EP1190086A2 - Modification genetique de cellules germinales males pour la production d'especes transgeniques et aux fins de therapies geniques - Google Patents

Modification genetique de cellules germinales males pour la production d'especes transgeniques et aux fins de therapies geniques

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Publication number
EP1190086A2
EP1190086A2 EP00937536A EP00937536A EP1190086A2 EP 1190086 A2 EP1190086 A2 EP 1190086A2 EP 00937536 A EP00937536 A EP 00937536A EP 00937536 A EP00937536 A EP 00937536A EP 1190086 A2 EP1190086 A2 EP 1190086A2
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European Patent Office
Prior art keywords
vertebrate
cell
male
transgenic
promoter
Prior art date
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EP00937536A
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German (de)
English (en)
Inventor
Carol W. Readhead
Robert Winston
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.)
Ip2ipo Innovations Ltd
Cedars Sinai Medical Center
Original Assignee
Imperial College Innovations Ltd
Cedars Sinai Medical Center
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Priority claimed from US09/311,599 external-priority patent/US6734338B1/en
Application filed by Imperial College Innovations Ltd, Cedars Sinai Medical Center filed Critical Imperial College Innovations Ltd
Publication of EP1190086A2 publication Critical patent/EP1190086A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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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
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to the medical arts, particularly to the field of transgenics and gene therapy.
  • the invention is particularly directed to in vitro and in vivo methods for genetically modifying male germ cells and support cells (i.e., Leydig and Sertoli cells), which methods incorporate a method of incorporating exogenous genetic material into the genome of a vertebrate to produce transgenic vertebrates and transgenic vertebrate animal lines.
  • transgenics The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction. This technology has been used to produce models for various diseases in humans and other animals. Transgenic technology is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. It is also used to study the relationship between genes and diseases. About 5,000 diseases are caused by a single genetic defect. More commonly, other diseases are the result of complex interactions between one or more genes and environmental agents, such as viruses or carcinogens. The understanding of such interactions is of prime importance for the development of therapies, such as gene therapy and drug therapies, and also treatments such as organ transplantation. Such treatments compensate for functional deficiencies and/or may eliminate undesirable functions expressed in an organism.
  • Transgenesis has also been used for the improvement of livestock, and for the large scale production of biologically active pharmaceuticals.
  • transgenic animals have been produced almost exclusively by microinjection of the fertilized egg.
  • the pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic DNA or hybrid DNA molecules.
  • the microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female.
  • P.J.A Krimpenfort et al, Transgenic mice depleted in mature T-cells and methods for making transgenic mice, U.S. Pat. Nos. 5,175,384 and 5,434,340; P.J.A.
  • mice While small animals such as mice have proved to be suitable models for certain diseases, their value in this respect is limited. Larger transgenic animals would be much more suitable than mice for the study of the effects and treatment of most human diseases because of their greater similarity to humans in many aspects, and better for studying organ systems or behavior. Larger mammals are also more suitable than mice as potential organ donors to humans due to the comparable size of their organs. Now that transgenic animals with the potential for human xenotransplantation are being developed, more of these larger animals will be required. Transgenic technology will allow that such donor animals will be immunocompatible with the human recipient.
  • transgenic mice have not been validated (Brinster, R. L., et al, No simple solution for making transgenics, Cell 59:239-241 [1989]). Recently, transgenic mice were produced after the injection of exogenous DNA together with sperm heads into oocytes ( Perry, A.C., et al, Mammalian transgenesis by intracytoplasmic sperm injection, Science 284: 1180- 1183 [1999]). Following uterine transfer, 20% of these embryos developed into transgenic offspring.
  • the vector was introduced into the perivitelline space of metaphase II oocytes (Chan, A.W., et al, Transgenic cattle produced by reverse-transcribed gene transfer in oocytes, Proc. Natl. Acad. Sci. USA 95: 14028-14033 [1998]).
  • spermatogenesis is the process by which a diploid spermatogonial stem cell provides daughter cells which undergo dramatic and distinct morphological changes to become self- propelling haploid cells (male gametes) capable, when fully mature, of fertilizing an ovum.
  • primordial germ cells are first seen in the endodermal yolk sac epithelium at E8 and are thought to arise from the embryonic ectoderm (A. McLaren and M. Buehr, Cell Diff. Dev. 31: 185 [1992]; Y. Matsui etal, Nature 353:750 [1991]). They migrate from the yolk sac epithelium through the hindgut endoderm to the genital ridges and proliferate through mitotic division to populate the testis.
  • spermatogonium goes through 5 or 6 mitotic divisions before it enters meiosis.
  • the primitive spermatogonial stem cells (AO/As) proliferate and form a population of intermediate spermatogonia types Apr, Aal, A 1-4 after which they differentiate into type B spermatogonia.
  • the type B spermatogonia differentiate to form primary spermatocytes which enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine.
  • the states of meiosis that are morphologically distinguishable are; preleptotene, leptotene, zygotene, and pachytene; secondary spermatocytes and the haploid spermatids are later stages.
  • Spermatids undergo great morphological changes during spermatogenesis, such as reshaping the nucleus, formation of the acrosome and assembly of the tail (A.R. Bellve et al. , Recovery, capacitation, acrosome reaction, and fractionation of sperm, Methods Enzymol. 225: 113-36 [1993]).
  • the spermatocytes and spermatids establish vital contacts with the Sertoli cells through unique hemi-junctional attachments with the Sertoli cell membrane.
  • the final changes in the maturing spermatozoan i.e., spermatozoon
  • testis of the recipient vertebrate of untransfected male germ cells before transferring transfected male germ cells into it.
  • Depopulation of testis has commonly been done by exposing the whole vertebrate to gamma irradiation (X-ray), or localizing irradiation to the testis.
  • X-ray gamma irradiation
  • G. Pinon-Lataillade et ah Endocrinological and histological changes induced by continuous low dose gamma-irradiation of rat testis, ActaEndocrinol. (Copenh) 109(4):558-62 [1985]; G.
  • Kangasniemi et ah Cellular regulation of basal and FSH- stimulated cyclic AMP production in irradiated rat testes, Anat. Rec. 227(l):32-36 [1990]; G. Pinon- Lataillade et al. , Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions, Reprod. Nutr. Dev. 31(6):617-29 [1991]).
  • Another method of depopulating a vertebrate testis is by administering a composition containing an alkylating agent, such as busulfan (Myleran).
  • an alkylating agent such as busulfan (Myleran).
  • Myleran alkylating agent
  • F.X. Jiang Behaviour of spermatogonia following recovery from busulfan treatment in the rat, Anat. Embryol. 198(1):53-61 [1998]
  • L.D. Russell and R.L. Brinster Ultrastructural observations of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules, J. Androl. 17(6):615-27 [1996]; N.
  • the present invention addresses the need for spermatogenic genetic modification, either in vitro or in vivo, that is highly effective in transferring allogeneic as well as xenogeneic genes into the animal' s germ cells and in producing transgenic vertebrate animals.
  • the present technology addresses the requirements of germ line and stem cell line gene therapies in humans and other vertebrate species, including the need for a superior method of depopulating a testis of untransfected male germ cells.
  • the present technology is of great value in producing transgenic animals in large species as well as for repairing genetic defects that lead to male infertility. Male germ cells that have stably integrated the DNA are selectable.
  • the present invention arose from a desire by the present inventors to improve on existing methods for the genetic modification of an animal's germ cells and for producing transgenic animals.
  • the pre-existing art methods rely on direct injection of DNA into zygotes produced in vitro or in vivo, or by the production of chimeric embryos using embryonal stem cells incorporated into a recipient blastocyst. Following this, such treated embryos are transferred to the primed uterus or oviduct.
  • These prior methods are extremely slow and costly, rely on several invasive steps, and only produce transgenic progeny sporadically and unpredictably.
  • the present inventors devised the present method which relies on the in vivo or in vitro (ex vivo) genetic modification of vertebrate male germ cells with a nucleic acid segment, i.e., a polynucleotide, encoding a desired trait or product.
  • a nucleic acid segment i.e., a polynucleotide
  • the present invention relates to the in vivo and in vitro (ex vivo) genetic modification, for example, by transfection or transduction, of vertebrate animal germ cells with a desired genetic material.
  • the in vivo method involves injection of genetic material together with a suitable vector directly into the testicle of the animal.
  • all or some of the male germ cells within the testicle are genetically modified in situ, under effective conditions.
  • the in vitro method involves obtaining germ cells from the gonad (i.e., testis) of a suitable donor or from the animal's own testis, using a novel isolation or selection method, transfecting or otherwise genetically altering them in vitro, and then returning them to the substantially depopulated testis of the donor or of a different recipient male vertebrate under suitable conditions where they will spontaneously repopulate the depopulated testis.
  • the in vitro method has the advantage that the transfected germ cells can be screened by various means before being returned to the testis of the same or a different suitable recipient male to ensure that the transgene is incorporated into the genome in a stable state. Moreover, after screening and cell sorting only enriched populations of germ cells can be returned. This approach provides a greater chance of transgenic progeny after mating.
  • the inventive in vivo method of incorporating exogenous genetic material into the genome of a vertebrate involves administering to a male vertebrate's testis(es) a gene delivery mixture comprising a viral vector, such as, but not limited to, a retroviral vector, that comprises at least one polynucleotide defining a gene encoding a desired trait or product and, optionally, a gene encoding a genetic selection marker.
  • a viral vector such as, but not limited to, a retroviral vector, that comprises at least one polynucleotide defining a gene encoding a desired trait or product and, optionally, a gene encoding a genetic selection marker.
  • the gene(s) are operatively linked to a promoter sequence (all the individual genes used are not necessarily linked to a single promoter sequence), such that a transcriptional unit is formed, and are administered under conditions effective to reach at least one of the spermatozoa, or precursors of spermatozoa, residing in the vertebrate's testis.
  • the delivery mixture, including the polynucleotide(s) are administered in amounts and under conditions effective such that a polynucleotide encoding a desired trait or product is incorporated into the genome of at least one male germ cell, such as a spermatozoan or precursor cell, so that a genetically modified male gamete is produced by the male vertebrate.
  • the invention also includes an in vitro method of incorporating at least one polynucleotide encoding a desired trait or product into the genome of a vertebrate.
