Compositions and methods for culturing cells

Abstract

The compositions and methods described herein include culturing an oocyte, an ooplast, or an embryo (e.g., an early-stage embryo) in a culture media with a GPCR ligand. Such oocytes can be used for somatic cell nuclear transfer (SCNT) and subsequent cloning and implantation into a recipient female.

Description

[0001] ATTORNEY DOCKET NO. 51488-002WO2

[0002] PATENT

[0003] COMPOSITIONS AND METHODS FOR CULTURING CELLS

[0004] Background of the Invention

[0005] Animal cloning, such as by somatic cell nuclear transfer (SCNT), has broad agricultural, medical, and societal applications. One of the significances of animal cloning in each of these applications is that it also provides the ability to genetically engineer the animal genome, which could be used to improve disease resistance and performance of livestock, to develop large animals as bioreactors to produce human therapeutics, and to resurrect extinct species.

[0006] Currently, however, animal cloning is extremely inefficient, making it economically unfeasible for large scale production of animals with this method. To develop genetically engineered large animals as bioreactors it also requires the development of novel techniques to precisely engineer the animal genome to achieve maximal levels of transgene expression and the desired posttranslational modifications of the produced therapeutic proteins. Accordingly, improved methods of engineered animal production are needed.

[0007] Summary of the Invention

[0008] In one aspect, featured is a method for producing a mature oocyte from an immature oocyte. The method includes contacting one or more immature oocyte(s) with a G-protein coupled receptor (GPCR) ligand.

[0009] In another aspect, featured is a method for maintaining an oocyte. The method includes contacting one or more immature oocyte with a GPCR ligand.

[0010] In some embodiments, contacting the immature oocyte with the ligand produces intracellular cyclic AMP (cAMP).

[0011] In some embodiments, intracellular cyclic AMP (cAMP) is increased due to direct activation of adenylate cyclase. In some embodiments, intracellular cAMP is increased by inhibition of cAMP- phosphodiesterase (PDE).

[0012] In some embodiments, the intracellular environment of the immature oocyte is hypoxic and cAMP rich.

[0013] In some embodiments, the intracellular environment has an oxygen concentration of 0-4% (e.g., 0%, 0.5%, 1 %, 1 .5%, 2%, 2.5%, 3%, 3.5%, or 4%) oxygen.

[0014] In some embodiments, the intracellular environment of the immature oocyte has a cAMP concentration of at least 1 pM (e.g., at least 1 pM, 2 pM, 3 pM, 5 pM, or more).

[0015] In some embodiments, the method includes incubating the one or more immature oocytes under conditions which are substantially free of oxygen.

[0016] In some embodiments, the method further includes incubating one or more immature oocytes with an inhibitor of prolyl hydroxylase (PHD).

[0017] In some embodiments, the PHD is a HIF-prolyl hydroxylase.

[0018] In some embodiments, the inhibitor of PHD is CoCI2, DFO (deferoxamine), or DMOG (dimethyloxalylglycine), Roxadustat (FG-4592), Vadadustat (AKB-6548), Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934). ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0019] In another aspect, featured is method of animal cloning. The method includes contacting an oocyte with a culture medium comprising a GPCR ligand.

[0020] In some embodiments, the method further includes fertilizing the oocyte.

[0021] In some embodiments, the fertilizing step includes in vitro fertilization.

[0022] In some embodiments, the in vitro fertilization includes somatic cell nuclear transfer (SCNT).

[0023] In another aspect, featured is a method for making an SCNT embryo. The method includes contacting a somatic cell with a GPCR ligand and transferring the nucleus of the somatic cell to an enucleated oocyte, thereby making the SCNT embryo.

[0024] In another aspect, featured is a method for increasing the efficiency of SCNT. The method includes contacting a donor mammalian cell, recipient mammalian oocyte, or a mammalian SCNT embryo with a GPCR ligand, thereby increasing the efficiency of the SCNT.

[0025] In another aspect, featured is method of producing an embryo. The method includes the steps of:

[0026] (a) injecting or fusing one or more nuclei into an ooplast; and (b) culturing the ooplast in the presence of a GPCR ligand to produce a viable embryo.

[0027] In some embodiments, the method further includes transferring the embryo to an oviduct of a female to produce a cloned animal.

[0028] In another aspect, featured is a method for culturing an embryo in vitro. The method includes (a) collecting one or more early-stage embryo(s) of less than 3 cells; (b) placing the one or more embryos in a culture system; (c) contacting the one or more embryos with a GPCR ligand; and (d) culturing the embryos until blastocyst stage (e.g., culturing the embryo(s) until they have at most 100 cells). In some embodiments, the method includes culturing the embryo(s) until they have at most 100 cells, 95 cells, 90 cells, 85 cells, 80 cells, 75 cells, 70 cells, 65 cells, 60 cells, 55 cells, 50 cells, 45 cells, 40 cells, 35 cells, 30 cells, 25 cells, 20 cells, 15 cells, 10 cells, 9 cells, 8 cells, 7 cells, 6 cells, 5 cells, 4 cells, 3 cells, 2 cells, or 1 cell.

[0029] In another aspect, featured is a method of culturing an embryo for implantation. The method includes culturing the embryo in one or more culture media (or medium) comprising a GPCR ligand.

