EP1234021A2 - Procedes et compositions ameliorant le potentiel de developpement d'oocytes et de zygotes - Google Patents

Procedes et compositions ameliorant le potentiel de developpement d'oocytes et de zygotes

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Publication number
EP1234021A2
EP1234021A2 EP00972510A EP00972510A EP1234021A2 EP 1234021 A2 EP1234021 A2 EP 1234021A2 EP 00972510 A EP00972510 A EP 00972510A EP 00972510 A EP00972510 A EP 00972510A EP 1234021 A2 EP1234021 A2 EP 1234021A2
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EP
European Patent Office
Prior art keywords
rephcative
oocytes
donor cell
mitochondna
donor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00972510A
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German (de)
English (en)
Inventor
Robert F. Casper
Ian Rogers
Jonathan Tilly
Andrea Jurisicova
Gloria I. Perez
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mount Sinai Hospital Corp
General Hospital Corp
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Mount Sinai Hospital Corp
General Hospital Corp
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Application filed by Mount Sinai Hospital Corp, General Hospital Corp filed Critical Mount Sinai Hospital Corp
Publication of EP1234021A2 publication Critical patent/EP1234021A2/fr
Withdrawn legal-status Critical Current

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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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • TITLE Methods and Compositions for Enhancing Developmental Potential of Oocytes and Zygotes FIELD OF THE INVENTION
  • the invention relates to compositions and methods for enhancing the developmental potential of oocytes, zygotes, and preimplantation embryos.
  • IVF in vitro fertilization
  • other assisted reproductive technologies about 50% of human embryos undergo a suicide program of active cell death and become fragmented.
  • zygote development and the first cleavage divisions depend upon maternal RNA and protein products accumulated during oogenesis. Reproductive failure can be attributed to the lack of cleavage in the developing embryo. This phenomenon can be traced to a defect in the composition of the oocyte cytoplasm. Maternal cytoplasmic components are involved in embryonic arrest, because the "2-cell block" in mice can be overcome by transplantation of ooplasm from zygotes of non-arresting strains into the zygotes of arresting strains (Muggleton-Harns et al. Nature. 1982 Sep 30;299 (5882):460-2).
  • the invention relates to a method for enhancing developmental potential of oocytes comprising increasing intracellular levels of rephcative mitochondria in the oocytes.
  • the intracellular levels of rephcative mitochondria are increased by introducing rephcative mitochondria into the oocytes.
  • a method of the invention may additionally comp ⁇ se fertilizing the oocytes to obtain a zygote with mcreased intracellular levels of rephcative mitochond ⁇ a.
  • the invention also relates to a method for enhancing developmental potential of zygotes comp ⁇ sing increasing intracellular levels of rephcative mitochond ⁇ a in the zygotes.
  • the intracellular levels of rephcative mitochond ⁇ a are increased by introducing rephcative mitochondria into zygotes.
  • the invention further relates to an oocyte or a zygote with increased intracellular levels of rephcative mitochondria obtamed from a method of the invention.
  • the invention relates to a composition comp ⁇ sing rephcative mitochond ⁇ a for enhancing developmental potential of oocytes or zygotes, and for treating and preventing he ⁇ table mitochond ⁇ al diseases.
  • the composition may comp ⁇ se cryopreserved mitochondna.
  • the invention provides a method for fertilizing oocytes comp ⁇ sing removing oocytes from a follicle of an ovary, introducing rephcative mitochrond ⁇ a into the oocytes, and fertilizing the resulting oocytes with spermatozoa.
  • the invention provides a method for stonng and then enhancing the developmental potential of oocytes comp ⁇ sing cryopreservmg immature oocytes, thawing the cryopreserved oocytes, and introducing rephcative mitochond ⁇ a into the oocytes.
  • a method is also contemplated for enhancing the developmental potential of oocytes comp ⁇ sing cryopreservmg rephcative mitochond ⁇ a, thawing the mitochond ⁇ a, and introducing the rephcative mitochond ⁇ a into oocytes.
  • the methods and compositions of the invention improve the quality of the oocytes that are being fertilized and the quality of zygotes, to increase the rate of success m embryo development and ongoing pregnancy
  • the methods and compositions are particularly useful m enhancing the developmental potential of oocytes or zygotes with mitochond ⁇ al DNA mutations or abnormal mitochond ⁇ al metabolic activity.