  • the in vitro method involves obtaining from a donor male vertebrate a male germ cell, such as a spermatozoan cell or a precursor cell, and genetically modifying the cell in vitro with at least one polynucleotide encoding a desired trait or product other than an immortalizing molecule, and a polynucleotide defining a gene encoding a genetic selection marker, in the presence of a gene delivery mixture comprising a viral vector, at about or below the vertebrate's body temperature and for an effective period of time such that the polynucleotide encoding a desired trait or product is incorporated into the genome of the cell.
  • the genetically modified germ cell is isolated or selected, with the aid of the genetic selection marker expressed in the genetically modified cell, and transferred to a testis of a recipient male vertebrate such that the cell lodges in a seminiferous tubule of the testis, such that a genetically modified male gamete is produced therein.
  • the male vertebrate is bred with a female vertebrate of its species such that a transgenic progeny is thereby produced that carries the polynucleotide in its genome.
  • This invention also relates to a non-human transgenic male vertebrate produced in accordance with either the in vivo or in vitro method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate.
  • the transgenic vertebrate is the recipient of the gene delivery mixture.
  • the transgenic vertebrate is the recipient of the genetically modified male germ cell that was transferred to its testis.
  • the transgenic male vertebrate can be bred with a female of its species, because it comprises a native male germ cell carrying in its genome a polynucleotide of exogenous origin defining a gene encoding a desired trait or product. But somatic cells in tissues outside the testis of the transgenic vertebrate lack the polynucleotide.
  • This invention also relates to a non-human transgenic vertebrate produced in accordance with either the in vivo or in vitro method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate.
  • the non-human transgenic vertebrate is the direct or indirect progeny of the male vertebrate that received the gene delivery mixture, in accordance with the in vivo method.
  • the non-human transgenic vertebrate is the direct or indirect progeny of the recipient of the genetically modified male germ cell that was transferred to its testis, in accordance with the in vitro method.
  • the transgenic progeny is the immediate offspring of the transgenic male vertebrate, or is an offspring thereof separated by one or more generations.
  • the transgenic vertebrate includes one or more cells carrying in their genome a polynucleotide of exogenous origin that encodes a desired trait or product.
  • the invention includes a transgenic cell derived from the transgenic progeny.
  • the cell is a germ cell, such as a spermatozoan (i.e., spermatozoon) or ovum, a precursor cell of either of these, or a somatic cell.
  • the invention also relates to vertebrate semen containing a plurality of the inventive transgenic male germ cell.
  • the invention is also directed to a method of producing a non-human transgenic vertebrate animal line comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide.
  • the method involves breeding of the transgenic progeny with a member of the opposite sex of the same species; and selecting its progeny for the presence of the polynucleotide.
  • This technology is applicable to the production of transgenic animals for use as animal models, and to the modification of the genome of an animal, including a human, by addition, modification, or subtraction of genetic material, often resulting in phenotypic changes.
  • the present methods are also applicable to altering the carrier status of an animal, including a human, where that individual is carrying a gene for a recessive or dominant gene disorder, or where the individual is prone to pass a multigenic disorder to his offspring.
  • Figure 1 illustrates the process of microinjection of gene delivery mixture into a mammalian (mouse) testis.
  • Figure 1(a) shows a preferred site of microinjection into a vas efferens; in mammals, the vasa efferentia connect to the lumen of all seminiferous tubules.
  • Figure 1(b) shows a vas efferens supported by a pipette tip, 1mm diameter.
  • Figure 1(c) shows a mouse testis perfused with bromophenol blue after being injected in the vas efferens.
  • Figure 1(d) shows air bubbles in the testis, confirming satisfactory delivery of viral particles.
  • Figure 2 shows testicular cells transduced by a pseudotyped lentiviral vector expressing Green Fluorescent Protein (GFP) in Zeiss 410 confocal images (wavelength 488 nm; 19 stacked images) of a cryosection of mouse testis.
  • Figure 2(a) shows a transduced Sertoli cell expressing GFP.
  • Figure 2(b) shows transduced spermatogonia; GFP expression is visible in the cytoplasm surrounding large dark nuclei.
  • GFP Green Fluorescent Protein
  • Figure 3 shows a DNA analysis from three consecutive litters of progeny from one male treated in accordance with the in vivo method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate.
  • the top panel shows GFP-specific PCR amplification products separated on an agarose gel from embryonic DNA of 22 individual progeny. In this run, there was an absence of amplification from fetus No.2, but other PCR assays confirmed the presence of the transgenic reporter gene.
  • the bottom panel shows a Southern blot analysis of the same DNA. The Southern blot was probed with a radiolabed GFP DNA fragment.
  • a first method an in vivo method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate, delivers a polynucleotide using known gene delivery systems to male germ cells in situ in the testis of the male vertebrate (e.g., in vivo transfection or transduction), allows the genetically modified germ cells to differentiate in their own milieu, and then selects for progeny animals exhibiting the nucleic acid's integration into its germ cells (transgenic animals).
  • the thus selected progeny can be mated, or their sperm utilized for insemination or in vitro fertilization to produce further generations of transgenic progeny.
  • the in vitro method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate involves obtaining male germ cells from the testis of a suitable donor or from the animal's own testis, genetically modifying them in vitro, isolating or selecting genetically modified germ cells, and then transferring them to the testis under suitable conditions where they will spontaneously repopulate it.
  • the inventive method is suitable for application to a variety of vertebrate animals, all of which are capable of producing gametes, i.e. sperm or ova.
  • novel genetic modification(s) and/or characteristic(s) can be imparted to vertebrates, including mammals, such as humans, non-human primates, for example simians, marmosets, domestic agricultural animals such as ovines (e.g., sheep), bovines (e.g., cattle), porcines (e.g., pigs), equines (e.g., horses), particularly race horses, marine mammals, feral animals, rodents such as mice and rats, gerbils, hamsters, rabbits, and the like.
  • mammals such as humans, non-human primates, for example simians, marmosets, domestic agricultural animals such as ovines (e.g., sheep), bovines (e.g., cattle), porcines (e.g., pigs), equines (e.g., horses
  • vertebrate animals include fowl such as chickens, turkeys, ducks, ostriches, emus, geese, guinea fowl, doves, quail, rare and ornamental birds, and the like.
  • fowl such as chickens, turkeys, ducks, ostriches, emus, geese, guinea fowl, doves, quail, rare and ornamental birds, and the like.
  • endangered species of wild animal such as rhinoceros, tigers, cheetahs, species of condor, and the like.
  • Gene delivery (or transfection) mixture in the context of this patent, means selected genetic material together with an appropriate vector mixed, for example, with an effective amount of lipid transfecting agent, for example, a cationic or polycationic lipid, such as polybrene.
  • lipid transfecting agent for example, a cationic or polycationic lipid, such as polybrene.
  • a cationic or polycationic lipid such as polybrene.
  • the amount of each component of the mixture is chosen so that the genetic modification, e.g., by transfection or transduction, of a specific species of male germ cell is optimized. Such optimization requires no more than routine experimentation.
  • the ratio of DNA to lipid is broad, preferably about 1: 1, although other proportions can also be utilized depending on the type of lipid transfecting agent used. (E.g., Banerjee, R. et al. [1999] ; Jaaskelainen, I. et al. , A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides, Eur. J. Pharm. Sci.
  • Geneetic material means DNA sequences capable of imparting novel genetic modification(s), or biologically functional characteristic(s), to the recipient animal.
  • novel genetic modification(s) or characteristic(s) can be encoded by one or more genes or gene segments defined by a polynucleotide, or can be caused by removal or mutation of one or more genes, and can additionally contain regulatory sequences, such as, but not limited to enhancers, promoters, or activator/suppressor binding sites.
  • the transfected genetic material is preferably functional, that is it expresses a desired trait by means of a product or by suppressing the production of another.
  • genomic imprinting i.e. inactivation of one of a pair of genes (alleles) during very early embryonic development, or inactivation of genetic material by mutation or deletion of gene sequences, or by repression of a dominant negative gene product, among others.
  • the desired product is any preselected product other than an immortalizing molecule, such as
  • An immortalizing molecule can transform cells into "cancer-like" cells. "Immortalization” resulting from the expression of an immortalizing molecule can cause a male germ cell to lose many of its important germ cell characteristics, for instance the ability to undergo meiosis, which is crucial for the production of normally functioning male gametes. (E.g., see, Wolkowicz, M.J., Coonrod, S. M., Reddi, P.P. Millan, J. L., Hofmann, M-C, Herr, J.C.,
  • novel genetic modification(s) can be artificially induced mutations or variations, or natural allelic mutations or variations of a gene(s).
  • Mutations or variations can be induced artificially by a number of techniques, all of which are well known in the art, including chemical treatment, gamma irradiation treatment, ultraviolet radiation treatment, ultraviolet radiation, the use of specific chimeric DNA/RNA oligonucleotides (chimeraplasty), and the like.
  • Chemicals useful for the induction of mutations or variations include carcinogens such as ethidium bromide and others known in the art.
  • DNA segments of specific sequences can also be constructed to thereby inco ⁇ orate any desired mutation or variant or to disrupt a gene or to alter genomic DNA.
  • the genetic material is inheritable and is, therefore, present in almost every cell of future generations of the progeny, including the germ cells.
  • Transfecting agent means a composition of matter added to the genetic material for enhancing the uptake of exogenous DNA segment(s) into a eukaryotic cell, preferably a mammalian cell, and more preferably a mammalian germ cell.
  • the enhancement is measured relative to the uptake in the absence of the transfecting agent.
  • transfecting agents include adenovirus-transferrin-polylysine-DNA complexes. These complexes generally augment the uptake of DNA into the cell and reduce its breakdown during its passage through the cytoplasm to the nucleus of the cell.
  • These complexes can be targeted to the male germ cells using specific ligands which are recognized by receptors on the cell surface of the germ cell, such as the c-kit ligand or modifications thereof.
  • transfecting agents include lipofectin, lipfectamine, DLMR E C, Superfect, and Effectin (Qiagen), unifectin , maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE ( 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl- N,N-dihydroxyethylammonium bromide), polybrene, or poly(ethylenimine) (PEI).
  • non-viral agents have the advantage that they facilitate stable integration of xenogeneic DNA sequences into the vertebrate genome, without size restrictions commonly associated with virus-derived transfecting agents.
  • Virus means any virus, or transfecting fragment thereof, which can facilitate the delivery of the genetic material into male germ cells.
  • viruses which are suitable for use herein are adenoviruses, adeno-associated viruses, retroviruses such as human immune-deficiency virus, lentiviruses, mumps virus, and transfecting fragments of any of these viruses, and other viral DNA segments that facilitate the uptake of the desired DNA segment by, and release into, the cytoplasm of germ cells and mixtures thereof.