[0030] In some embodiments, the method further includes cryopreserving the embryo.

[0031] In some embodiments of any of the above aspects, the G-protein coupled receptor is linked to stimulatory G (Gs) proteins (GsPCR) or to inhibitory G (Gi) proteins (GiPCR).

[0032] In some embodiments of any of the above aspects, the ligand is an agonist or an antagonist.

[0033] In some embodiments, the GsPCR is an adenosine receptor.

[0034] In some embodiments of any of the above aspects, the adenosine receptor is an adenosine 1 (A1 ) receptor, an adenosine 2A (A2A) receptor, an adenosine 2B (A2B) receptor, or an adenosine 3 (A3) receptor.

[0035] In some embodiments, the adenosine receptor is an A2A or A2B receptor.

[0036] In some embodiments, the GsPCR is a prostaglandin receptor.

[0037] In some embodiments, the prostaglandin receptor is a prostaglandin E2 receptor.

[0038] In some embodiments, the prostaglandin E2 receptor is a prostaglandin E receptor EP2 or a prostaglandin E receptor EP4. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0039] In some embodiments, the GiPCR is an adenosine receptor.

[0040] In some embodiments, the adenosine receptor is an A1 receptor, an A2A receptor, an A2B receptor, or an A3 receptor.

[0041] In some embodiments, the adenosine receptor is an A1 receptor or an A3 receptor.

[0042] In some embodiments of any of the above aspects, the ligand is an adenosine receptor agonist or antagonist.

[0043] In some embodiments, the ligand is an A1 , A2A, A2B, or A3 agonist or antagonist.

[0044] In some embodiments, the ligand is an A2B-A3 dual agonist or antagonist.

[0045] In some embodiments of any of the above aspects, the ligand is an agonist or antagonist of a prostaglandin receptor.

[0046] In some embodiments, the antagonist is an EP2 antagonist. In some embodiments, the EP2 antagonist is PF-04418948.

[0047] In some embodiments, the antagonist is an EP4 receptor antagonist. In some embodiments, the EP4 receptor antagonist is AH6809 or AH23848.

[0048] In some embodiments, the agonist is an EP2 receptor agonist.

[0049] In some embodiments, the agonist is an EP4 receptor agonist. In some embodiments, the EP4 receptor agonist is 11 -Deoxy-PGE1 , gamma-Lactam PGE analog 2a, or gamma-Lactam analog 4. In some embodiments, the EP4 receptor agonist is AGN205203 or TCS 2510 gamma-Lactam PGE analog 3.

[0050] In another aspect, featured is a culture medium obtained from media used in the method of any of the above embodiments.

[0051] In another aspect, featured is a culture medium for culturing an oocyte. The culture medium includes a GPCR ligand, e.g., in an amount effective to support culture of an oocyte therein.

[0052] In some embodiments, the culture medium is suitable for maintaining, freezing, or cryopreservation of an SCNT embryo.

[0053] In some embodiments, the culture medium includes one or more of the following: the mammalian SCNT embryo is at 1 -cell or 2-cell stage, or the recipient mammalian oocyte is an enucleated recipient mammalian oocyte, or the mammalian SCNT embryo, recipient mammalian oocyte or a blastocyst is a human SCNT embryo, recipient human oocyte or a human blastocyst, or the mammalian SCNT embryo, recipient mammalian oocyte or blastocyst is from a non-human mammal.

[0054] Definitions

[0055] As used herein, an environment that is “cAMP rich” has a concentration of cAMP that is higher than a basal level.

[0056] As used herein, a “subject” is a mammal, such as a bovine (cow), pig, horse, sheep, goat, camel, hamster (e.g., Syrian hamster), guinea pig, rodent (e.g., Peromyscus), rabbit, rat, mouse, cat, dog, monkey, domesticated pet, or human. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0057] Detailed Description

[0058] Animal cloning, such as by somatic cell transfer (SCNT), has broad agricultural, medical, and societal applications. One of the significances of animal cloning in each of these applications is that it also provides the ability to genetically engineer the animal genome, which could be used to improve disease resistance and performance of livestock, to develop large animals as bioreactors to produce biologies, such as human therapeutics (e.g., antibodies or antigen binding fragments, enzymes for enzyme replacement, or other therapeutics), resurrect extinct or endangered species, and reduce environmental footprints.

[0059] The present invention features improved methods for culturing oocytes, ooplasts, and embryos. The methods described herein employ the use of a G-protein coupled receptor (GPCR) ligand in cell culture to produce fertilized oocytes and embryos that can be subsequently used for implantation into a recipient female (e.g., a mammal, such as a bovine).

[0060] The methods described herein ensure epigenetic integrity of donor cells during cell culture and genetic engineering of donor cells and cloned embryos. Moreover, the present invention also provides a platform for genetic manipulation to introduce gene expression cassettes into nuclear donor genomes and other genetic modifications in order to establish single cell-derived cell colonies, which are the integral parts of SCNT, to produce genetically engineered animals. Typically, SCNT and related procedures tend to introduce epigenetic errors in nuclear door cells. However, the present invention provides improved culturing conditions that reduces epigenetic errors, e.g., during SCNT.