  • the invention provides a method for improving embryo development after in vitro fertilization or embryo transfer in a female mammal comp ⁇ smg implanting into the female mammal an embryo de ⁇ ved from an ooctye or zygote containing mcreased intracellular levels of rephcative mitochond ⁇ a.
  • the invention also provides a method for reducmg the detrimental effects of mitochond ⁇ al DNA mutations (e g. deletion or rmssense mutations) m the progeny of an individual affected by such mutations comp ⁇ smg introducing mto oocytes or zygotes from the individual rephcative mitochondna that does not contain the DNA mutations (i.e. healthy mitochond ⁇ a).
  • the invention further provides an oocyte or a zygote comp ⁇ sing both mitochond ⁇ a with mitochond ⁇ al DNA mutations, and punfied and isolated rephcative mitochondna that do not contain the mitochond ⁇ al DNA mutations (i.e. healthy mitochond ⁇ a).
  • the mvention also relates to a method for treating he ⁇ table mitochond ⁇ al diseases m the progeny of an individual affected by such diseases compnsmg mtroducmg mto oocytes or zygotes from the individual rephcative mitochond ⁇ a comp ⁇ smg mitochond ⁇ a that does not contain the DNA mutations (i.e. healthy mitochond ⁇ a).
  • the oocyte is a recipient ooctye m a nuclear transfer method.
  • the mvention relates to a method for enhaincing developmental potential of recipient oocytes in a nuclear transfer method compnsmg mtroducmg rephcative mitochondna mto the recipient oocytes.
  • the mvention also contemplates recipient oocytes comp ⁇ smg rep cattve mitochondna, and blastocyts, embryos, and non-human animals formed from the nuclear transfer methods of the invention.
  • the donor nucleus is placed in an enucleated oocyte obtamed from a different mdividual.
  • mitochondria in the recipient oocyte have not-co-existed with the donor nucleus. Since mitochondria are always maternally inhe ⁇ ted, their replication, transc ⁇ ption, translation, and function does not only depend on mitochond ⁇ al DNA, but is tightly intercalated with the nuclear genome that co-exists with the mitochrondna.
  • the invention by introducing rephcative mitochondna mto recipient oocytes enhances the developmental potential of the recipient oocytes. This is expected to increase the live birth rate in nuclear transfer methods.
  • the invention provides a method of cloning a non-human mammalian embryo by nuclear transfer comp ⁇ smg
  • the method may further compnse permitting the embryo to develop mto a cloned mammal.
  • the invention also provides a method of cloning a non-human mammal by nuclear transfer comp ⁇ sing
  • a method of clonmg a non-human mammalian fetus by nuclear transfer is provided compnsmg the following steps:
  • mtroducmg a donor cell nucleus from a donor cell of a non-human mammal, and rephcative mitochondria preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the non-human mammal from which the donor cell nucleus is denved, mto an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit,
  • (c) transfemng the cultured nuclear transfer unit to a host non-human mammal of the same species such that the nuclear transfer unit develops into a fetus.
  • the method may also compnse developing the fetus into an offspring.
  • the invention provides a recipient oocyte compnsmg a penvitelline space and a donor cell nucleus and rephcative mitochondria preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same individual from which the donor cell nucleus is dervied, deposited in the penvitelline space
  • FIG. 1 is a bar graph showing the effect of mitochond ⁇ a injection on preimplantaion embryo development DETAILED DESCRIPTION OF THE INVENTION
  • oocytes refers to the gamete from the follicle of a female animal, whether vertebrate or invertebrate.
  • the animal is a mammal, and more preferably is a non-human p ⁇ mate, a bovme, equine, porcme, ovme, caprine, buffalo, guinea pig, hamster, rabbit, mice, rat, dog, cat, or a human Suitable oocytes for use in the invention include immature oocytes, and mature oocytes from ovanes stimulated by administering to the oocyte donor, in vitro or in vivo, a fertility agent or fertility enhancmg agent (e g.
  • the oocytes are aged (e.g. from humans 40 years +, or from animals past their reproductive prime).
  • the oocytes some embodiments of the invention contain mitochond ⁇ al DNA mutations. Methods for isolating oocytes are known in the art.