  • a preferred viral vector is Moloney murine leukemia virus and the retrovirus vector derived from Moloney virus called vesicular-stomatitis-virus- glycoprotein (VSV-G)-Moloney murine leukemia virus.
  • VSV-G vesicular-stomatitis-virus- glycoprotein
  • a most preferred viral vector is a pseudotyped (VSV-G) lentiviral vector derived from the HIV virus (Naldini et al. [1996]).
  • the mumps virus is particularly suited because of its affinity for immature sperm cells including spermatogonia. All of the above viruses may require modification to render them non-pathogenic or less antigenic.
  • Other known vector systems are also useful within the confines of the invention.
  • a gene delivery mixture involves the in vivo introduction of the gene delivery mixture to the germ cells by direct delivery into at least one of the animal's testes, where it is distributed to male germ cells at various stages of development.
  • the in vivo method employs injection of the gene delivery mixture, preferably into the seminiferous tubules, or into the rete testis, and most preferably into the vas efferens or vasa efferentia, using, for example, a micropipette.
  • the injection is made through the micropipette with the aid of a picopump delivering a precise measured volume under controlled amounts of pressure.
  • the micropipette is made of a suitable material, such as, metal or glass, and is usually made from glass tubing which has been drawn to a fine bore at its working tip, e.g. using a pipette puller. The tip can be angulated in a convenient manner to facilitate its entry into the testicular tubule system.
  • the micropipette can be provided with a beveled working end to allow a better and less damaging penetration of the fine tubules at the injection site.
  • This bevel can be produced by means of a specially manufactured grinding apparatus.
  • the diameter of the tip of the pipette for the in vivo method of injection is typically about 15 to 45 microns, although other sizes can be used as needed, depending on the animal's size.
  • the tip of the pipette can be introduced into the rete testis or the tubule system of the testicle, with the aid of a binocular microscope with coaxial illumination, with care taken not to damage the wall of the tubule opposite the injection point, and keeping trauma to a minimum.
  • a magnification of about 25x to 80x is suitable, and bench mounted micromanipulators are not severally required as the procedure can be carried out by a skilled artisan without additional aids.
  • a small amount of a suitable, non-toxic dye can be added to the gene delivery mixture (fluid) to confirm delivery and dissemination to the seminiferous tubules of the testis. It can include a dilute solution of a suitable, non-toxic dye, which can be visualized and tracked under the microscope. In this manner, the gene delivery mixture reaches and is brought into intimate contact with the male germ cells.
  • Male germ cells include spermatozoa (i.e., male gametes) and developmental precursors thereof.
  • primordial germ cells are thought to arise from the embryonic ectoderm, and are first seen in the epithelium of the endodermal yolk sac at the E8 stage. From there they migrate through the hindgut endoderm to the genital ridges.
  • spermatozoa In the sexually mature male vertebrate animal, there are several types of cells that are precursors of spermatozoa, and which can be genetically modified, including the primitive spermatogonial stem cells, known as AO/As, which differentiate into type B spermatogonia. The latter further differentiate to form primary spermatocytes, and enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine.
  • Useful precursor cells at several mo ⁇ hological/developmental stages are also distinguishable: preleptotene spermatocytes, leptotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, secondary spermatocytes, and the haploid spermatids.
  • the latter undergo further mo ⁇ hological changes during spermatogenesis, including the reshaping of their nucleus, the formation of acrosome, and assembly of the tail.
  • the final changes in the spermatozoan i.e., male gamete
  • the polynucleotide contained in the gene delivery mixture administered in the in vivo method to the testis will reach germ cells that are at any one of the above described stages, and will be taken up preferentially by those that are at a relatively more receptive stage.
  • the male germ cells are preferably, but not exclusively, diploid spermatogonia, which are exposed to or contacted with the gene delivery mixture.
  • the gene delivery mixture once in contact with the male germ cells, facilitates the uptake and transport of exogenous genetic material into the appropriate cell location for integration into the genome and expression.
  • a number of known gene delivery methods can be used for the uptake of nucleic acid sequences into the cell.
  • the gene delivery mixture typically comprises the polynucleotide encoding the desired trait or product, together with a suitable promoter sequence, and optionally agents which increase the uptake of or comprise the polynucleotide sequence, such as liposomes, retroviral vectors, adenoviral vectors, adenovirus enhanced gene delivery systems, or combinations thereof.
  • a reporter construct including a genetic selection marker, such as the gene encoding for Green Fluorescent Protein, can further be added to the gene delivery mixture.
  • Targeting molecules such as c-kit ligand, can be added to the gene delivery mixture to enhance the transfer of genetic material into the male germ cell.
  • An immunosuppressing agent such as cyclosporin or a corticosteroid can also be added to the gene delivery mixture as known in the art.
  • the male germ cells are obtained or collected from the donor male vertebrate, by means known in the art.
  • the thus obtained germ cells are then exposed to the gene delivery mixture, preferably within several hours, or cryopreserved for later use.
  • obtaining the male germ cells from the donor vertebrate can be accomplished by transection of the testes.
  • Transection of the isolated testicular tissue can be accomplished, for example, by isolation of the vertebrate's testes, decapsulation and teasing apart and mincing of the seminiferous tubules.
  • the separated cells can then be incubated in an enzyme mixture comprising enzymes known for gently breaking up the tissue matrix and releasing undamaged cells such as, for example, pancreatic trypsin, collagenase type I, pancreatic DNAse type I, as well as bovine serum albumin and a modified DMEM medium.
  • the cells can be incubated in the enzyme mixture for a period of about 5 min to about 30 min, more preferably about 15 to about 20 min, at a temperature of about 33 °C to about 37°C, more preferably about 36 to 37°C.
  • an incubation medium such as DMEM, and the like, and plated on a culture dish for genetic modification by exposure to the gene delivery mixture.
  • This transection method is not suitable when the donor and recipient male vertebrates are intended to be the same animal, in which case induced a less destructive biopsy method or induced ejaculation by means known in the art is preferred. Any of a number of commercially available gene delivery mixtures can be used, to which the polynucleotide encoding a desire trait or product is further admixed.
  • the final gene delivery mixture comprising the polynucleotide can then be admixed with the cells and allowed to interact for a period of about 2 hrs to about 16 hrs, preferably about 3 to 4 hrs, at a temperature of about 33 °C to about 37 °C, preferably about 36°C to 37 °C, and more preferably in a constant and/or controlled atmosphere.
  • the cells are preferably placed at a lower temperature of about 33°C to about 34°C, preferably about 30-35 °C for a period of about 4 hrs to about 20 hrs, preferably about 16 to 18 hrs.
  • Other conditions which do not deviate radically from the ones described can also be utilized as an artisan would know.
  • a most preferred embodiment employs a retroviral vector system, which was developed for gene therapy (Naldini, L., et ah, In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science 272: 263-267 [1996]), which is used to transduce male germ cells in vivo or in vitro.
  • This gene delivery system employs retroviral particles generated by a three-plasmid expression system.
  • a packaging construct contains the human cytomegalovirus (hCMV) immediate early promoter, driving the expression of all viral proteins.
  • the construct' s design eliminates the cis-acting sequences crucial for viral packaging, reverse transcription and integration of these transcripts.
  • the second plasmid encodes a heterologous envelope protein (env), namely the G glycoprotein of the vesicular stomatitis virus (VSV-G).
  • the third plasmid contains cis-acting sequences of human immunodeficiency virus (HIV) required for packaging, reverse transcription and integration, as well as unique restriction sites for cloning heterologous complementary DNAs (cDNAs).
  • HIV human immunodeficiency virus
  • cDNAs unique restriction sites for cloning heterologous complementary DNAs
  • a genetic selection marker such as the enhanced green fluorescent protein (EGFP), and/or a gene encoding another preselected or desired trait or product is cloned downstream of the hCMV promoter in the HR' vector, and is operatively linked so as to form a transcriptional unit.
  • EGFP enhanced green fluorescent protein
  • a gene encoding another preselected or desired trait or product is cloned downstream of the hCMV promoter in the HR' vector
  • a VSV-G pseudotyped retroviral vector system is capable of infecting a wide variety of cells including cells from different species and of integrating into the genome.
  • Some retroviruses i.e., lenti viruses, such as HF/, have the ability to infect non-dividing cells. They have a limited capacity for heterologous DNA sequences, the size limit for this vector being 7-7.5 kilobases (Verma, I.M. and Somia, N., Gene Therapy -promises, problems and prospects, Nature 389:239-242 [1997]).
  • a promoter sequence is operatively linked to a polynucleotide sequence encoding the desired trait or product.
  • "operatively linked" means that, within a transcriptional unit, the promoter sequence, is located upstream (i.e., 5' in relation thereto) from the coding sequence and the coding sequence, is 3' to the promoter, or alternatively is in a sequence of genes 3' to the promoter and expression is coordinately regulated thereby.
  • Both the promoter and coding sequences are oriented in a 5' to 3' manner, such that transcription can take place in vitro in the presence of all essential enzymes, transcription factors, co- factors, activators, and reactants, under favorable physical conditions, e.g., suitable pH and temperature. This does not mean that, in any particular cell, conditions will favor transcription. For example, transcription from a tissue-specific promoter is generally not favored in heterologous cell types from different tissues.
  • a promoter sequence is chosen that operates in the cell type of interest and/or under the physiologic or developmental conditions of interest.
  • Useful promoter sequences include constitutive promoters, such as, but not limited to, cytomegalovirus (CMV) promoter, or inducible promoters, such as, but not limited to, the human C-reactive protein (CRP) promoter (e.g., Kanzler, S., et ah, TGF- betal in liver fibrosis: an inducible transgenic mouse model to study liver fibro gene sis, Am. J.
  • CMV cytomegalovirus
  • CRP human C-reactive protein
  • IGF-I insulin-like growth factor
  • Useful promoters include those that promote transcription in cells of diverse tissues, such as, but not limited to, an insulin receptor (IR) gene promoter (e.g., Tewari, D.S., et ah, Characterization of the promoter region and 3' end of the human insulin receptor gene, J. Biol. Chem.
  • GHR growth hormone receptor
  • P2 or P3 promoters e.g., Jiang, H., et a , Isolation and characterization of a novel promoter for the bovine growth hormone receptor gene, J. Biol. Chem. 274(12):7893-900 [1999]
  • a leptin promoter e.g., Chen, XL., et ah, Analysis of a 762-bp proximal leptin promoter to drive and control regulation of transgene expression of growth hormone receptor in mice, Biochem. Biophys. Res. Commun. 262(1): 187-92 [1999]).