[0061] Methods of Culture

[0062] The methods of culturing described herein include culturing, for example, an oocyte (e.g., an immature oocyte or a mature oocyte), an ooplast, or an embryo (e.g., an early-stage embryo). The culture may be employed using any of the culture media as described herein. The methods described herein employ the use of a GPCR ligand in the cell culture.

[0063] The culture step includes seeding one or more cells (e.g., oocyte, ooplast, or embryo) in culture medium in a suitable culture container, such as a flask or culture dish.

[0064] The culturing step may be performed, e.g., for 1 hour to 1 year (e.g., 1 minute to 10 minutes, e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, e.g., 10 minutes to 1 hour, e.g., 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, e.g., 1 hour to 1 day, e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 1 day, e.g., 1 day to 1 week, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, e.g., 1 week to 1 month, e.g., 2 weeks, 3 weeks, or 4 weeks, e.g., 1 month to 1 year, e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months).

[0065] The culturing may be performed at any suitable temperature, e.g., from 30 °C to 42 °C, e.g., 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, 40 °C, 40.5 °C, 41 °C, 41 .5 °C, or 42 °C, e.g., from 35 °C to 40 °C, e.g., 38.5 °C. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0066] In some embodiment, the method includes culturing the cells (e.g., oocyte (e.g., an immature or mature oocyte), ooplast, or embryo) in a first culturing step and then culturing in the presence of a GPCR ligand in a second culturing step. In another embodiment, the method may include culturing the oocyte, ooplast, or embryo in a first culturing step in the presence of a GPCR ligand and then culturing in the absence of a GPCR ligand in a second culturing step.

[0067] The culture medium may be exchanged one or more times during culture. For example, in some embodiments, the culture medium is changed, e.g., once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, or longer. The culture medium may be exchanged every two days. The cells may be cultured until the cells reach about 8-0-100% confluency. Such culturing may take, e.g., from 1 to 7 days, e.g., 3 to 6 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0068] Cells may be harvested from the culture dish or flask in order to collect the cells for subsequent use. For example, the cells may be harvested by treating them with trypsin-EDTA (e.g., 0.25%) and collecting the cells. The cells may be used directly for genetic engineering, subculturing, or cryopreservation for future use.

[0069] In some embodiments, the method further includes cloning an animal, e.g., by fertilizing the oocyte. In some embodiments, the fertilizing step includes in vitro fertilization. In some embodiments, the in vitro fertilization includes somatic cell nuclear transfer (SCNT).

[0070] Also featured herein is a method for culturing one or more embryos in vitro. The method includes collecting one or more early-stage embryos of less than 3 cells (e.g., 3 cells, 2 cells, or 1 cell) and placing the one or more embryos in a culture system. The method may further include contacting the one or more embryos with a GPCR ligand and culturing the embryos until blastocyst stage (e.g., culturing the embryo(s) until they have at most 100 cells). In some embodiments, the method includes culturing the embryo(s) until they have at most 100 cells, 95 cells, 90 cells, 85 cells, 80 cells, 75 cells, 70 cells, 65 cells, 60 cells, 55 cells, 50 cells, 45 cells, 40 cells, 35 cells, 30 cells, 25 cells, 20 cells, 15 cells, 10 cells, 9 cells, 8 cells, 7 cells, 6 cells, 5 cells, 4 cells, 3 cells, 2 cells, or 1 cell. The method may further include cryopreserving the embryo.

[0071] Hypoxic conditions

[0072] The culture methods described herein may include culturing the cells under hypoxic conditions or in the presence of a hypoxic mimetic. Hypoxic conditions, as described herein, include any condition where oxygen is present in concentrations below normal oxygen concentrations (normoxic conditions). Hypoxic mimetics mimic hypoxia by inducing the accumulation of hypoxiainducible factor one alpha (HiF1a), which is a protein subunit of a transcription factor that responds to decreases in available oxygen.

[0073] In some embodiments, culturing includes culturing the cells under hypoxic conditions. Hypoxic conditions may include, e.g., an oxygen level of less than 20%. In some embodiments, the culturing under hypoxic conditions includes culturing the cells at an oxygen level of between 1% to 19% (e.g., between 1% and 10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). In some embodiments, the culturing under hypoxic conditions ATTORNEY DOCKET NO. 51488-002WO2 PATENT includes culturing the cells at an oxygen level of between 1 % to 19%. In some embodiments, the culturing includes culturing the cells at an oxygen level of between 1 % to 10%. In some embodiments, the culturing includes culturing the cells at an oxygen level of 5%. In some embodiments, the culturing includes culturing the cells under normoxic conditions. In some embodiments, the method includes culturing under conditions which are substantially free of oxygen.

[0074] In some embodiments, culturing includes culturing the cells in the presence of a hypoxic mimetic (e.g., a HIF-1 a stabilizer or a prolyl hydroxylase domain (PHD) inhibitor). Exemplary hypoxia mimetics include, but are not limited to, an iron chelator (e.g., deferoxamine mesylate (DFO), compound A, deferasirox, and 2,2'-dipyridyl (DP)), an ion competitor (e.g., cobalt chloride (C0CI2, e.g., from 100 pM to 1 mM, e.g., about 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, or 1 mM C0CI2,) or a divalent metal ion such as Ni2+, Mn2+, Co2+, or Zn2+), and a 2 oxoglutarate (20G) analog (e.g., dihydroxybenzoic acid (DHB), N-oxalylglycine, dimethyloxalylglycine (DMOG)), a PHD inhibitor (e.g., FG-4497, GSK360A, TM6008, or folic acid), or a HIF-1 a stabilizer (e.g., miR-335, isoflurane, N-acetylcysteine, MG-132, BSc21 18, and tilorone). Hypoxia mimetics are also described, e.g., in Davis et al. Front. Cell Dev. Biol. 6:175, 2018. In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of cobalt chloride. In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of desferrioxamine (e.g., DFO). In some embodiments, the culturing in the presence of a hypoxic mimetic includes culturing the cells in the presence of DMOG. In some embodiments, the culturing includes culturing the cells in the absence of a hypoxic mimetic.

