryos were then washed three times in WB with BSA, and either cultured in microdrops under oil for 24 hours or 48 hours, or placed into PBSAZ for I-PCR analysis.
  • the number of cells per embryo was quantified after 24 hours or 48 hours, by staining with bisbenzamide . (The embryos were incubated with 5 ⁇ g/ml bisbenzamide in WB medium for 30 minutes at 37°C) .
  • Fig. 6 shows the I-PCR analysis of B6.K1 preimplantation embryos painted with exogenous Qa-2 protein.
  • B6.K1 8 -cell embryos were incubated in a 1:100 dilution of B6.K2 lymphocyte lysate containing Qa-2 protein, or in a 1:100 dilution of B6.K1 lymphocyte lysage (control), in WB medium without BSA, for one hour at room temperature. The embryos were then washed three times in PBSAZ, and the zona pellucidae were removed by a 3-5 minute incubation in Acid Tyrode's solution (pH 2.5) .
  • Lane 6 the lanes show the following: Lane 1: Blank; Lane 2: 5 embryos + B6.K1 lysate; Lane 3: 3 embryos + B6.K1 lysate; Lane 4: 2 embryos + B6.K1 lysate; Lane 5: 3 embryos + B6.K2 lysate; Lanes 6-8: single embryos + B6.K2 lysate; Lane 9: Chimeric protein - DNA complex (positive control); Lane 10: PBSAZ (negative control) .
  • Fig. 6 demonstrates that after a one-hour incubation in a 1:100 dilution of the B6.K2 lymphocyte lysate, Qa-2 protein was detectable on the surface of B6.K1 8-cell embryos (lanes 5-8) . Embryos incubated with a 1:100 dilution of lysate from B6.K1 lymphocytes did not incorporate Qa-2 protein, as expected (lanes 1-4) . The results show that preimplantation embryos, like T-cells, can be painted with GPI-linked Qa-2 protein.
  • substantially purified Qa-2 protein in the method of the invention should increase the development rate at least as much or even more than what was observed by using partially purified Qa-2. It is also possible that Qa-2 could increase preimplantation embryo development more substantially if it is painted onto oocytes or onto embryos at earlier stages of development, such as the 2-cell stage.
  • K2 lysate from B6.K2 splenocytes, used for painting of Qa- 2 protein.
  • Qa-2 antigen is encoded by the Q6, Q7, Q8 and Q9 genes, which contain 8 exons .
  • GPI-anchored Qa-2 has a signal in exon 5 for the GPI "tail.”
  • the Q9 gene has 117 nucleotides in exon 5, and there are 18 sites for known restriction enzymes. It would therefore be possible to cut exon 5 with different restriction enzymes, using routine procedures, to create varying lengths of the Qa-2 GPI anchor signal sequence and engineer different DNA constructs that could produce Qa-2 with tails of variable lengths to test for functionality.
  • PCR products of any length could be generated by using appropriate primers based on the known sequence of the Q9 gene (Cai et al . , 1996) .
  • the constructs After the constructs were generated, they would be cloned and transfected into a mammalian cell line to generate a large amount of the modified Qa-2 protein. Cell lysates from the transfected cells could then be purified by immunoaffinity chromatography and painted onto tester cells. Function would be assessed by crosslinking the Qa-2 antigen on the tester cells with anti-Qa-2 monoclonal antibody. Functional GPI-linked Qa-2 protein will result in cell proliferation, which can be measured by incorporation of a radioactively labeled isotope into DNA.
  • the GPI anchor from the Qa-2 molecule can be added to "Ped- like" molecules, such as HLA-G (i.e., members of the class I major histocompatibility complex family of molecules) , to remedy the first problem with embryonic viability, slow rate of development.
  • HLA-G i.e., members of the class I major histocompatibility complex family of molecules
  • Another use of this technology would be to enhance implantation of the embryo in the uterus.
  • a member of the adhesion family of proteins could be painted on the embryos to enhance "sticking," or implantation, to the uterus lining.
  • suitable proteins could be painted on embryos to prevent rejection of the embryos by the maternal immune system, a phenomenon thought to occur in some cases of spontaneous abortion.
  • Two possible candidate proteins for such a use are HLA-G and DAF, both of which may be immunoprotective molecules.
  • the protein painting procedure as described herein could also be used to selectively target embryos with proteins that enhance cell death, thereby providing a novel contraceptive technique.
  • Some members of the Bcl-2 family enhance cell death via apoptotic mechanisms and a second gene family, the caspase family of proteins, also mediate cell death via apoptotic mechanisms.
  • members of the TNF superfamily may also be candidates to mediate the death of the embryo.
  • Reagents would be designed that specifically target the embryos and not the maternal tissues.
  • One approach is to put the GPI-linked proteins into liposomes or other carriers that could be made to recognize only embryos and not the maternal tissues.
  • a liposome or carrier could be designed to specifically recognize zona pellucida proteins that are found only on embryos and not on maternal tissues, thereby allowing the chosen GPI-linked protein to attach to and kill only the embryos.
  • Bolton et al . "Development of spare human preimplantation embryos in vitro: An analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst," J. In Vitro Fert . Embryo

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  • Immunology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