  • tissue-selective (i.e., tissue-specific) promoters i.e., promoters from which expression occurs preferentially in cells of a particular kind of tissue, compared to one or more other types of tissue.
  • tissue-specific promoters are particularly useful in applications directed to gene therapy or to the genetic enhancement of non-human vertebrates.
  • a promoter sequence which is only active in cycling spermatogonial stem cell populations can be used for differential expression in male germ cells, for example, B-Myb or a male germ cell-specific promoter, such as the c-kit promoter region, c-raf-1 promoter, ATM (ataxia- telangiectasia) promoter (also active in cerebellar cells and thymocytes), vasa promoter, cyclin A 1 promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, or FRMI (from fragile X site) promoter.
  • B-Myb or a male germ cell-specific promoter, such as the c-kit promoter region, c-raf-1 promoter, ATM (ataxia- telangiectasia) promoter (also active in cerebellar cells and thymocytes), vasa promote
  • useful promoters include cyclin Al promoters (e.g., Miiller, C, et ah, Cloning of the cyclin Al genomic structure and characterization of the promoter region, J. Biol. Chem. 276(16): 11220-28 [1999]); CD34 promoters (e.g., Burn, T.C., etal. , Hematopoietic stem cell specific gene expression, U.S. Patent No.
  • cyclin Al promoters e.g., Miiller, C, et ah, Cloning of the cyclin Al genomic structure and characterization of the promoter region, J. Biol. Chem. 276(16): 11220-28 [1999]
  • CD34 promoters e.g., Burn, T.C., etal. , Hematopoietic stem cell specific gene expression, U.S. Patent No.
  • a c-kit promoter e.g., Wilcox, D.A., et ah, Integrin alphal lb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus- transduced human hematopoietic cells, Proc. Natl. Acad. Sci. USA 96(17):9654-59 [1999]).
  • integrin alphallb promoter e.g., Wilcox, D.A., et ah, Integrin alphal lb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus- transduced human hematopoietic cells, Proc. Natl. Acad. Sci. USA 96(17):9654-59 [1999]).
  • Cartilage-selective promoters for expression in chondrocytes for example, an osteocalcin (OC) promoter (e.g., Newberry, E.P., et a , The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter, Biochemistry 38(33): 10678-90 [1999]); a SOX9 promoter, aggrecan gene promoter (AGC1), or collagen oligomeric matrix protein (COMP) gene promoter (e.g., Kanai, Y. & Koopman, P., Structural and functional characterization of the mouse Sox9 promoter: implications for campomelic dysplasia, Hum. Mol.
  • OC osteocalcin
  • AAC1 aggrecan gene promoter
  • COMP collagen oligomeric matrix protein
  • a cartilage-derived retinoic acid-sensitive protein (CD-RAP) gene promoter is also useful for cartilage-selective expression by chondrocytes.
  • CD-RAP cartilage-derived retinoic acid-sensitive protein
  • useful promoter sequences include, an albumin gene promoter (e.g., Pastore, L., et al. , Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors, Hum. Gen. Ther. 10(11): 1773-81 [ 1999]); a CYP7A or CYP7A1 promoter (e.g., Nitta, M., et ah, CPF: an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7 alpha-hydroxylase gene, Proc. Natl. Acad. Sci.
  • an albumin gene promoter e.g., Pastore, L., et al. , Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors, Hum. Gen. Ther. 10(11): 1773-81 [ 1999]
  • a CYP7A or CYP7A1 promoter e.g., Nit
  • a GHR Pl promoter e.g., Zou, L., et ah, Isolation of a liver-specific promoter for human growth hormone receptor gene, Endocrinology 138(4): 1771-74 [1997]; Jiang, H., et al. [1999]; Adams, T.E., Differential expression of growth hormone receptor messenger RNA from a second promoter, Mol. Cell Endocrinol.
  • TAFI thrombin-activatable fibrinolysis inhibitor
  • Neuronal specific promoters are also useful, for example, a neurofilament promoter or a neural-specific enolase promoter. Many other tissue specific promoters are useful for tissue specific expression of a preselected gene for phenotypic expression of a desired trait or product in the various tissues or organs of the vertebrate body.
  • cytokine-inducible proteins include promoters that regulate the expression of products and modulators of the Jak-STAT signaling cascade, for example, a SOCS-3 promoter (C.J. Auernhammer et a , Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter, Proc. Natl. Acad Sci. USA 96:6964-69 [1999]), a STAT-3 promoter (C. Bousquet & S. Melmed, J. Biol. Chem. 274:10723-30 [1999]), a POMC promoter (C.J. Auernhammer et al. [1998b]), or Spi 2.1 promoter (T.E. Adams et ah [1995]).
  • SOCS-3 promoter C.J. Auernhammer et a , Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter
  • Useful promoters also include exogenously inducible promoters. These are promoters that can be "turned on” in response to an exogenously supplied agent or stimulus, which is generally not an endogenous metabolite or cytokine. Examples include an antibiotic-inducible promoter, such as a tetracycline-inducible promoter; a heat-inducible promoter; a light-inducible promoter; or a laser- inducible promoter. (E.g., Halloran, M.C. et ah, Laser-induced gene expression in specific cells of transgenic zebrafish, Development. 127(9): 1953-1960 [2000]; Gerner, E.W.
  • et ah Heat-inducible vectors for use in gene therapy, Int. J. Hyperthermia 16(2): 171-81 [2000]; Rang, A., and Will, H., The tetracycline-responsive promoter contains functional interferon-inducible response elements, Nucleic Acids Res. 28(5): 1120-5 [2000]; Hagihara Y. et ah, Long-term functional assessment of encapsulated cells transfected with Tet-On system, Cell Transplant. 8(4):431-4 [1999]; Huang, C.J.
  • et ah Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system, Mol. Med. 5(2): 129-37 [1999]; Forster, K. et ah, Tetracycline-inducible expression systems with reduced basal activity in mammalian cells, Nucleic Acids Res. 27(2):708-10 [1999]; Liu, H.S. et ah, Lac/Tet dual-inducible system functions in mammalian cell lines, Biotechniques 24(4):624-8, 630-2 [1998]).
  • promoters include developmentally or temporally regulated promoters. Examples include the myelin P0 promoter (P. Thatikunta et ah, Reciprocal Id expression and myelin gene regulation in Schwann cells, Mol. Cell Neurosci. 14(6):519-28 [1999]), Gabra3 or GABRA3 promoters (W. Mu and D.R. Burt, the mouse GABA(A) receptor alpha3 subunit gene and promoter, Brain Res. Mol. Brain Res. 73(1-2): 172-80 [1999]), tyrosine hydroxylase promoter (J.J.
  • Schimmel et ah 4.5 kb of the rat tyrosine hydroxylase 5 'flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors, Brain Res. Mol. Brain Res. 74(1-2): 1-14 [1999]), vimentin promoters (A. Benazzouz and P. Duprey, The vimentin promoter as a tool to analyze the early events of retinoic acid-induced differentiation of cultured embryonal carcinoma cells, Differentiation (65(3): 171-80 [1999]), GATA-6 promoters (A. Brewer et ah, The human and mouse GATA-6 genes utilize two promoters and two initiation codons, J. Biol. Chem.
  • SH P1 or SHJP2 promoters S. Schurmans et ah, The mouse SH1P2 (Inppll)gene: complementary DNA, genomic structure, promoter analysis, and gene expression in the embryo and adult mouse, Genomics 62(2):260-71 [1999]
  • hGH-N promoters B.M. Shewchuk et ah, Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II, of the human growth hormone locus control region are essential for in vivo hGH-N gene activation, J. Biol. Chem.
  • the in vivo and in vitro methods of inco ⁇ orating exogenous genetic material into the genome of a vertebrate involve inco ⁇ orating the polynucleotide encoding a desired trait or product into the genome of at least one spermatozoan or precursor thereof, so that a genetically modified male gamete is produced by the male vertebrate.
  • the genetically modified germ cells of the vertebrate animal now transgenic, have the non-endogenous (exogenous) genetic material integrated into their chromosomes.
  • Isolating and/or selecting of genetically modified cells is by any suitable means, such as, but not limited to, physiological and/or mo ⁇ hological phenotypes of interest using any suitable means, such as biochemical, enzymatic, immunochemical, histologic, electrophysiologic, biometric or like methods; and analysis of cellular nucleic acids, for example the presence or absence of specific DNAs or RNAs of interest using conventional molecular biological techniques, including hybridization analysis, nucleic acid amplification (such as but not limited to, polymerase chain reaction [PCR], reverse transcriptase-mediated polymerase chain reaction [RT-PCR], transcription-mediated amplification [TMA], reverse transcriptase-mediated ligase chain reaction [RT-LCR]), and/or electrophoretic technologies.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-mediated polymerase chain reaction
  • TMA transcription-mediated amplification
  • R-LCR reverse transcriptase-mediated ligase chain reaction
  • the gene delivery mixture includes at least one polynucleotide comprising a gene encoding a genetic selection marker that is operatively linked to a promoter sequence such that a transcriptional unit is formed.
  • the promoter sequence can be the same or different from the promoter regulating the expression from the gene encoding the desired trait or product.
  • the genetic selection marker also known as a reporter gene, is, for example, a gene encoding an enzyme, such as ⁇ -galactosidase, or encoding a fluorescent protein, such as Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP), Yellow Fluorescent Protein, Blue Fluorescent Protein, a phycobiliprotein, such as phycoerythrin or phycocyanin, or any other protein which fluoresces under a suitable wave-length.
  • GFP Green Fluorescent Protein
  • EGFP enhanced Green Fluorescent Protein
  • Yellow Fluorescent Protein Yellow Fluorescent Protein
  • Blue Fluorescent Protein a phycobiliprotein, such as phycoerythrin or phycocyanin, or any other protein which fluoresces under a suitable wave-length.
  • Another preferred genetic selection marker or reporter gene suitable for some applications is a gene encoding a protein that can enzymatically lead to the emission of light from a substrate(s); for pu ⁇ oses of the present invention, such a protein is a "light-emitting" or luminescent protein.
  • a light-emitting protein includes proteins such as luciferase or apoaequorin.
  • Transgenic cells expressing a fluorescent or luminescent protein encoded by the reporter construct can be sorted with the aid of, for example, a flow activated cell sorter (FACS) set at the appropriate wavelength(s), or they can be selected by chemical methods.