[0075] In some embodiments, the methods include incubating one or more immature oocytes with an inhibitor of prolyl hydroxylase domain (PHD). In some embodiments, the PHD is a HIF-prolyl hydroxylase. In some embodiments, the inhibitor of PHD is CoCI2, DFO (deferoxamine), or DMOG (dimethyloxalylglycine), Roxadustat (FG-4592), Vadadustat (AKB-6548), Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[0076] In some embodiments, the intracellular environment of an immature oocyte is hypoxic. In some embodiments, the intracellular environment has an oxygen concentration of 0 to 4% (e.g., 0%, 0.5%, 1 %, 1 .5%, 2%, 2.5%, 3%, 3.5%, or 4%) oxygen. cAMP regulation

[0077] Cyclic nucleotide phosphodiesterases (PDE) catalyze the hydrolysis of cyclic adenosine 3’, 5’ monophosphate (cAMP). PDEs are responsible for the decrease of cAMP in order to regulate the levels of cAMP in mammalian cells by which many biological processes are regulated. PDE inhiibotors inhibit such regulation, thereby increasing intracellular cAMP.

[0078] The methods described herein may include culturing cells in the presence of a PDE inhibitor. Suitable PDE inhibitors include, for example, caffeine and cilostamide. One or both of these PDE inhibitors may be introduced into the culture media to increase intracellular cAMP.

[0079] In some embodiments, contacting the immature oocyte with the ligand produces intracellular cyclic AMP (cAMP). In some embodiments, intracellular cyclic AMP (cAMP) is increased due to direct activation of adenylate cyclase. In some embodiments, intracellular cAMP is increased by inhibition of ATTORNEY DOCKET NO. 51488-002WO2 PATENT cAMP-phosphodiesterase (PDE). In some embodiments, the PDE inhibitor is caffeine. In some embodiments, the method includes culturing in the presence of 0.1 mM to 5 mM caffeine, e.g., from 0.5 mM to 2.5 mM, e.g., 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mM caffeine. In some embodiments, the PDE inhibitor is cilostamide. In some embodiments, the method includes culturing in the presence of 1 pM to 50 pM, e.g., 2 pM to 20 pM, e.g., 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM cilostamide.

[0080] In some embodiments, the intracellular environment of the immature oocyte is hypoxic and cAMP rich. In some embodiments, the intracellular environment has an oxygen concentration of 0-4% (e.g., 0%, 0.5%, 1 %, 1 .5%, 2%, 2.5%, 3%, 3.5%, or 4%) oxygen.

[0081] In some embodiments, the intracellular environment of the immature oocyte has a cAMP concentration of at least 1 pM (e.g., at least 1 pM, 2 pM, 3 pM, 5 pM, or more).

[0082] Culture Media

[0083] The methods described herein include culturing an oocyte (e.g., an immature oocyte), an ooplast (e.g., an enucleated oocyte), or an embryo (e.g., a fertilized oocyte). The methods may employ any suitable culture medium (e.g., a basal medium) sufficient to support viability, e.g., of the oocyte. The culture medium may include a GPCR ligand. The culture medium may include any suitable culture supplements and a GPCR ligand. In some embodiments, the culture medium is suitable for maintaining, freezing, or cryopreservation of the oocyte (e.g., an immature oocyte), ooplast (e.g., an enucleated oocyte), or embryo (e.g., a fertilized oocyte, e.g., an SCNT embryo).

[0084] The culture medium may contain one or more salts, amino acids, vitamins, or other supplements. The one or more salts may include, for example, sodium chloride, potassium chloride, sodium biocarbonate, calcium chloride, magnesium chloride, cobalt chloride, sodium lactate, and the like. The one or more amino acids may include, for example, asparagine, cysteine, histidine, lysine, proline, serine, aspartic acid, glycine, and glutamic acid. In some embodiments, the culture medium is supplemented with essential amino acids (e.g., histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). The culture medium may further include pantothenate or pantothenic acid (Vitamin B5). The culture medium may further include taurine (2- aminoethanesulfonic acid). The culture medium may include one or more acids, e.g., hydrochloric acid, e.g., to modulate the pH of the culture. The culture medium may include GLUTAMAX™ supplement or L-glutamine.

[0085] In one embodiment, the basal culture medium is Taurgen-Medium, the components thereof are shown in Table 1 below. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0086] Table 1. Taurgen-Medium

[0087] Another medium contemplated herein is an intro maturation (IVM) medium. Oocyte maturation may be achieved using Taurgen-IVM, e.g., under hypoxic conditions. For example, the oocytes may be matured under hypoxic conditions e.g., with 0.2-3.0% oxygen levels under 5% CO2, 38.50 °C for 24 hours.