  • oocytes are used as recipient cells (such cells are referred to herein as "recipient oocytes")
  • the recipient ooctyes are obtamed from non-human mammals, in particular domestic, sports, zoo, and pet animals mcludmg but not limited to bovme, ovine, porcine, equme, capnne, buffalo, and gumea pigs, rabbits, mice, hamsters, rats, primates, etc.
  • zygote refers to a fertilized oocyte p ⁇ or to the first cleavage division.
  • the expression "enhancing the developmental potential of oocytes” refers to increasing the quality of the oocyte so that it will be more capable of bemg fertilized and/or enhancmg mitochond ⁇ al function or activity m the oocyte for subsequent development and reproduction. Increasmg the quality of the oocyte, and thus the fertilized oocyte (e.g. zygote), preferably results m enhanced development of the oocyte into an embryo and its ability to be implanted and form a healthy pregnancy.
  • the expression "enhancing the developmental potential of zygotes” refers to increasing the quality of the zygotes and/or enhancmg rmtochondnal function or activity m the zygotes for subsequent development and reproduction.
  • Increasing the quality of the zygotes preferably results in enhanced development of the zygotes into an embryo and their ability to be implanted and form a healthy pregnancy.
  • Quality can be assessed by the appearance of the developing embryo by visual means and by the IVF or nuclear transfer success rate.
  • Cntena to judge quality of the developing embryo by visual means include, for example, their shape, rate of cell division, fragmentation, appearance of cytoplasm, and other means recognized in the art of IVF and nuclear transfer.
  • “Spermatozoa” refers to male gametes that can be used to fertilize oocytes.
  • “Heritable mitochond ⁇ al diseases” refers to diseases caused by defects in mitochond ⁇ al DNA or by defects in nuclear genes that are important to mitochondnal function. Examples of rmtochondnal diseases include but are not limited to Kearns-Sayre syndrome, MERRF syndrome (Myoclonic Epilepsy with Ragged Red Fibres), MELAS syndrome (Mitochondnal Encephalopathy, Myopathy, Lactic Acidosis and Stroke-like episodes), and Leber's disease (I. Nonaka, Cu ⁇ ent Opinion in Neurology and Neurosurgery, 5 (1992) 622)
  • rephcative microchondna refers to a preparation of punfied mitochondna that are capable of replicating during embryo development and increasing mitochondnal copy number or function.
  • the rephcative mitochondna is substantially free of other cytoplasmic components mcludmg nuclear DNA, mRNA, protems, antioxidants, and organelles other than mitochond ⁇ a.
  • the rephcative mitochondria preparations are at least 60% free, preferably 75% free, and most preferably 90% free from other cytoplasmic components.
  • the rephcative mitochond ⁇ a preparations contam greater than 70%, more preferably greater than 80%, most preferably greater than 90% functional mitochondria.
  • a rephcative mitochondna preparation typically contams about 2,000 to 20,000 mitochondna in a volume of 5 to 15 picoL.
  • rephcative mitochondna are preferably denved from any stem cell (e.g. hematopoietic, embryonic, trophoblastic, primordial germ cells) or from any immortalized cell lme (e.g. cancer, or intentionally transformed somatic cells) of any species, preferably human.
  • the cells are preferably free of the common mitochondnal deletion mutation found clinically m patients with KSS syndrome (i.e. deleted 4799bp region at nt 8470-13,447; see Simonnetti et al, 1992) and any other pathologic mitochond ⁇ al DNA mutation.
  • Stem cells used to prepare the rephcative mitochond ⁇ a can be genetically modified by genetic engineering techniques.
  • a transgene may be introduced mto the cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, hpofection, electroporation, or micromjection. Suitable methods for transforming and transfecting cells can be found m Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. (See also Nolta et al Blood. 1995 Jul 1 ,86(1):101-10; and Nolta et al Proc Natl Acad Sci U S A.
  • a transgene may be introduced mto cells using an appropnate expression vector including but not limited to cosmids, plasmids, or modified viruses (e g replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtamed usmg standard methods including cultunng the cells on a monolayer of virus-producing cells (Van der Putten, Proc Natl Acad Sci U S A. 1985 Sep;82(18):6148-52; Stewart et al (1987) EMBO J 6:383-388). Examples of genes that may be introduced into the stem cells include genes encoding cell death protectors such as Bcl-xL and McL- 1.