  • FACS flow activated cell sorter
  • a preferred method of isolating or selecting male germ cell populations comprises obtaining specific male germ cell populations, such as spermatogonia, from a mixed population of testicular cells by extruding the cells from the seminiferous tubules and gentle enzymatic disaggregation.
  • the spermatogonia or other male germ cell populations can be isolated from a mixed cell population by a method including the utilization of a promoter sequence, which is specifically or selectively active in cycling male germ line stem cell populations, for example, B-Myb or a specific promoter, such as the c-kit promoter region, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, vasa promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, cyclin A 1 promoter, or FRMI (from fragile X site) promoter, linked to a reporter construct, for example, a construct comprising a gene encoding Green Fluorescent Protein (or EGFP), Yellow Fluorescent
  • the in vitro method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate in which male germ cells are obtained from a donor animal and genetically modified in vitro to impart a gene encoding a desired trait or product, male germ cells which exhibit any evidence that the DNA has been modified in the desired manner are isolated or selected, and transferred to the testis of a suitable recipient animal. After transfer, further selection can be attempted after biopsy of one or both of the recipient male' s testes, or after examination of the animal' s ejaculate amplified by the polymerase chain reaction to confirm whether the desired nucleic acid sequence was actually inco ⁇ orated.
  • the initial gene delivery can have included a reporter gene, such as a gene encoding the Green Fluorescent Protein, enhanced Green Fluorescent Protein (EGFP),
  • Yellow Fluorescent Protein Blue Fluorescent Protein
  • a phycobiliprotein such as phycoerythrin or phycocyanin, or any other protein which fluoresces under light of suitable wave-lengths, or encoding a light-emitting protein.
  • the genetically modified germ cells are preferably transferred to a testis of a recipient male vertebrate, which can be, but need not be, the same donor animal.
  • the testes of the recipient animal are preferably depopulated of native germ cells.
  • Substantial depopulation of the endogenous male germ cells facilitates the colonization of the recipient testis by the genetically modified germ cells from the donor animal.
  • the depopulation can be done by any suitable means, including by gamma irradiation, by chemical treatment, by means of infectious agents such as viruses, or by autoimmune depletion or by combinations thereof.
  • vertebrate testes are most preferably depopulated by a combined treatment of the vertebrate with an alkylating agent and gamma irradiation in accordance with the inventive method of substantially depopulating a vertebrate testes.
  • the method involves a treatment with a cytotoxic alkylating agent, such as, but not limited to, busulfan ( 1 ,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome), chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic acid, combined with gamma irradiation, to be administered in either sequence.
  • a cytotoxic alkylating agent such as, but not limited to, busulfan ( 1 ,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome), chlorambucil, cyclophosphamide, melphal
  • the combination of a dose of an alkylating agent and a dose of gamma radiation yields unexpectedly superior results in depopulating the testes of germ cells, compared to either treatment alone.
  • the dose of the alkylating agent and the dose of gamma radiation are in an amount sufficient to substantially depopulate the testis.
  • the preferred dose of alkylating agent is about 4 to 10 milligrams per kilogram of body weight, and about 6 to 8 milligrams per kilogram of body weight is most preferred.
  • the alkylating agent can be administered by any pharmaceutically acceptable delivery system, including but not limited to, intraperitoneal, intravenous, or intramuscular injection, intravenous drip, implant, transdermal or transmucosal delivery systems.
  • a recovery period between the administration of alkylating agent and irradiation is not essential, and the two treatments are most preferably done within zero to 24 hours of each other.
  • the time between the two treatments should not exceed 2 weeks, because this yields less than optimal results for pu ⁇ oses of transferring genetically modified or heterologous male germ cells to recipient testes.
  • the recipient vertebrate is gamma irradiated with a dose of about 200 to 800 Rads, most preferably about 350 to 450 Rads, directed locally to the testis to be depopulated. Less than 200 Rad yields little effect; greater than 800 Rad commonly produces symptoms of radiation sickness, particularly in the gastrointestinal tract.
  • male germ cells can be transferred thereto as described herein. Prior to three days, traces of cytotoxic alkylating agent or endogenous apoptotic signal molecules may remain in the recipient testis to harm the male germ cells transferred thereto. After two months, the endogenous population of male germ cells will typically begin to restablish itself, yielding less than optimal results when transfected, genetically altered, or heterologous male germ cells are transferred to a recipient testes for breeding pu ⁇ oses.
  • the gene delivery mixture is administered to the male germ cells of the donor vertebrate, in vitro, in sufficient amount and under effective conditions such that one or more of them is genetically modified.
  • Genetically modified male germ cells from the donor male vertebrate can then be transferred to the testis(es) of the recipient male such that they lodge in a seminiferous tubule of the testis, where they then mature into genetically modified gametes.
  • Transferring the isolated or selected genetically modified germ cells into the recipient testis can be accomplished by direct injection using a suitable micropipette.
  • Support cells such as Leydig or Sertoli cells that provide hormonal stimulus to spermatogonial differentiation, can be transferred to a recipient testis along with the modified germ cells.
  • These transferred support cells can be unmodified, or, alternatively, can themselves have been genetically modified, together with- or separately from the germ cells.
  • These transferred support cells can be autologous or heterologous to either the donor or recipient testis.
  • a preferred concentration of cells in the transfer fluid can easily be established by simple experimentation, but will likely be within the range of about 1 x 10 5 - 10 x
  • This micropipette can be introduced into the vasa efferentia, the rete testis or the seminiferous tubules, optionally with the aid of a picopump to control pressure and/or volume, or this delivery can be done manually.
  • the micropipette employed is in most respects similar to that used for the in vivo injection, except that its tip diameter generally will be about 45 to about 70 microns.
  • the microsurgical method of introduction is similar in all respects to that used for the in vivo method described above.
  • a suitable dyestuff or bubbles (less than 1 mm in diameter) can also be inco ⁇ orated into the carrier fluid for easy identification of satisfactory delivery of the transfected germ cells to at least one seminiferous tubule of the testis ( Figure 1).
  • breeding the male vertebrate with a female vertebrate of its species means causing the union of male and female gametes so that fertilization occurs and a transgenic zygote is formed; a transgenic progeny or offspring is thereafter produced during gestation of the developing fetus.
  • a union of male and female gametes is brought about by natural mating, i.e., copulation by the male and female vertebrates of the same species, or by in vitro or in vivo artificial means. If artificial means are chosen, then inco ⁇ orating into the genome a genetic selection marker that is expressed in male germ cells is particularly useful.
  • expression of the genetic selection marker is regulated from a constitutive or male germ-cell specific promoter, operatively linked to the gene encoding the genetic selection marker.
  • Artificial means include, but are not limited to, artificial insemination, in vitro fertilization (rVF) and/or other artificial reproductive technologies, such as intracytoplasmic sperm injection (ICSI), subzonal insemination (SUZI), or partial zona dissection (PZD).
  • ICSI intracytoplasmic sperm injection
  • SUZI subzonal insemination
  • PZD partial zona dissection
  • others such as cloning and embryo transfer, cloning and embryo splitting, and the like, can also be employed.
  • transgenic vertebrate progeny can, in turn, also be bred, whether by natural mating, artificial insemination, or by in vitro fertilization (IVF) and/or other artificial reproductive technologies, such as intracytoplasmic sperm injection (ICSI), subzonal insemination (SUZI), or partial zona dissection (PZD), to obtain further generations of transgenic progeny.
  • IVF in vitro fertilization
  • ICSI intracytoplasmic sperm injection
  • SUZI subzonal insemination
  • PZD partial zona dissection
  • a "transgenic" vertebrate is one that has had foreign or exogenous DNA permanently introduced into its cells.
  • the exogenous genes which have been introduced into the animal's cells are called “transgenes” and are xenogeneic and/or allogeneic transgenic genetic material, including biologically functional genetic material.
  • the present invention is applicable to the production of transgenic animals containing xenogeneic, i.e., exogenous DNA from a different species, either in its native, undisturbed form, or in artificially mutated form.
  • the genetic material is "allogeneic" genetic material, exogenous transgenic material obtained from a different strain, race, breed, or individual of the same species, for example, from an animal having a "normal” form of a gene, or a desirable allele, variant, or mutation thereof.
  • the gene can be a hybrid construct consisting of promoter DNA sequences and DNA coding sequences operatively linked together. These sequences can be obtained from different species or DNA sequences from the same species that are not normally juxtaposed.
  • the DNA construct can also contain DNA sequences from prokaryotic organisms, such as bacteria, or from viruses.
  • the transfected germ cells of the transgenic vertebrate animal preferably have the non-endogenous (exogenous) genetic material integrated into their chromosomes.
  • non-endogenous (exogenous) genetic material integrated into their chromosomes.
  • the transgenic vertebrate is the recipient of the gene delivery mixture.
  • the non-human transgenic male vertebrate is the recipient of the genetically modified male germ cell that was transferred to its testis, in accordance with the in vitro method.
  • the transgenic male vertebrate can be bred with a female of its species, because it comprises a native male germ cell carrying in its genome a polynucleotide of exogenous origin defining a gene encoding a desired trait or product.
  • transgenic male vertebrate will continue to produce genetically modified gametes for an indefinite period.
  • the transgenic state is temporary, lasting for at least several weeks or months, after which non-modified gametes are again exclusively or predominantly produced by the animal.
  • non-human transgenic vertebrate produced in accordance with the in vivo or in vitro method of inco ⁇ orating exogenous genetic material into the genome of a vertebrate, wherein the non-human vertebrate is the direct or indirect progeny of the transgenic male vertebrate described above.
  • this transgenic progeny is the immediate offspring, male or female, of the transgenic male vertebrate or is an offspring thereof separated by one or more generations.
  • the transgenic vertebrate includes one or more cells carrying in their genome a polynucleotide of exogenous origin that encodes a desired trait or product.
  • the invention includes a transgenic cell derived from the transgenic vertebrate progeny.
  • the cell is a germ cell, such as a spermatozoan or ovum, a precursor germ cell of either of these, or a somatic cell.
  • Male germ cells are obtained from a male animal's semen, or spermatozoa, spermatogonia, or immature spermatocytes are separated from whole biopsies of testicular tissue containing the male germ cells. Alternatively, male germ line stem cells can be isolated from embryonic tissue.
  • Female germ cells are obtained by known means, including hormonally induced "ripening" and harvesting from the oviducts or aspiration by way of the cervix or by way of a laparoscopic incision.
  • Somatic cells include stem cells.