[0088] Taurgen-IVM can be made by supplementing a basal medium, such as Tissue Culture Medium 199 (TCM199), with Earle’s salts (e.g., containing sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate), fetal bovine serum (FBS), e.g., 5% to 20% (v / v), e.g., 10% FBS, sodium pyruvate, e.g., 1 pg / ml to 50 pg / ml, e.g., 22 pg / ml sodium pyruvate, estradiol, e.g., 1 pg / ml to 10 pg / ml, e.g., 2 pg / ml estradiol, follicle-stimulating hormone (FSH), e.g., 1 pg / ml to 50 pg / ml, e.g., 20 pg / ml FSH, gentamicin sulfate, e.g., 1 pg / ml to 100 pg / ml gentamicin sulfate, e.g., 50 pg / ml gentamicin sulfate, and glutamine, e.g., 0.1 mM to 10 mM glutamine, e.g., 1 mM glutamine. The IVM may further include Taurgen-Supplements. One example of Taurgen-Supplements includes acteoside, e.g., in a range of concentrations of 2 pM-250 pM, and adenosine, e.g., in a range of concentrations of 1 mM-50 mM.

[0089] An exemplary formulation of TCM199 is shown below in Table 2. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0090] Table 2. TCM199 components ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0091] GPCR ligands

[0092] The methods of culturing and culture media described herein include the use of a G-protein coupled receptor (GPCR) ligand. The GPCR ligand may be used, e.g., to contact an oocyte (e.g., an immature oocyte), ooplast, or embryo. The GPCR may be linked to stimulatory (Gs) protein (GsPCR) or an inhibitory (Gi) protein (GiPCR). The ligand may be an agonist. Alternatively, the ligand may be an antagonist. ATTORNEY DOCKET NO. 51488-002WO2

[0093] PATENT

[0094] In some embodiments, the GsPCR is an adenosine receptor. The adenosine receptor may be an adenosine 1 (A1 ) receptor, an adenosine 2A (A2A) receptor, an adenosine 2B (A2B) receptor, or an adenosine 3 (A3) receptor. In some embodiments, the adenosine receptor is an A2A or A2B receptor. The ligand may be an A1 , A2A, A2B, or A3 agonist or antagonist. The ligand may be an A2B-A3 dual agonist or antagonist.

[0095] In some embodiments, the GiPCR is an adenosine receptor. The adenosine receptor may be an A1 receptor, an A2A receptor, an A2B receptor, or an A3 receptor. In some embodiments, the adenosine receptor is an A1 receptor or an A3 receptor. The ligand may be an A1 , A2A, A2B, or A3 agonist or antagonist. The ligand may be an A2B-A3 dual agonist or antagonist.

[0096] Suitable adenosine receptors that may be targeted are shown in Table 3 below. Also shown are agonists and antagonists targeting the same.

[0097] Table 3. Adenosine receptors and ligands

[0098] A GsPCRs agonist may be provided in a suitable culture medium in a range of concentrations of 0.1 pM to 10 pM, e.g., 0.1 pM to 1 pM, e.g., 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM, e.g., 1 pM to 10 pM , e.g., 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM, e.g., 0.1 pM to 2.5 pM, e.g., 0.1 pM to 2. 5 pM, e.g., 0. 5 pM, 1 pM, 1 .5 pM, 2 pM, or 2.5 pM). The specific concentration of the GsPCR agonist to be added may be adjusted according to the level of oxygen (e.g., 0.2%-3.0%) employed during culturing.

[0099] A GsPCR antagonist may be provided in a suitable culture medium in a range of concentration of 1 pM to 25 pM (e.g., 2 pM to 20 pM, e.g., 1 pM, to 10 pM, e.g., 1 pM, 2 pM, 3 pM, 4 ATTORNEY DOCKET NO. 51488-002WO2

[0100] PATENT pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM, e.g., 10 pM to 25 pM, e.g., 1 1 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, or 30 pM). The specific concentration of the GsPCR antagonist to be added may be adjusted according to the level of oxygen (0.2%-3.0%) employed during culturing.

[0101] In some embodiments, the GsPCR is a prostaglandin receptor. The prostaglandin receptor may be a prostaglandin E2 receptor. For example, the prostaglandin E2 receptor may be a prostaglandin E receptor EP2 or a prostaglandin E receptor EP4. The ligand may be an agonist or antagonist of a prostaglandin receptor.

[0102] In some embodiments, the antagonist is an EP2 antagonist (e.g., PF-04418948).

[0103] In some embodiments, the antagonist is an EP4 receptor antagonist (e.g., AH6809 or AH23848).

[0104] In some embodiments, the agonist is an EP2 receptor agonist.

[0105] In some embodiments, the agonist is an EP4 receptor agonist (e.g., 1 1 -Deoxy-PGE1 , gamma-Lactam PGE analog 2a, gamma-Lactam analog 4, AGN205203, TCS 2510 gamma-Lactam PGE analog 3).