  • Cryoprotective methods can be used to maintain maximum viability of the rephcative mitochond ⁇ a.
  • Cryopreservation can be earned out in a medium contammg for example dimethylsulphoxide, ethylene glycol, or glycerol or sucrose with 1,2-propanediol, or the mitochondna can be vitnfied using cryoprotectants such as ethylene glycol and dimethyl sulphoxide.
  • the cryopreservation procedure involves cooling the mitochond ⁇ a in a cryoprotective solution to an appropnate temperature (e.g -176°)
  • Scanning and transmission electron microscopy can be used to assess the pu ⁇ ty and morphology of a preparation.
  • the preparation can be analyzed for membrane mitochondnal potential and the total number and concentration of functional mitochond ⁇ a present can be determined in accordance with conventional methods as described herem.
  • Rephcative ability of the mitochondna in a preparation can be determined usmg conventional techniques mcludmg restnction fragment polymorphism methods as desc ⁇ bed herem.
  • the present invention generally involves the use of rephcative mitochondna to enhance the developmental potential of animal oocytes, especially mammals, mcludmg sports, zoo, pet, and farm animals, in particular dogs, cats, cattle, pigs, horses, goats, buffalo, rodents (e.g. mice, rats, guinea pigs), monkeys, sheep, and humans.
  • rephcative mitochond ⁇ a are used to enhance the developmental potential of non-human recipient oocytes.
  • a method of the mvention involves removmg the oocytes from follicles m the ovary.
  • rephcative mitochondna are introduced into the oocytes, or the oocytes can be cryopreserved for storage m a gamete or cell bank. If the oocytes are not cryopreserved the oocytes should be treated m accordance with the method of the mvention preferably within 48 hours after aspiration. If the oocytes are frozen, they can be thawed when it is desired to use them and treated in accordance with a method of the invention.
  • Rephcative mitochond ⁇ a may be introduced mto the oocytes (or zygotes) by conventional micromjection techniques or by other techniques such as electrofiision of mitochond ⁇ a contained withm hposomes or other suitable means.
  • the oocytes are fertilized with suitable spermatozoa from the same species.
  • the fertilization can be earned out by known techniques mcludmg sperm injection.
  • Suitable human m vitro fertilization and embryo transfer procedures that can be used mclude m vitro fertilization (IVF) (Trounson et al. Med J Aust. 1993 Jun 21 ;158(12):853-7, Trouson and Leeton, m Edwards and Purdy, eds..
  • the methods and compositions of the invention can be used to mcrease the success rate of embryo development.
  • they can be used to reduce the detrimental effects of mitochond ⁇ al DNA mutations (e.g. deletion or missense mutations) or abnormal or deficient mitochondnal function in the progeny of an individual affected by such mutations or abnormal or deficient function, by introducing in oocytes or zygotes from the mdividual rephcative mitochond ⁇ a that compnses healthy mitochondna.
  • Mitochond ⁇ al DNA deletions or mutations usually result m impaired oxidative phosphorylation and clinical pathology related to muscle or neurologic tissues.
  • KSS Kearns- Sayre syndrome
  • progressive extemal ophthalmoplegia is the result of a common 4799 bp deletion (Holt et al, Ann Neurol.
  • the invention also contemplates unproved nuclear transfer methods usmg rephcative mitochondna.
  • Nuclear transfer methods or nuclear transplantation methods are known in the literature and are descnbed m for example, Campbell et al, The ⁇ ogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420.
  • This process generally requires collectmg immature (prophase I) oocytes from mammalian ovaries, and maturing the oocytes m a maturation medium pnor to fertilization or enucleation until the oocyte attains the metaphase II stage.
  • Metaphase II stage oocytes which have been matured in vivo, may also be used in nuclear transfer techniques.
  • Enucleation of the recipient oocytes may be earned out by known methods, such as desc ⁇ bed in U.S Pat. No. 4,994,384
  • metaphase II oocytes may be placed in HECM, optionally containing cytochalasin B, for immediate enucleation, or they may be placed in a suitable medium, (e.g an embryo culture medium), and then enucleated later, preferably not more than 24 hours later.