  • a stem cell is an undifferentiated mother cell that is self- renewable over the life of the organism and is multipotent, i.e., capable of generating various committed progenitor cells that can develop into fully mature differentiated cell lines. (E.g., T. Zigova and P.R. Sanberg, The rising star of neural stem cell research, Nature Biotechnol. 16(11): 1007-08 [1998]). All vertebrate tissues arise from stem cells, including hematopoietic stem cells, from which various types of blood cells derive; ectodermal stem cells; neural stem cells, for example, neural progenitors from which brain and nerve tissues derive. Somatic cells also include progenitor cells or terminally differentiated cells of any kind associated with any tissue or organ of the vertebrate body.
  • Somatic cells are obtained by known sampling or biopsy means from any bodily tissue, organ, or fluid, including but not limited to, blood, heart, kidney, ureter, bladder, urethra, brain, thyroid, parotid gland, pancreas, hypothalamus, pituitary gland, submaxillary gland, sublingual gland, lymph node, bone, bone marrow, cartilage, lung, mediastinum, breast, uterus, ovary, testis, prostate, cervix uteri, endometrium, liver, spleen, adrenal, esophagus, stomach, intestine, hair root, muscle, nerve, urine, amniotic fluid, chorionic villus, skin, vascular or oral epithelium, or spinal fluid.
  • the inventive transgenic cells can be cultured or stored by well known means.
  • the invention also relates to vertebrate semen containing a plurality of the inventive transgenic male germ cell.
  • the inventive vertebrate semen is useful for breeding or other suitable pu ⁇ oses.
  • the semen is obtained from ejaculate produced by the inventive transgenic male vertebrate or its transgenic male progeny (either immediate progeny or progeny separated by one or more generations), and methods of inducing ejaculation by a male vertebrate and capturing the semen are well known.
  • the semen can be processed, e.g., by washing, and/or stored by means such as are known in the art.
  • storage conditions include the use of cryopreservation using programmed freezing methods and/or the use of cryoprotectants, for example, dimethyl sulfoxide (DMSO), glycerol, trehalose, or propanediol-sucrose, and the use of storage in substances such as liquid nitrogen.
  • cryoprotectants for example, dimethyl sulfoxide (DMSO), glycerol, trehalose, or propanediol-sucrose
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • trehalose trehalose
  • propanediol-sucrose propanediol-sucrose
  • the invention is also includes a method of producing a non-human transgenic vertebrate animal line, the individuals of which comprise native germ cells carrying in their genome at least one xenogeneic polynucleotide.
  • the transgenic vertebrates bred with other transgenic or non-transgenic animals of the same species will produce some transgenic progeny, including fertile individuals.
  • the method involves breeding of the fertile transgenic progeny with a member of the opposite sex of the same species as described above; and selecting its progeny for the presence of the polynucleotide.
  • the inventive method of producing a non-human transgenic vertebrate animal line is simple and efficient, and is more easily accomplished in large mammals than in mice because of the larger size of the testicular ducts.
  • the inventive technology is applicable to the field of gene therapy, since it permits the introduction of genetic material encoding and regulating specific genetic traits.
  • the inventive technology permits the introduction of genetic material encoding and regulating specific genetic traits.
  • by treating parents it is possible to correct many single gene disorders which otherwise might affect their children.
  • This technology can also be applied in a similar way to correct disorders in animals other than human primates.
  • the germ cells of the animal it may be necessary to introduce one or more "gene(s)" into the germ cells of the animal to attain a desired therapeutic effect, as in the case where multiple genes are involved in the expression or suppression of a defined trait.
  • multigenic disorders include diabetes mellitus caused by deficient production of, or response to, insulin, inflammatory bowel disease, certain forms of atheromatous cardiovascular disease and hypertension, schizophrenia and some forms of chronic depressive disorders, among others.
  • one gene can encode an expressible product, whereas another gene encodes a regulatory function, as is known in the art.
  • a specific reproductive application of the present invention is to the treatment of animals, particularly humans, with disorders of spermatogenesis.
  • Defective spermatogenesis or spermiogenesis frequently has a genetic basis, that is, one or mutations in the genome can result in failure of production of normal sperm cells. This can happen at various stages of the development of germ cells, and may result in male infertility or sterility.
  • the present invention is applicable, for example, to the insertion or inco ⁇ oration of nucleic acid sequences into a recipient's genome and, thereby, establish spermatogenesis in the correction of oligozoospermia or azoospermia in the treatment of infertility.
  • the present methods are also applicable to males whose subfertility or sterility is due to a motility disorder with a genetic basis.
  • the present invention is additionally applicable to the generation of transgenic animals expressing agents which are of therapeutic benefit for use in human and veterinary medicine or well being.
  • agents which are of therapeutic benefit for use in human and veterinary medicine or well being. Examples include the production of pharmaceuticals in domestic cows' milk, such as factors which enhance blood clotting for patients with types of haemophilia, or hormonal agents such as insulin and other peptide hormones.
  • the present method is further applicable to the generation of transgenic animals, for example pigs, of a suitable anatomical and physiological phenotype for human xenograft transplantation.
  • the inventive transgenic technology permits the generation of animals which are immune-compatible with a human recipient. Appropriate organs, for example, can be removed from such animals to allow the transplantation of, for example, the heart, lung and kidney.
  • male germ cells genetically modified in accordance with this invention can be obtained from the transgenic animal, and stored under conditions effective for later use, as is known in the art.
  • the adenovirus enhanced transferrin-polylysine-mediated gene delivery system has been described and patented by Curiel et al. (Curiel D.T.,et ah, Adenovirus enhancement of transferrin- polylysine-mediated gene delivery, PNAS USA 88: 8850-8854 (1991).
  • the delivery of DNA depends upon endocytosis mediated by the transferrin receptor (Wagner et ah, Transferrin-polycation conjugates as carriers for DNA uptake into cells, PNAS (USA) 87: 3410-3414 (1990).
  • this method relies on the capacity of adenoviruses to disrupt cell vesicles, such as endosomes and release the contents entrapped therein.
  • This system can enhance the gene delivery to mammalian cells by as much as 2,000 fold over other methods.
  • Human transferrin was conjugated to poly (L-lysine) using EDC (l-ethyl-3-(3- dimethyl aminopropyl carbodiimide hydrochloride) (Pierce), according to the method of Gabarek and Gergely (Gabarek & Gergely, Zero-length cross-linking procedure with the use of active esters, Analyt. Biochem 185 : 131 (1990)).
  • EDC l-ethyl-3-(3- dimethyl aminopropyl carbodiimide hydrochloride
  • Gabarek and Gergely Gabarek and Gergely, Zero-length cross-linking procedure with the use of active esters, Analyt. Biochem 185 : 131 (1990)
  • EDC reacts with a carboxyl group of human transferrin to form an amine-reactive intermediate.
  • the activated protein was allowed to react with the poly (L-lysine) moiety for 2 hrs at room temperature, and the reaction was quenched by adding
  • the Green Lantern- 1 vector (Life Technologies, Gibco BRL, Gaithersberg, MD) is a reporter construct used for monitoring gene transfection in mammalian cells. It consists of the gene encoding the Green Fluorescent Protein (GFP) driven by the cytomegalovirus (CMV) immediate early promoter. Downstream of the gene is a SV40 polyadenylation signal. Cells transfected with Green Lantern-1 fluoresce with a bright green light when illuminated with blue light. The excitation peak is 490 nm.
  • GFP Green Fluorescent Protein
  • CMV cytomegalovirus
  • Adenovirus dI312 a replication-incompetent strain deleted in the Ela region, was propagated in the Ela trans-complementing cell line 293 as described by Jones and Shenk (Jones and Shenk, PNAS USA (1979) 79: 3665-3669).
  • a large scale preparation of the virus was made using the method of Mittereder and Trapnell (Mittereder et al., "Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy", J. Urology, 70: 7498-7509 (1996)).
  • the virion concentration was determined by UV spectroscopy, 1 absorbance unit being equivalent to 10 viral particles /ml.
  • the purified virus was stored at -70 °C.
  • Example 5 Comparison of Adenovirus-enhanced Transferrin-polylysine & Lipofectin Mediated Transfection Efficiency
  • the conjugated adenovirus particle complexed with DNA were tested on CHO cells in vitro prior to in vivo testing.
  • a luciferase reporter gene was used due to the ease of quantifying luciferase activity.
  • the expression construct consists of a reporter gene encoding luciferase, is driven by the CMV promoter (Invitrogen, Carlsbad, CA 92008).
  • CHO cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum.
  • DMEM Dulbecco's modified Eagle's medium
  • CHO cells were seeded into 6 cm tissue culture plates and grown to about 50% confluency (5x 10 5 cells). Prior to transfection the medium was aspirated and replaced with serum free
  • DMEM fetal calf serum
  • Cells were either transfected with transferrin-polylysine-DNA complexes or with lipofectin DNA aggregates.
  • transferrin-polylysine mediated DNA transfer the DNA-adenovirus complexes were added to the cells at a concentration of 0.05-3.2 x 10 4 adenovirus particles per cell. Plates were returned to the 5 % C0 2 incubator for 1 hour at 37 ° C. After 1 hour 3 ml of complete media was added to the wells and the cells were allowed to incubate for 48 hours before harvesting. The cells were removed from the plate, counted and then lysed for measurement of luciferase activity.
  • Table 1 The data included in Table 1 below show that the adenovirus-enhanced transferrin-polylysine gene delivery system is 1,808 fold more efficient than lipofection for transfection of CHO cells.
  • Table 1 Comparison of Lipofection & Adenovirus Enhanced
  • Example 6 In Vivo Delivery of DNA to Animal's Germ Cells via Transferrin-L-lvsine-DNA-Viral Complexes
  • the GFP DNA-transferrin-polylysine viral complexes prepared as described in Example 4 above, were delivered into the seminiferous tubules of three (3)-week-old B6D2F1 male mice.
  • the DNA delivery by transferrin receptor-mediated endocytosis is described by Schmidt et a and Wagner et al. (Schmidt et ah, Cell 4: 41-51 (1986); Wagner, E., et al. PNAS (1990), (USA) 81: 3410-3414 [1990]).
  • this delivery system relies on the capacity of adeno viruses to disrupt cell vesicles, such as endosomes and release the contents entrapped therein. The transfection efficiency of this system is almost 2,000 fold higher than lipofection.
  • mice were anesthetized with 2% Avertin ( 100% Avertin comprises 10 g 2,2,2- tribromoethanol (Aldrich) and 10 ml t-amyl alcohol (Sigma), and a small incision made in their skin and body wall, on the ventral side of the body at the level of the hind leg.