[0106] In some embodiments, the culture media includes adenosine or an adenosine analog. Adenosine may be present at a concentration of 0.1 pM to 1 mM, e.g., 0.1 pM to 1 pM, e.g., 0.1 pM to 1 pM, e.g., 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM, e.g., 1 pM to 10 pM , e.g., 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM, e.g., 10 pM to 100 pM, e.g., 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 90 pM, or 100 pM, e.g., 100 pM to 1 mM, e.g., 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, or 1 mM.

[0107] In some embodiments, the adenosine analog is 5'-Nethylcarboxamidoadenosine (NECA), a non-selective agonist of adenosine receptors A1 , A2A, A2B and A3. The NECA may be provided in a range of 0.1 pM to 20 pM, e.g., 0.1 pM to 1 pM, e.g., 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM, e.g., 1 pM to 10 pM , e.g., 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM, e.g., 10 pM to 20 pM, e.g., 1 1 pM, 12 pM, 13 pM, 14 pM 5 pM, 16 pM, 17 pM, 18 pM, 19 pM, or 20 pM.

[0108] In some embodiments, the culture medium includes acteoside. The acteoside may be present at a concentration 1 pM to 500 pM, e.g., 2 pM to 250 pM, e.g., 1 pM to 10 pM , e.g., 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, or 10 pM, e.g., 10 pM to 100 pM, e.g., 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 90 pM, or 100 pM, e.g., 100 pM to 500 pM, e.g., 200 pM, 300 pM, 400 pM, or 500 pM.

[0109] In some embodiments, the medium includes a basal medium, such as a commercially available basal medium. The medium may include or be composed of TCM199 medium. The medium may include one or more salts, e.g., Earle’s salts, e.g., containing one or more of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium dihydrogen phosphate, sodium bicarbonate, and dextrose (glucose). The medium may further include fetal bovine serum (FBS) e.g., 10% (v / v) FBS.

[0110] The medium may further include sodium pyruvate. For example, the medium may contain 1 pg / mL to 50 pg / mL sodium pyruvate, e.g., 22 pg / mL sodium pyruvate. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0111] The medium may further include estradiol. For example, the medium may contain 0.1 pg / mL to 5 pg / mL estradiol, e.g., 2 pg / ml estradiol.

[0112] The medium may further include follicle-stimulating hormone. For example, the medium may contain 1 pg / mL to 50 pg / mL follicle-stimulating hormone, e.g., 20 pg / ml follicle-stimulating hormone.

[0113] The medium may further include gentamicin sulfate. For example, the medium may contain 10 pg / mL to 100 pg / mL gentamicin sulfate, e.g., 50 pg / ml gentamicin sulfate.

[0114] The medium may further include glutamine. For example, the medium may contain 0.1 mM to 10 mM glutamine, e.g., 1 mM glutamine.

[0115] Somatic Cell Nuclear Transfer (SCNT)

[0116] SCNT is a process by which an enucleated oocyte is implanted in its cytoplasm with a donor nucleus from a somatic cell. The SCNT technique allows cloning an animal with a desired nucleus. After the somatic cell transfer, the cytoplasmic factors affect the nucleus to become a zygote. The blastocyst stage is developed by the egg to help create embryonic stem cells from the inner cell mass of the blastocyst.

[0117] The process of somatic cell nuclear transfer involves two different cells, a female gamete (oocyte). An oocyte may be obtained by any technique known in the art, e.g., using ovarian stimulation. The second cell is a somatic cell, which is a non-oocyte. The somatic cell may be, for example, a skin cell, fat cell, or liver cell. The nucleus and genetic material of the donor oocyte is removed and thereby deprogrammed, thus forming an ooplast. The somatic cell and the enucleated oocyte (ooplast) are then fused by inserting the somatic cell into the ooplast. After being inserted into the egg, the somatic cell nucleus is reprogrammed by the host ooplast. The ovum, now containing the nucleus of the somatic cell, may be stimulated with a shock or chemical stimulus in order to begin division. The egg is now viable and capable of producing an organism containing all necessary genetic information from just one parent. Development will ensue normally and after many mitotic divisions, the single cell forms a blastocyst, which is an early-stage embryo having about 100 cells, with an identical genome to the original somatic cell organism. The oocyte or embryo may be cultured in a culture medium as described herein, e.g., containing a GPCR ligand (e.g., agonist or antagonist). The cloned embryo may then be implanted into a host female for further development and birth.

[0118] Genetic Assays

[0119] The methods described herein may use one or more genetic tests to ascertain one or more genetic traits or features of a cell (e.g., oocyte) or embryo as described herein. Genetic tests, such as whole genome analysis (WGA), may be employed to identify a genetically desirable oocyte or embryo.

[0120] An aliquot (e.g., one or more cells) of the embryo may be subject to one or more genetic tests, including, but not limited to, whole genome analysis (WGA), gene expression profiling (e.g., gene expression profiling using microarray), sequencing of a coding region of a gene, sequencing of a non-coding region of a gene, and whole genome sequencing. The result of the genetic tests may be ATTORNEY DOCKET NO. 51488-002WO2

[0121] PATENT analyzed in order to determine the oocyte or embryo has one or more genetically desirable traits. Under such a scenario, the resulting offspring will likely demonstrate the genetically desirable trait.