  • Enucleation may be achieved microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm (McGrath and Solter, Science, 220:1300, 1983), or usmg functional enucleation (see U.S. 5,952,222).
  • the recipient oocytes may be screened to identify those which have been successfully enucleated.
  • the recipient oocytes may be activated on, or after nuclear transfer using methods known to a person skilled in the art. Suitable methods include cultu ⁇ ng at sub-physiological temperatures, applymg known activation agents (e.g. penetration by sperm, elect ⁇ cal and chemical shock), increasing levels of divalent cations, or reducing phosphorylation of cellular protems (see U.S. 5, 496,720) .
  • activation agents e.g. penetration by sperm, elect ⁇ cal and chemical shock
  • increasing levels of divalent cations e.g. penetration by sperm, elect ⁇ cal and chemical shock
  • increasing levels of divalent cations e.g. penetration by sperm, elect ⁇ cal and chemical shock
  • reducing phosphorylation of cellular protems see U.S. 5, 496,720.
  • a nucleus of a donor cell is introduced into the enucleated recipient oocyte.
  • the donor cell nucleus may be obtamed from any mammalian cells.
  • Donor cells may be differentiated mammalian cells denved from mesoderm, endoderm, or ectoderm.
  • the donor cell nucleus may be obtained from epithelial cells, neural cells, epidermal cells, kera ⁇ nocytes, hematopoietic cells, melanocytes, chondrocytes, B- lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.
  • Suitable mammalian cells may be obtamed from any cell or organ of the body.
  • the mammalian cells may be obtamed from different organs mcludmg skm, lung, pancreas, liver, stomach, mtestme, heart, reproductive organ, bladder, kidney and urethra.
  • the nucleus of the donor cell is preferably membrane-bounded.
  • a donor cell nucleus may consist of an entire blastomere or it may consist of a karyoplast.
  • a karyoplast is an aspirated cellular subset including a nucleus and a small amount of cytoplasm bounded by a plasma membrane. (See Methods and Success of Nuclear Transplantation m Mammals, A. McLaren, Nature, Volume 109, June 21, 194 for methods for preparing karyoplasts).
  • Rephcative mitochondna is introduced mto the enucleated recipient oocyte.
  • the rephcative mitochondria is preferably de ⁇ ved from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, and most preferably from the same mdividual from which the donor cell nucleus is de ⁇ ved. Methods for preparing rephcative mitochond ⁇ a are desc ⁇ bed herem.
  • Donor cells may be propagated, genetically modified, and selected m vitro pnor to extracting the nucleus, or the rephcative mitochondna.
  • the nucleus of a donor cell and/or the rephcative mitochond ⁇ a may be introduced mto an enucleated recipient oocyte usmg micromampulation or micro-surgical techmques known m the art (see McGrath and Solter, supra).
  • the nucleus of a donor cell may be transfe ⁇ ed to the enucleated recipient oocyte by depositing an aspirated blastomere or karyoplast under the zona pellucida so that its membrane abutts the plasma membrane of the recipient oocyte. This may be accomplished using a transfer pipette. Similar methods may be used to introduce the rephcative mitochondria.
  • Fusion of the donor nucleus and the enucleated oocyte may be accomplished according to methods known m the art. For example, fusion may be aided or induced with viral agents, chemical agents, or electro-induced. Electrofusion involves providing a pulse of electricity sufficient to cause a transient breakdown of the plasma membrane. (See U.S. 4, 994,384). In some cases (e.g. with small donor nuclei) it may be preferable to inject the nucleus directly into the oocyte rather than usmg electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264- 267 (1994).
  • the clones produced usmg the nuclear transfer methods as descnbed herem may be cultured either in vivo (e g in sheep oviducts) or in vitro (e.g. in suitable culture medium) to the morula or blastula stage.
  • the resulting embryos may then be transplanted mto the ute ⁇ of a suitable animal at a suitable stage of estrus usmg methods known to those skilled m the art.
  • a percentage of the transplants will initiate pregnancies in the stn ⁇ ogate animals.
  • the offspring will be genetically identical where the donor cells are from a single embryo or a clone of the embryo.