  • Avertin 100% Avertin comprises 10 g 2,2,2- tribromoethanol (Aldrich) and 10 ml t-amyl alcohol (Sigma), and a small incision made in their skin and body wall, on the ventral side of the body at the level of the hind leg.
  • the animal's testis was pulled out through the opening by grasping at the testis fat pad with forceps, and the vas efferens tubules exposed and supported by a glass syringe.
  • the GFP DNA-transferrin-polylysine viral complexes were injected into a single vasa efferentia using a glass micropipette attached to a hand held glass syringe or a pressurized automatic pipettor (Eppendorf), and Trypan blue added to visualize the entry of the mixture into the seminiferous tubules. The testes were then placed back in the body cavity, the body wall was sutured, the skin closed with wound clips, and the animal allowed to recover on a warm pad.
  • GFP DNA was present in the testes of the animals that had received the DNA complexes, but was absent from sham operated animals.
  • RT PCR reverse transcriptase PCR
  • the DNA detected above by PCR analysis is episomal GFP DNA.
  • the transfected gene was being transiently expressed.
  • GFP transfected males were mated with normal females. The females were allowed to complete gestation, and the pups to be born. The pups (FI offspring or progeny) were screened for the presence of the novel genetic material(s).
  • the testes were decapsulated and the seminiferous tubules were teased apart and minced with sterile needles. The cells were incubated in enzyme mixture for 20 minutes at 37 °C.
  • the enzyme mixture was made up of 10 mg bovine serum albumin (embryo tested), 50 mg bovine pancreatic trypsin type IJJ, Clostridium collagenase type I, 1 mg bovine pancreatic DNAse type I in 10 mis of modified HTF medium (Irvine Scientific, Irvine, CA).
  • the enzymes were obtained from Sigma Company (St. Louis, Missouri 63178).
  • the cells were washed twice by centrifugation at 500 x g with HTF medium and resuspended in 250 ⁇ l HTF medium. The cells were counted, and 0.5 x 10 6 cells were plated in a 60mm culture dish in a total volume of 5ml DMEM (Gibco-BRL, Life Technologies, Gaithesburg, MD 20884).
  • a transfection mixture was prepared by mixing 5 ⁇ g Green Lantern DNA (Gibco-BRL, Life Technologies, Gaithesburg, MD 20884) with 20 ⁇ l Superfect (Qiagen, Santa Clarita, CA 91355) and 150 ⁇ l DMEM.
  • the transfection mix was added to the cells and they were allowed to incubate for 3 hours at 37 ° C, 5% C0 2 The cells were transferred to a 33 ° C incubator and incubated overnight.
  • the testicular cells transfected with Green Lantern viewed with Nomaski optics x20 show the same cells viewed with FITC. Nearly all the cells were fluorescent, which is confirmation of their successful transfection.
  • Example 11 Preparation of a Cell Suspension from Testicular Tissue for Cryopreservation A cell suspension was prepared from mice of different ages as described below. Group I: 7-10 day olds
  • mice' s testes were dissected, placed in phosphate buffered saline (PBS) decapsulated, and the seminiferous tubules were teased apart. Seminiferous tubules from groups I and II were transferred to HEPES buffered culture medium (D-MEM) (Gibco-BRL, Life Technologies, Gaithersberg, MD 20884) containing lmg/ml Bovine serum albumin (BSA) (Sigma, St. Louis, MO 63178) and Collagenase Type I (Sigma) for the removal of interstitial cells. After a 10 minute incubation at 33 C, the tubules were lifted into fresh culture medium.
  • D-MEM HEPES buffered culture medium
  • BSA Bovine serum albumin
  • Collagenase Type I Sigma
  • the enzyme mix was made up in D-DMEM with HEPES (Gibco-BRL) and consisted of lmg/ml bovine serum albumin (BSA) (Sigma, embryo tested), lmg/ml collagenase I (Sigma) and 5 mg/ml bovine pancreatic trypsin (Sigma) and 0. lmg/ml deoxyribonuclease I (DN-EP,
  • the cells were injected into the testis via the vasa efferentia using a micropipette. 3 x 10 5 cells in a total volume of 50 l were used for the injection. The cells were mixed with Trypan blue prior to the injection.
  • the recipient mice were anesthetized with 0.017mL/g body wt. Avertin. An incision was made across the lower abdominal wall and the testis was gently pulled to the exterior through the incision by pulling on the fat pad associated with the testis.
  • the vas efferens was exposed and approximately 20 ⁇ h of cell suspension was injected into the vas efferens using a glass micropipette held in a steel micropipette holder (Leitz). The cells were expelled from the pipette using air pressure from a 20 mL glass syringe. Prior to the transfer of transfected germ cells to the recipient animals, the recipient testes were depopulated of endogenous male germ cells.
  • Example 13 Depopulating the Recipient Testis of Male Germ Cells- Comparison of Depopulating Treatments. Eight-week-old C57BL/6J mice were allowed to acclimatize for a few days and then were assigned to one of the following three treatment groups. They received: (1) 400 Rad gamma irradiation; (2) 4 ⁇ G/g body weight of busulfan (1,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome); or (3) a combination treatment of busulfan (4 ⁇ g/g body wt) followed one week later by 400 Rad of gamma irradiation ("busulfan/400 Rad" treatment).
  • a fourth group of untreated C57BL/6J mice of the same age as the treatment groups was used as a control. There were 24 mice in each treatment group, and 3 mice were mice sacrificed at each of the following time intervals after treatment: 5 hours, 24 hours, 48 hours, 72 hours, 1 week, 2 weeks, 1 month and 2 months after treatment.
  • mice receiving the combined busulfan/400 Rad treatment were examined histologically at time points up to five months after treatment (the testes of these other mice were fixed overnight in 4% paraformaldehyde in PBS, pH 7.4 at 4°C, dehydrated and embedded in paraffin before sectioning and H&E staining).
  • the male mice receiving busulfan received a dose of 4 ⁇ g busulfan per g body wt.
  • the busulfan was first dissolved 8mg/mL in 100% dimethyl sulfoxide (DMSO) then, immediately before injection, was diluted 1: 1 in phosphate buffered saline, pH 7.4. The mice were injected with the diluted busulfan solution intraperitoneally.
  • DMSO dimethyl sulfoxide
  • mice were anesthetized with 0.017 mL/g body wt. of 2.5% Avertin.
  • Gamma irradiation was specifically directed to the testis in the following manner. Each mouse was placed in a lead chamber with only the testis and lower abdomen exposed through elliptical holes to the irradiating source ( 137 Cs Gammacell 40 irradiator [Nordion] ) . There were six aligned holes in the floor and roof of the chamber through which the gamma radiation passed unobstructed. After irradiation the animals were allowed to recover from the anesthesia on a warm heating pad or water bed until they regained consciousness.
  • mice from each treatment group were euthanized, and testicular tissues to be examined were fixed in 10% formalin in PBS, pH 7.4, at 4°C for 24 hours. Small slits in the testis capsule were made to allow penetration of the fixative. Fixed samples were washed four times with PBS, and embedded in paraffin using a Tissue Tek-II tissue processor (MET). Sections of 8 ⁇ m thickness were cut, stained with haemotoxylin and eosin (H&E), and mounted with Aquamount (Lerner Laboratories) on glass slides with coverslips. The sections were viewed on a Zeiss or Olympic light microscope with a 40X objective lens (total magnification 400X).
  • H&E haemotoxylin and eosin
  • Quantitative Histologic Analysis Quantitative data were collected from the testes of two animals for each of the treatment groups at two months after treatment. (Table 2). For the control group only one mouse was used. The seminiferous tubules in a single section were counted using a 5X objective on a Zeiss light microscope (50x total magnification). Individual seminiferous tubules were examined at 400X total magnification. Seminiferous tubules were considered severely damaged if hardly any cells remained in the tubule, and the tubule consisted of a basement membrane with a single layer of cells, mostly spermatogonia, lying along the basement membrane.
  • Example 14 In vivo Transduction Using a Viral Vector
  • a retroviral vector was used to transduce (genetically alter or modify) male germ cells of mice in vivo.
  • a pseudo-typed HlV-derived viral vector (L. Naldini et al. , In vivo gene delivery and stable transduction of nondividing cells by a Lentiviral vector, Science 272:263-67 [1996]), was used, as modified by Carlos Lois to express Green Flourescent Protein (GFP) instead of the LacZ reporter gene, under the transcriptional control of the CMV promoter (HR'GFP).
  • GFP Green Flourescent Protein
  • C57BL/6J mice were treated with busulfan 44 days prior to viral infection.
  • C57BL/6J male mice were injected intraperitoneally with 0.1 ml busulfan at a concentration of 1 mg/ml. The dose was 4 ⁇ g busulfan/gm body wt.
  • One pretreated mouse was anesthetized with
  • Avertin (0.017mls/gmbody wt.), and a ventral midline incision was made and the right testis exposed.
  • vas efferentia were dissected away from the fat, and ten microlitres of HJV-derived GFP vector, HR'GFP, at a titer of lxlO 9 particles/ml were injected into the seminiferous tubules of the right testis via the vas efferens of a busulfan-treated C57 BL/6J mouse. Injection was done with a quartz glass micropipette attached to a Picospritzer LT. The Picospritzer was set at 80psi and gave 1 second bursts upon manual depression of a foot pedal.
  • All the seminiferous tubules of the testis can be reached with a single injection as the vas efferens leads to a common chamber, the rete testis, from which all the tubules radiate.
  • the left testis was not injected and was used as a control. Transduction of the testicular cells within the tubules was widespread.
  • testes Twenty one days after infection, the mouse was sacrificed and the testes were fixed overnight in 4% paraformaldehyde in PBS, pH 7.4 at 4°C. The testes were washed three times in PBS and placed in 20% sucrose overnight at 4°C. The testes were frozen in OCT and sectioned at 8 ⁇ m on a cryostat. The sections were thawed to room temperature immersed in phosphate saline buffer and viewed on a Zeiss 310 confocal microscope. The laser was set at a wavelength of 488 nm.
  • Example 15 Optimization Microinjection of Gene Delivery Mixture through the Vas Efferens.
  • Busulfan an alkylating cytotoxic agent, depopulates the testis (Bucci, L. and Meistrich, M., Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations, Mut. Res. 176:259-268 [1987]).