[0122] Genetic tests may include analyzing the DNA in the cells of the embryos. In one embodiment, microsatellite marker panels may be used to identify genetic traits that are highly polymorphic and amenable to standardization among laboratories performing these tests (see, e.g., Sherman et al. Anim Genet. 35: 220-6; Heyen et al. Arnim Genet. 28: 21 -27; U.S. Pat. No. 5,874,217; Ostrander et al. Mammalian Genome. 6: 192-195; Franscisco et al. Mammalian Genome. 7: 359-362, each of which is hereby incorporated by reference). In another embodiment, single nucleotide polymorphisms (SNPs) may be utilized in the genetic test. The genetic information may include, for example, single nucleotide polymorphisms (SNPs), insertions, deletions, inversions and other mutations, e.g., associated with a genetically desirable trait.

[0123] Examples

[0124] Example 1. SCNT Procedure

[0125] Somatic cell nuclear transfer (SCNT) procedure

[0126] SCNT may be performed by demecolcine induced enucleation. Matured oocytes with extruded first polar bodies (1 PB) are transferred to Taurgen-IVM medium supplemented with demecolcine (0.04 Ig / mL) and incubated for 2 h and then are denuded of granulosa cells in the presence of hyaluronidase (300 U / mL) in TCM-199 medium. Denuded oocytes may be incubated in Hoechst 33352 at a concentration of 5 ug / ml and then enucleated by aspiration of the metaphase II plate in 5-IL drops of TCM-199 medium with 4mg / mL bovine serum albumin (BSA), gentamicin (5 mg / mL), and 7.5 Ig / mL cytochalasin B, under UV light.

[0127] Construction of cloned embryos

[0128] Enucleated oocytes may be transferred to Taurgen-Medium with Taurgen-Supplements for 15 min and individually injected with a donor cell into the perivitelline space under mineral oil. Donor cells may be kept in 11% polyvinylpyrrolidone (PVP) solution. Reconstructed oocytes may be activated in vitro with ionomycin (5 IM, 5 min) and 6-dimethylaminopurine (10mM) with 7.5 Ig / mL cytochalasin B for 3 to 5 h. The in vitro development may be recorded every 24 h.

[0129] Example 2. In Vitro Maturation

[0130] Bovine oocytes maturation may be achieved using Taurgen-IVM under hypoxic condition with 0.2-3.0% oxygen levels under 5% CO2, 38.50 °C for 24 hours.

[0131] Taurgen-IVM can be made by supplementing a basal medium, such as Tissue Culture Medium 199 (TCM199), with Earle’s salts, 10% (v / v) FBS, 22 pg / ml sodium pyruvate, 2 pg / ml estradiol, 20 pg / ml follicle-stimulating hormone, 50 pg / ml gentamicin sulfate, and 1 mM glutamine, plus Taurgen-Supplements.

[0132] One example of Taurgen-Supplements includes acteoside in a range of concentrations of 2 pM-250 pM, and adenosine in a range of concentrations of 1 mM-50 mM. ATTORNEY DOCKET NO. 51488-002WO2 PATENT

[0133] Ovaries may be obtained from slaughterhouses and transferred to the laboratory at 37 °C within 1-2 h after animal slaughter. Oocytes may be aspirated in Taurgen-Medium and selected for those having at least two to three layers of compact cumulus cells and uniform cytoplasm for in vitro maturation. Oocytes may be matured in vitro in Taurgen-IVM in a humidified atmosphere of 5% CO2 and hypoxic conditions (0.2-3.0% oxygen) at 38.5 °C (for cattle; different temperatures may be used for other species) for 24 h.

[0134] Other Embodiments While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0135] Other embodiments are within the claims.

Claims

ATTORNEY DOCKET NO. 51488-002WO2 PATENTCLAIMS1 . A method for producing a mature oocyte from an immature oocyte, the method comprising contacting one or more immature oocyte with a G-protein coupled receptor (GPCR) ligand.

2. A method for maintaining an oocyte, the method comprising contacting one or more immature oocyte with a GPCR ligand.

3. The method of claim 1 or 2, wherein contacting the immature oocyte with the ligand produces intracellular cyclic AMP (cAMP).

4. The method of claim 3, wherein intracellular cyclic AMP (cAMP) is increased due to direct activation of adenylate cyclase.

5. The method of claim 3 or 4, wherein intracellular cAMP is increased by inhibition of cAMP- phosphodiesterase (PDE).

6. The method of any one of claims 3-5, wherein the intracellular environment of the immature oocyte is hypoxic and cAMP rich.

7. The method of claim 6, wherein the intracellular environment has an oxygen concentration of 0-4% oxygen.

8. The method of any one of claims 3-7, wherein the intracellular environment of the immature oocyte has a cAMP concentration of at least 1 uM.

9. The method of any one of claims 1 -8, wherein the method comprises incubating the one or more immature oocytes under conditions which are substantially free of oxygen.

10. The method of any one of claims 1 -9, further comprising incubating one or more immature oocytes with an inhibitor of prolyl hydroxylase (PHD).11 . The method of claim 10, wherein the PHD is a HIF-prolyl hydroxylase.

12. The method of claim 10 or 11 , wherein the inhibitor of PHD is CoCI2, DFO (deferoxamine), or DMOG (dimethyloxalylglycine), Roxadustat (FG-4592), Vadadustat (AKB-6548), Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

13. A method of animal cloning, the method comprising contacting an oocyte with a culture medium comprising a GPCR ligand.ATTORNEY DOCKET NO. 51488-002WO2 PATENT14. The method of any one of claims 1 -13, further comprising fertilizing the oocyte.