  • Example 1 The following non-limiting examples are illustrative of the present invention: Example 1
  • Example 2 Assessment of mitochondrial function, mtDNA copy number and mtDNA deletion rates in human oocytes of various ages and in human embryos showing preimplantation developmental defects.
  • oocytes and embryos will be incubated with a fluorochrome (DePsipher, R&D Systems) that allows simultaneous detection of mitochond ⁇ a with disrupted (non- functional) and maintained mitochondnal potential.
  • Samples will be analyzed usmg a deconvolution microscope and the amount of fluorescence will be recorded using Delta Vision software package (Silicon Graphics).
  • the dye In dy g cells or those with disrupted membrane potential, the dye will remain in its monomenc form in the cytoplasm and the mitochond ⁇ a will appear green, whereas m healthy cells the dye aggregates m the mitochondna will appear red.
  • this technique can be used to estimate mitochondnal copy number based on the total amount of fluorescence emitted on both channels.
  • the immature (GV and MI stage) oocytes obtamed from the ICSI program, unfertilized oocytes from IVF, and spare embryos donated to research will be analyzed.
  • C/ mtDNA deletions Although the above studies will determine the viability and abundance of the mitochondna, a further assessment can be done using PCR to semi-quantitatively assess mtDNA deletions in the same population of human oocytes and embryos used above. Different PCR p ⁇ mer sets, encompassmg all regions of the mitochondnal chromosome, have been designed and the proportion of mitochondria with a deletion m any part of the chromosome will be determined usmg the approach of Zhang et al. (Biochem Biophys Res Commun 1996 Jun 14,223(2).450-5).
  • ES and TS cells will be grown in vitro under standard culture conditions (Hadjantonakis et al Mech Dev. 1998 Aug;76(l-2)-79-90, Tanaka et al Science.
  • the nucleated cells obtained from human umbilical cord blood of healthy donors will be isolated using a Ficoll gradient.
  • CD34+/CD38- cells will be separated usmg a cell depletion magnetic column.
  • Equivalent (but adult rather than fetal) cells can also be obtained from munne bone manow of adult animals (Ploemacher et al Exp Hematol 1989 Mar, 17(3).263-6)
  • the somatic cell source will be luteimzed granulosa/cumulus cells isolated from folhcular fluid during oocyte retneval for IVF or from ova ⁇ es of hormonally pruned mice (Trbovich et al Cell Death Differ.
  • mitochondnal fraction can be isolated from all stem cell types and from granulosa cells usmg the method of Rickwood (Darley-Usmer VM., Rickwood D, Willson MT Mitochondna, a Practical Approach, Oxford Washmgton DC, IRL Press,
  • cells are suspended m a sucrose-based buffer and lysed usmg a glass homogenizer.
  • the nuclei are pelleted and the mitochondnal fraction is further ennched and punfied usmg a continuous Percoll gradient to separate damaged from intact mitochond ⁇ a and to eliminate most cellular deb ⁇ s Scanning and transmission electron microscopy will be used to assess the pu ⁇ ty and morphology of the mitochond ⁇ al fraction.
  • the maintenance of membrane mitochond ⁇ al potential will be analyzed by DePsipher dye as desc ⁇ bed above m Example 1, coupled with FACS analysis for rapid calculation of the total number and concentration of both functional and damaged mitochond ⁇ a present.
  • Ovulated oocytes will be snipped of their cumulus cells and will be injected with mitochondna ennched fraction m a dose response fashion accordmg to the technique of Van Blerkom et al . (Hum Reprod. 1998 Oct;13(10):2857-68). It has been estimated that mature oocytes contain about 100,000 mitochondna (Jansen and de Boer, Mol Cell Endocnnol. 1998 Oct 25;145(l-2):81-8). Between 2000 and 20,000 mitochondna m a volume of 5 to 15 picoL will be mjected.
  • a control group of oocytes will be left intact or mjected with either buffer used for suspension of mitochondna, or with the mitochond ⁇ a depleted fraction. Damaged mitochond ⁇ a obtamed from the percoll gradient will also be injected to determine possible negative effects of damaged mitochond ⁇ a on oocyte survival. All oocytes will then be cultured and scored for fragmentation at 24 and 48 hours. This model will be used to confirm the optimal number and type of mitochondria to inject to protect against fragmentation Expected Outcome- It is expected that mitochond ⁇ a de ⁇ ved from stem cells will be successful in preventing fragmentation, and will have the benefit of potential rephcative ability. b) Does injection of mitochondria from stem cells into normal mouse zygotes fertilized in vitro provide long-lasting protection from cell death ?