  • IP intraperitoneal
  • Stem cell spermatogonia are known to be resistant to insults, often surviving when other germ cell types are destroyed (Huckins, C. & Oakberg, W.F., Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. 11. The irradiated testes, Anat. Rec. 192:529-42 [1978]).
  • Microsurgery After depopulation of the testis as described in Example 15, viral particles were delivered to the seminiferous tubules as follows: Mice were anaesthetised with isofluorane (0.5-2% in oxygen). Each testis was exposed through a midline abdominal incision. Using a microsurgical approach (Winston [1975]; Zeiss microscope at magnification 4 to 50x) the tissue bundle containing the vasa efferentia was visualised (Fig la-lb). Dissection from the surrounding fat was aided by a stream of phosphate buffered saline forced through a fine needle.
  • a quartz glass micropipette was back-filled with 10 ⁇ L viral particles (10 9 pfu/ml) mixed with 1 ⁇ L polybrene (80mg/mL). This was attached to a micropipette (Eppendorf) and the particles introduced into the vas efferens under 2.2 bar pressure in pulses of 1.5 seconds, controlled by foot pedal.
  • a micropipette Eppendorf
  • 1% Bromophenol dye showed that most seminiferous tubules could be filled (Fig. lc), but during treatments, no dye was used and small air bubbles were introduced into the liquid containing viral particles to confirm dispersion into the seminiferous tubules (Fig. Id). To preserve fertility, only single vasa efferentia were injected, reducing injury to the remaining ducts.
  • the plasmid pHR'-CMVLacZ (L. Naldini et al. [1996]), was modified by replacing the Bam - Xhol fragment containing the LacZ gene with a fragment containing the EGFP gene ('humanised' GFP, Clontech).
  • 40 ⁇ g plasmid DNA was used to transfect a 10-cm plate of 293T cells.
  • the 40 ⁇ g of plasmid DNA was made up of 10 ⁇ g pCMV R9, 20 ⁇ g of modified pHR' and 10 ⁇ g envelope plasmid.
  • VSV-G Vesicular-stomatitis-virus-glycoprotein pseudotyped vectors were produced by con transfection of the vector plasmid with the Moloney murine leukemi a virus (MLV) gag-pol packaging plasmid pCMV-GAGPOL and the VSV-G plasmid. The supernatant was harvested 48-60 hours after transfection, subjected to high speed centrifugation, filtered through 0.45 ⁇ m filters and assayed. The transducing viral particles had the MLV restricted envelope protein, env, substituted with a broad-spectrum env protein from the vesicular stomatitis virus. In Vivo Transduction of Male Germ Cells.
  • mice Six mice were now treated with viral particles containing the transducing vector pHR' (10 9 particles/mL). A single vas efferens was injected with a volume of lO ⁇ L retroviral concentrate together with l ⁇ L (80mg/mL) polybrene. After 24 days the mice were sacrificed and the testes removed and fixed for cryosectioning and histological examination. Testes were fixed for 48 hours in 4% Paraformaldehyde pH 7.4, and placed in 20% sucrose in phosphate saline buffer pH 7.4 at 4°C for 24 hours. They were embedded in OCT and stored at-70°C. They were cryosectioned at 8 ⁇ m and viewed in a Zeiss 410 confocal microscope (Fig. 2).
  • B6D2F1 females were introduced into cages with the males. Transduced males fathered at least two successive litters. Litters were conceived 14, 15, 19 and 20 weeks after transduction. All the males, except one dying immediately after surgery, fathered transgenic offspring. (Table 3).
  • PCR and Southern blot analysis of DNA from embryonic offspring were screened for presence of the transgene by polymerase chain reaction (PCR) and Southern blot analysis.
  • PCR polymerase chain reaction
  • GFP specific primers were used and a radiolabeled GFP cDNA probe was used for the Southern blot analysis (Fig. 3).
  • DNA was purified from embryos using the Gentra purification system. The presence of the transgene was ascertained using PCR amplification with the following GFP specific primers:
  • the PCR cycling conditions were: denaturing 94°C for 1 minute, annealing at 60°C for 1 minute and extension at 72°C for 3 minutes.
  • PCR ran for 35 cycles and yielded a specific GFP product 470 base pairs in length.
  • Each cycle step can be reduced to one second - "one second PCR” to yield a distinct 470-bp PCR amplification product.
  • Southern blot analysis was also done on the same embryo DNA extracts. The DNA was cut with Bam I-Xhol, run on a 0.8% agarose gel and blotted overnight in 20x SSC onto Hydrobond XL paper.
  • the blots were hybridised overnight at 65°C with a 32 P -radiolabeled Bam -Xhol GFP fragment isolated from the pHR'plasmid. The blots were washed at 65°C (30 minutes) each in 2x SSC with 0.1% SDS, lx SSC with 0.1 % SDS, 0.1 X SSC with 0.1% SDS and exposed to X-ray film. PCR and Southern analysis showed that a high percentage of transgenic offspring were obtained in litters conceived within 15 weeks. The results are summarized in Table 3.
  • mice untreated with busulfan also generated transgenic offspring.
  • Male germ cells take 60 days to differentiate from spermatogonia (Russell, L.D., et al. In: Histological and Histopathological evaluation of the testis, Cache River Press [1990]), undergo meiosis and form spermatozoa. Since conception was more than 60 days after transduction, it is presumed that the transgenic offspring were conceived from differentiated daughter cells of transduced spermatogonia.
  • EGFP expression was driven by the CMV promoter and was evident in the testicular cells of the founder males 24 days after infection. The animals that were infected did not appear to have toxic side effects (Verma, I.M. and Somia, N. [1997]) with the possible exception of one dying 17 weeks after surgery.

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Abstract

Cette invention a trait à une technique in vivo d'incorporation de matériel génétique exogène au génome d'un vertébré, consistant à administrer au testicule de ce vertébré mâle un mélange d'apport de gène comportant un vecteur viral, tel qu'un vecteur rétroviral, pour apporter un polynucléotide codant un trait ou un produit désiré. L'invention porte également sur une technique in vitro d'incorporation de matériel génétique exogène au génome d'un vertébré, les cellules germinales étant obtenues à partir d'un donneur vertébré mâle et modifiées par voie génétique in vitro avant leur transfert vers le vertébré mâle destinataire. Après transfert, le vertébré mâle porteur des cellules germinales modifiées par voie génétique est mis à s'accoupler avec un vertébré femelle de manière à produire une descendance transgénique porteuse du polynucléotide dans son génome. L'invention concerne également des vertébrés transgéniques non humains produits grâce à cette technique, y compris la descendance transgénique. Cette invention porte, en outre, sur une cellule transgénique dérivée du vertébré transgénique, en l'occurrence une cellule germinale, telle qu'un spermatozoïde ou un embryon, une cellule précurseur de l'un ou de l'autre de ces derniers ou une cellule somatique. L'invention concerne, de surcroît, une technique de production de lignée d'animal vertébré transgénique comportant des cellules germinales d'origine porteuses dans leur génome d'au moins un polynucléotide exogène, la semence transgénique contenant les cellules germinales mâles utilisées dans la mise en oeuvre de cette technique.
EP00937536A 1999-05-13 2000-05-12 Modification genetique de cellules germinales males pour la production d'especes transgeniques et aux fins de therapies geniques Withdrawn EP1190086A2 (fr)

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US09/311,599 US6734338B1 (en) 1997-11-14 1999-05-13 Transfection, storage and transfer of male germ cells for generation of transgenic species and genetic therapies
US311599 1999-05-13
PCT/US2000/013000 WO2000069257A2 (fr) 1999-05-13 2000-05-12 Modification genetique de cellules germinales males pour la production d'especes transgeniques et aux fins de therapies geniques

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FR2814370B1 (fr) * 2000-09-22 2004-08-20 Centre Nat Rech Scient Utilisation d'un complexe acide nucleique/pei pour le ciblage de cellules souches du cerveau
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
EP1438401B1 (fr) 2001-09-18 2011-06-15 Synageva BioPharma Corp. Production d'oiseaux transgeniques par injection cytoplasmique
KR100569168B1 (ko) * 2003-08-08 2006-04-07 (주)아비코아생명공학연구소 조류 정원줄기세포의 배양방법 및 이에 의해 수득한 조류정원줄기세포
GB0325140D0 (en) * 2003-10-28 2003-12-03 Babraham Inst Methods for selecting gametes and for producing genetically modified non-human animals
JP2007275004A (ja) * 2006-04-10 2007-10-25 Kaneka Corp レンチウイルスベクターの精巣への感染による遺伝子組換え鳥類作製法
GB0802980D0 (en) * 2008-02-19 2008-03-26 Atazoa Ltd Biological materials and uses thereof
AU2013331101B2 (en) 2012-10-19 2019-05-30 Trans Ova Genetics, L.C. Methods for generating genetically superior animals
US20140359796A1 (en) * 2013-05-31 2014-12-04 Recombinetics, Inc. Genetically sterile animals
CN111713450B (zh) * 2020-05-26 2022-07-12 上海交通大学医学院 一种慢性粒细胞白血病的pdx模型的建立方法

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GB8906214D0 (en) * 1989-03-17 1989-05-04 Nat Res Dev Introducing an exogenous gene into birds
JPH10304790A (ja) * 1995-09-29 1998-11-17 Hoechst Japan Ltd トランスジェニック動物の作成方法
JPH09220039A (ja) * 1996-02-14 1997-08-26 Chihiro Koike 精子又は卵子への外来遺伝子の導入方法及びトランスジェニック動物の作製方法
EP1030929B1 (fr) * 1997-11-14 2009-03-18 Cedars-Sinai Medical Center Transfection et transfert de cellules germinales non-humaines males pour produire des mammifères non-humains transgeniques
WO1999038991A1 (fr) * 1998-01-28 1999-08-05 Takara Shuzo Co., Ltd. Procede relatif au transfert de gene dans des cellules germinales
EP1053340A4 (fr) * 1998-02-09 2003-04-09 Tranxenogen Inc Manipulation genetique des spermatogonies
JP2003525581A (ja) * 1998-11-13 2003-09-02 セダーシナイ メディカル センター 脊椎動物精巣の細胞減失（ｄｅｐｏｐｕｌａｔｉｎｇ）方法およびトランスジェニック種の作製方法
CA2350829A1 (fr) * 1998-11-13 2000-05-25 Cedars-Sinai Medical Center Transfection de cellules germinales males permettant de generer des cellules souches transgeniques selectionnables

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WO2000069257A3 (fr) 2001-08-09
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JP2002543814A (ja) 2002-12-24
AU5268800A (en) 2000-12-05

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