15. The method of claim 14, wherein the fertilizing step comprises in vitro fertilization.

16. The method of claim 15, wherein the in vitro fertilization comprises somatic cell nuclear transfer (SCNT).

17. A method for making an SCNT embryo, the method comprising contacting a somatic cell with a GPCR ligand and transferring the nucleus of the somatic cell to an enucleated oocyte, thereby making the SCNT embryo.

18. A method for increasing the efficiency of SCNT, the method comprising contacting a donor mammalian cell, recipient mammalian oocyte, or a mammalian SCNT embryo with a GPCR ligand, thereby increasing the efficiency of the SCNT.

19. A method of producing an embryo, the method comprising the steps of:(a) injecting or fusing one or more nuclei into an ooplast;(b) culturing the ooplast in the presence of a GPCR ligand to produce a viable embryo.

20. The method of any one of claims 17-19, further comprising transferring the embryo to an oviduct of a female to produce a cloned animal.21 . A method for culturing an embryo in vitro comprising:(a) collecting one or more early-stage embryos of less than 3 cells;(b) placing the one or more embryos in a culture system;(c) contacting the one or more embryos with a GPCR ligand; and(d) culturing the embryos until blastocyst stage.

22. A method of culturing an embryo for implantation, the method comprising culturing the embryo in one or more culture media comprising a GPCR ligand.

23. The method of any one of claims 17-22, further comprising cryopreserving the embryo.

24. The method of any one of claims 1 -23, wherein the G-protein coupled receptor is linked to stimulatory G (Gs) proteins (GsPCR) or to inhibitory G (Gi) proteins (GiPCR).

25. The method of any one of claims 1 -24, wherein the ligand is an agonist or an antagonist.

26. The method of claim 24 or 25, wherein the GsPCR is an adenosine receptor.ATTORNEY DOCKET NO. 51488-002WO2PATENT27. The method of claim 26, wherein the adenosine receptor is an adenosine 1 (A1 ) receptor, an adenosine 2A (A2A) receptor, an adenosine 2B (A2B) receptor, or an adenosine 3 (A3) receptor.

28. The method of claim 27, wherein the adenosine receptor is an A2A or A2B receptor.

29. The method of claim 24 or 25, wherein the GsPCR is a prostaglandin receptor.

30. The method of claim 29, wherein the prostaglandin receptor is a prostaglandin E2 receptor.31 . The method of claim 30, wherein the prostaglandin E2 receptor is a prostaglandin E receptor EP2 or a prostaglandin E receptor EP4.

32. The method of claim 24 or 25, wherein the GiPCR is an adenosine receptor.

33. The method of claim 32, wherein the adenosine receptor is an A1 receptor, an A2A receptor, an A2B receptor, or an A3 receptor.

34. The method of claim 33, wherein the adenosine receptor is an A1 receptor or an A3 receptor.

35. The method of claim any one of claims 1 -34, wherein the ligand is an adenosine receptor agonist or antagonist.

36. The method of claim 35, wherein the ligand is an A1 , A2A, A2B, or A3 agonist or antagonist.

37. The method of claim 36, wherein the ligand is an A2B-A3 dual agonist or antagonist.

38. The method of claim any one of claims 1 -37, wherein the ligand is an agonist or antagonist of a prostaglandin receptor.

39. The method of claim 38, wherein the antagonist is an EP2 antagonist.

40. The method of claim 39, wherein the EP2 antagonist is PF-04418948.41 . The method of claim 38, wherein the antagonist is an EP4 receptor antagonist.

42. The method of claim 41 , wherein the EP4 receptor antagonist is AH6809 or AH23848.

43. The method of claim 38, wherein the agonist is an EP2 receptor agonist.

44. The method of claim 38, wherein the agonist is an EP4 receptor agonist.ATTORNEY DOCKET NO. 51488-002WO2 PATENT45. The method of claim 44, wherein the EP4 receptor agonist is 11 -Deoxy-PGE1 , gamma-Lactam PGE analog 2a, or gamma-Lactam analog 4.

46. The method of claim 45, wherein the EP4 receptor agonist is AGN205203 or TCS 2510 gamma- Lactam PGE analog 3.

47. A culture medium obtained from media used in the method of any one of claims 1 -46.

48. A culture medium for culturing an oocyte comprising a GPCR ligand in an amount effective to support culture of an oocyte therein.

49. The culture medium of claim 47 or 48, wherein the culture medium is suitable for maintaining, freezing, or cryopreservation of an SCNT embryo.

50. The culture medium of claim 48 or 49, wherein the medium comprises one or more of the following: the mammalian SCNT embryo is at 1 -cell or 2-cell stage, or the recipient mammalian oocyte is an enucleated recipient mammalian oocyte, or the mammalian SCNT embryo, recipient mammalian oocyte or a blastocyst is a human SCNT embryo, recipient human oocyte or a human blastocyst, or the mammalian SCNT embryo, recipient mammalian oocyte or blastocyst is from a non-human mammal.51 . An in vitro culture system for an embryo comprising a GPCR ligand.

52. The in vitro culture system of claim 51 , wherein the system comprises the culture medium of any one of claims 47-51 .

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