  • zygotes from aged mice will be mjected with an ennched fraction of mitochondna and then- development to the blastocyst stage will be observed in vitro.
  • the number of mitochondna to be injected will be estimated usmg the methods set out m the previous experiment, and the concentration will be fine tuned if necessary.
  • blastocyst cell numbers and cell death rates will be recorded, with particular attention to the inner cell mass.
  • DMBA which have all been shown to activate the cell death pathway during blastocyst formation, will be investigated.
  • zygotes injected with appropnate mitochondna will be cultured in KSOM medium until they reach the early blastocyst stage, when the experimental treatment will be performed m vitro with either doxorubicin (200nM), glucose (30mM) ennched medium or with DMBA ( l ⁇ M).
  • Zygotes injected with buffer or with mitochondna-depleted fractions that develop to the blastocyst stage will be used as controls.
  • blastocyst cell number and cell death mdex will be determined as previously descnbed (Junsicova et al . 1998, supra). Expected outcome. Somatic cell mitochondria have been shown to be diluted out by subsequent cell divisions of preimplantation embryos, and are non-detectable by the blastocyst stage (Ebert et al l 989, J Reprod Fertil Jan, 82(1) 145-9 9) Stem cell mitochondria should behave more like oocyte mitochondria, which have been demonstrated by Van Blerkom et al (Hum Reprod. 1998 Oct,13(10):2857-68) to be detectable at least 80 hours after injection mto mouse oocytes.
  • FVB zygotes will be injected with va ⁇ ous stem or somatic cell mitochond ⁇ a-en ⁇ ched fractions as described above and transfened into pseudopregnant females. At least 20 progeny in each group will be obtained The offspnng will be followed over an 18-month penod for detection of any developmental abnormalities, reproductive dysfunction, or reduced life span, that might be attributable to a deleterious effect of donor mitochondria injection on pre and postnatal development.
  • RFLP restriction fragment length polymorphism
  • the rephcative potential of injected mitochondna can then be confirmed m the offspnng by determining the RFLP status of the isolated mitochondna. Expected outcome.
  • the offspring created by donor stem-cell mitochond ⁇ al injection should be phenotypically normal, with normal hfespan. These mice may have improved reproductive function, and decreased oocyte apoptosis in vitro, if the donor mitochondna are rephcative and capable of creating heteroplasmy.
  • the ability to create heteroplasmy is c ⁇ tical to the success of any future clinical studies aimed at correcting he ⁇ table mitochond ⁇ al diseases.
  • mice when mated to these treated males, produce embryos with a high rate of fragmentation and low pregnancy rates secondary to chromosomal damage (Doerksen and Trasler, 1996, supra).
  • 5-AZC 5-AZC (4 mg/kg for 3 weeks)
  • sperm will be collected from the cauda epididimus and mjected together with stem cell mitochondna or buffer mto the oocytes of FVB strain mice.
  • Transfected lines will be selected based on their resistance to neomycm and will be assessed for protem levels of Mel- 1 or Bcl-x L within their mitochond ⁇ al fraction usmg western blot analysis.
  • Cytochrome C another mitochond ⁇ al- localized protem, will be used as a loadmg control m order to show enhanced levels of Bcl-xL and Mcl-1 m mitochondna ennched fractions.
  • mitochond ⁇ a Upon establishing mcreased levels of protem expression on the mitochond ⁇ al membranes within these cells, mitochond ⁇ a will be isolated and used in similar experiments to those descnbed above. Therefore, early embryos can be augmented with more functional mitochondria, but also with mitochondna containing a higher protem content of either Bcl- x L or Mcl-1.
  • Example 5 Injection of mitochondria into human oocytes at the time of ICSI and rescue of fragmented embryos.
  • Oocytes from each patient will be divided mto two groups. Oocytes m group one will be mjected with a smgle speim as previously descnbed (Casper et al , 1996, supra). Oocytes m group 2 will be mjected with a smgle sperm aspirated mto the injection pipette together with between 5,000 and 20,000 intact mitochondna from human umbilical cord blood- derived hematopoetic stem/progenitor cells prepared as descnbed above.