La présente invention concerne l'utilisation d'une 'peinture de protéines' (l'addition de protéines exogènes modifiées par lipides) pour améliorer la viabilité, le développement et l'implantation d'embryons. On a démontré ici qu'une protéine modifiée par un lipide, par exemple, une protéine Qa-2 exogène modifiée par GPI (glycosylephosphatydilinositol ajoutée de manière post-translationnelle), s'intègre spontanément à la membrane du plasma de lymphocytes T B6.K1 et sur des embryons de préimplantation Ped lent (développement d'embryons de préimplantation lent). On a également démontré que, lorsque suffisamment de temps s'est écoulé après l'incorporation, la protéine Qa-2 exogène augmente le taux de développement d'embryons précoces mis en culture in vitro, le plus probablement par diffusion dans la membrane plasmique jusqu'à ce qu'elle soit 'connectée' à son mécanisme de signalisation fonctionnelle. La peinture protéique d'embryons présente par conséquent la caractéristique potentielle d'être un outil de valeur dans l'amélioration du développement et de l'implantation d'embryons.
PCT/US1998/015124 1997-07-23 1998-07-22 Methodes d'augmentation ou de reduction des taux de survie d'embryons de preimplantation Ceased WO1999005255A1 (fr)

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AU85053/98A AU8505398A (en) 1997-07-23 1998-07-22 Methods for enhancing or reducing preimplantation embryo survival rates

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US5349997P 1997-07-23 1997-07-23
US60/053,499 1997-07-23

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087346A1 (fr) * 2002-04-05 2003-10-23 Kiwi Ingenuity Limited Modification et implantation d'embryon
EP1896052A4 (fr) * 2005-06-17 2010-02-24 Mount Sinai Hospital Corp Procedes et compositions servant a accroitre le potentiel de developpement d'ovocytes et d'embryons preimplantatoires
US8013131B2 (en) 2004-03-22 2011-09-06 Kode Biotech Limited Synthetic membrane anchors
WO2013078312A1 (fr) 2011-11-23 2013-05-30 Mezadata Medical Ip Holding Llp Méthode de fécondation in vitro avec délai du transfert d'embryon et utilisation de cellules mononucléaires du sang périphérique
EP2698636A1 (fr) 2012-08-13 2014-02-19 Fundació Institut d'Investigació Biomèdica de Bellvitge Procédés et réactifs pour la prévention et/ou le traitement d'un rejet de greffe
WO2014068408A2 (fr) 2012-10-23 2014-05-08 Caris Life Sciences Switzerland Holdings, S.A.R.L. Aptamères et leurs utilisations
US9939443B2 (en) 2012-12-19 2018-04-10 Caris Life Sciences Switzerland Holdings Gmbh Compositions and methods for aptamer screening
US10942184B2 (en) 2012-10-23 2021-03-09 Caris Science, Inc. Aptamers and uses thereof
WO2021078680A1 (fr) * 2019-10-25 2021-04-29 Intellexon Gmbh Molécules hla de classe i dans la fécondation in vitro et d'autres implications médicales

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAYLOR C. T., JOHNSON P. M.: "COMPLEMENT-BINDING PROTEINS ARE STRONGLY EXPRESSED BY HUMAN PREIMPLANTATION BLASTOCYSTS AND CUMULUS CELLS AS WELL AS GAMETES.", MOLECULAR HUMAN REPRODUCTION., OXFORD UNIVERSITY PRESS, GB - BE, vol. 02., no. 01., 1 January 1996 (1996-01-01), GB - BE, pages 52 - 59., XP002914917, ISSN: 1360-9947, DOI: 10.1093/molehr/2.1.52 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7819796B2 (en) * 2002-04-05 2010-10-26 Kode Biotech Limited Embryo modification and implantation
WO2003087346A1 (fr) * 2002-04-05 2003-10-23 Kiwi Ingenuity Limited Modification et implantation d'embryon
US8013131B2 (en) 2004-03-22 2011-09-06 Kode Biotech Limited Synthetic membrane anchors
EP1896052A4 (fr) * 2005-06-17 2010-02-24 Mount Sinai Hospital Corp Procedes et compositions servant a accroitre le potentiel de developpement d'ovocytes et d'embryons preimplantatoires
MD4526B1 (ro) * 2011-11-23 2017-11-30 Mezadata Medical Ip Holding Llp Metodă de fertilizare in vitro cu întârzierea transferului embrionar şi utilizarea celulelor mononucleare din sângele periferic
WO2013078312A1 (fr) 2011-11-23 2013-05-30 Mezadata Medical Ip Holding Llp Méthode de fécondation in vitro avec délai du transfert d'embryon et utilisation de cellules mononucléaires du sang périphérique
EP2698636A1 (fr) 2012-08-13 2014-02-19 Fundació Institut d'Investigació Biomèdica de Bellvitge Procédés et réactifs pour la prévention et/ou le traitement d'un rejet de greffe
WO2014068408A2 (fr) 2012-10-23 2014-05-08 Caris Life Sciences Switzerland Holdings, S.A.R.L. Aptamères et leurs utilisations
US9958448B2 (en) 2012-10-23 2018-05-01 Caris Life Sciences Switzerland Holdings Gmbh Aptamers and uses thereof
US10942184B2 (en) 2012-10-23 2021-03-09 Caris Science, Inc. Aptamers and uses thereof
EP4170031A1 (fr) 2012-10-23 2023-04-26 Caris Science, Inc. Aptamères et leurs utilisations
US9939443B2 (en) 2012-12-19 2018-04-10 Caris Life Sciences Switzerland Holdings Gmbh Compositions and methods for aptamer screening
WO2021078680A1 (fr) * 2019-10-25 2021-04-29 Intellexon Gmbh Molécules hla de classe i dans la fécondation in vitro et d'autres implications médicales
CN114846155A (zh) * 2019-10-25 2022-08-02 英特莱克森有限责任公司 体外受精中的hla i类分子和进一步的医学意义
EP4048816A1 (fr) * 2019-10-25 2022-08-31 Intellexon GmbH Molécules hla de classe i dans la fécondation in vitro et d'autres implications médicales

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