  • oocytes will be transfe ⁇ ed mto a 100 ⁇ l droplet of HTF medium supplemented with 5% human serum albumin m a plastic 60 x 15 mm petn dish, covered with mineral oil and mcubated m a humidified 5% CO, environment at 37°C Cultured oocytes will be assessed for the presence of two pronuclei, indicative of normal fertilization at 16-18 h after ICSI. Embryo development and gradmg according to the method of Veeck (1991; Acta Eur Fertil. 1992 Nov-Dec;23(6):275-88) will be performed daily.
  • the embryo score (cell number X 1/grade) will be determined for each embryo at 48, and 72 hours, and cell number estimated at 96 and 120 hours. Morphologically normal appearing expanded blastocysts will be transferred at day 5 post-fertilization. If normal embryo development occurs in any of the control injected oocytes, they will be transfe ⁇ ed first. The pregnancies obtamed by this technique will be followed closely and the patients advised to consider amniocentesis to rule out a gross chromosomal abnormality. Babies born as a result of this procedure will have their cord blood collected and stored for determination of mitochondnal heteroplasmy if possible (le.
  • a mtDNA mutation is detected in the unfertilized oocytes), and which may be responsible for the embryo fragmentation or delayed development seen initially in these patients.
  • the babies will also be followed with assessment for normal development at birth, and at mtervals thereafter for as long as the parents agree.
  • Group 1 oocytes should result m embryos with delayed development or which are completely fragmented, consistent with the patient's past history.
  • group 2 oocytes injection of an ennched fraction of stem cell mitochondna will allow normal development to the blastocyst stage with lntraute ⁇ ne transfer and pregnancy in some patients.

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Abstract

L'invention concerne des compositions et des procédés améliorant le potentiel de développement d'oocytes ou de zygotes par augmentation des niveaux intracellulaires de mitochondries réplicatives dans les oocytes ou les zygotes. Selon un aspect de l'invention, les niveaux intracellulaires de mitochondries réplicatives sont augmentés par introduction de mitochondries réplicatives dans les oocytes ou les zygotes. Les oocytes peuvent être fécondés afin d'obtenir un zygote à niveaux intracellulaires augmentés de mitochondries réplicatives. On peut utiliser les procédés et compositions selon l'invention pour améliorer les procédés de fécondation in vitro et de transfert d'embryon, ainsi que les techniques de transfert de matériel nucléaire.
EP00972510A 1999-10-27 2000-10-27 Procedes et compositions ameliorant le potentiel de developpement d'oocytes et de zygotes Withdrawn EP1234021A2 (fr)

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WO2019113743A1 (fr) * 2017-12-11 2019-06-20 清华大学 Procédé de modification génétique

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CA2468171C (fr) 2001-11-15 2015-10-06 Children's Medical Center Corporation Techniques d'isolation, de developpement et de differenciation de cellules souches provenant de villosites choriales, de liquide amniotique ainsi que de placenta et applications therapeutiques
GB0200804D0 (en) * 2002-01-14 2002-03-06 Univ Birmingham Cloning methods and other methods of producing cells
US7955846B2 (en) 2004-05-17 2011-06-07 The General Hospital Corporation Compositions comprising female germline stem cells and methods of use thereof
CA3009909A1 (fr) 2004-05-17 2005-12-22 The General Hospital Corporation Compositions comprenant des cellules souches germinales femelles et leurs procedes d'utilisation
AU2014202447B2 (en) * 2011-04-14 2015-05-07 The General Hospital Corporation Compositions and methods for autologous germline mitochondrial energy transfer
CA2832336C (fr) 2011-04-14 2016-08-09 The General Hospital Corporation Compositions et methodes de transfert d'energie autologue des mitochondries dans les cellules germinales
US9845482B2 (en) * 2011-06-29 2017-12-19 The General Hospital Corporation Compositions and methods for enhancing bioenergetic status in female germ cells
CN114214270B (zh) * 2021-12-17 2023-11-24 中国农业科学院北京畜牧兽医研究所 一种调控冷冻牛卵母细胞的发育能力的方法及其应用

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