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WO2017075276A2 - Compositions and methods for chimeric embryo-assisted organ production - Google Patents

Compositions and methods for chimeric embryo-assisted organ production Download PDF

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Publication number
WO2017075276A2
WO2017075276A2 PCT/US2016/059206 US2016059206W WO2017075276A2 WO 2017075276 A2 WO2017075276 A2 WO 2017075276A2 US 2016059206 W US2016059206 W US 2016059206W WO 2017075276 A2 WO2017075276 A2 WO 2017075276A2
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WO
WIPO (PCT)
Prior art keywords
human
embryo
endogenous genes
cells
organ
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Application number
PCT/US2016/059206
Other languages
French (fr)
Other versions
WO2017075276A3 (en
Inventor
Atsushi Asakura
Raj ARAVALLI
Maxin CHEERAN
James Dutton
Daniel J. GARRY
Mary G. GARRY
Melanie Graham
Ling Li
Walter Low
Angela Panoskaltsis-Mortari
Timothy O'brien
Clifford Steer
Jakup TOLAR
Daniel F. Carlson
Scott C. Fahrenkrug
Naoko KOYANO-NAKAGAWA
Ann PARR
Original Assignee
Regents Of The University Of Minnesota
Recombinetics, Inc.
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Publication date
Application filed by Regents Of The University Of Minnesota, Recombinetics, Inc. filed Critical Regents Of The University Of Minnesota
Priority to EP16860830.5A priority Critical patent/EP3367785A4/en
Priority to KR1020187014498A priority patent/KR20180100303A/en
Priority to CN201680074878.7A priority patent/CN108697067A/en
Priority to CA3003652A priority patent/CA3003652A1/en
Priority to JP2018542691A priority patent/JP2019502400A/en
Priority to AU2016344152A priority patent/AU2016344152A1/en
Publication of WO2017075276A2 publication Critical patent/WO2017075276A2/en
Publication of WO2017075276A3 publication Critical patent/WO2017075276A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • 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
    • A01K67/0276—Knock-out vertebrates
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation

Definitions

  • Organ transplant can be effective but there are far too few organs available and in many cases immunological mismatches lead to problems. For example, over 7,000 Americans have died while awaiting an organ transplant since 2003.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, excluding chimeric embryos wherein:
  • the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
  • the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RU X1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells; the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
  • the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
  • the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
  • the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous gene is PAX3 and the human organ and/or tissue comprises skeletal muscle cells; or
  • the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, crustacean, and fish.
  • the vertebrate non-human embryo is a cow, pig, sheep, goat, chicken or rabbit embryo.
  • a method of producing a chimeric embryo comprising:
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo.
  • a method of producing a chimeric embryo comprising: a) disrupting one or more endogenous genes responsible for the development of one or more endogenous organs or tissues in at least one non-human cell or non-human embryo; b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues;
  • the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a method of producing a chimeric embryo comprising
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
  • the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a method of producing a chimeric embryo comprising
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
  • the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a method of producing a chimeric embryo comprising a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
  • the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
  • the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a method of producing a chimeric embryo comprising
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
  • the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
  • the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
  • non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
  • the at least one human donor cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising:
  • step a) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
  • the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising:
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, excluding chimeric embryos, wherein:
  • the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising:
  • step a) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo;
  • step c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
  • the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, and wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
  • the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
  • the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising:
  • step a) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo;
  • step c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
  • the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, wherein:
  • the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
  • the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
  • the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
  • the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
  • the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
  • the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
  • the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising:
  • step a) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo;
  • step c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
  • the endogenous genes are RAG2 and IL2rg or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous genes are MYF5, MYOD and MRF4 or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
  • the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • a method of producing a human or humanized organ or tissue in a non-human host animal comprising
  • step a) if step a) is performed on a non-human cell, cloning the cell to produce an embryo;
  • step c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein: the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
  • the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
  • the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
  • non-human embryo is a non-human vertebrate embryo.
  • the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
  • non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
  • the at least one human cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
  • the method of paragraph 86 further comprising introducing a plurality of homology directed repair (HDR) template, each having a template sequence with homology to one of the endogenous genes, with each the template sequences replacing at least a portion of one of the endogenous gene sequences to disrupt the endogenous gene.
  • HDR homology directed repair
  • FIG. 1 is a schematic diagram illustrating the problem of tissue/organ transplantation and the solution provided by genome engineering of Human Cells and Animals for Organ Transplant.
  • FIG. 2A depicts a process for making animals homozygous for two knockouts using single edits.
  • FIG. 2B depicts a hypothetical process of making animals with multiple edits by making of a single edit at a time.
  • FIG. 3 depicts multiplex gene edits used to establish founders at generation F0.
  • FIGs. 4A-4D depict multiplex gene editing of swine RAG2 and IL2Ry (or IL2Rg).
  • FIG. 4A is a graph showing surveyor and restriction fragment length polymorphism (RFLP) analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection.
  • FIG. 4B is a graph showing RFLP analysis for homology dependent repair on cell populations 11 days post transfection.
  • FIG. 4C is a graph showing percentage of colonies positive for HDR at IL2Ry, RAG2 or both. Cells were plated from the population indicated by a "C" in FIG. 4A.
  • 4D is a graph showing colony analysis from cells transfected with Transcription Activator-Like Effector Nucleases (TALENS) mRNA 30 quantities of 2 and 1 ⁇ g for IL2Ry and RAG2 and HDR template at 1 ⁇ for each. Distribution of colony genotypes is shown below.
  • TALENS Transcription Activator-Like Effector Nucleases
  • FIGs. 5A - 5D depict Multiplex gene editing of swine APC and p53.
  • FIG. 5A is a graph showing surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair (HDR) on cell populations 3 days post transfection.
  • FIG. 5B is a graph showing RFLP analysis for homology dependent repair on cell populations 11 days post transfection.
  • FIGs. 5C and 5D are graphs showing percentage of colonies positive derived from the indicated cell population (indicated in FIG. 5 A, "C" and "D") for HDR at APC, p53 or both. Colonies with 3 or more HDR alleles are listed below.
  • FIGs. 5A is a graph showing surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair (HDR) on cell populations 3 days post transfection.
  • FIG. 5B is a graph showing RFLP analysis
  • FIG. 6A and 6B depict effect of oligonucleotide HDR template concentration on Five- gene multiplex HDR efficiency.
  • Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Ry, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 ⁇ (FIG. 6A) or 1 ⁇ (FIG. 6B) of each cognate HDR template.
  • Percent NHEJ and HDR were measured by Surveyor and RFLP assay.
  • FIGs. 7A and 7B are a five-gene multiplex data set that shows plots of experimental data for the effect of oligonucleotide HDR template concentration on 5 -gene multiplex HDR efficiency.
  • Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Ry, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 ⁇ or 1 uM of each cognate HDR template. Percent NHEJ and HDR were measured by Surveyor and RFLP assay.
  • Colony genotypes from 5-gene multiplex HDR Colony genotypes were evaluated by RFLP analysis.
  • each line represents the genotype of one colony at each specified locus.
  • FIG. 7B provides a tally of the number of colonies edited at 0-5 loci.
  • FIGs. 8A and 8B are another five-gene multiplex data set that shows plots of experimental data for a second experiment involving the effect of oligonucleotide HDR template concentration on Five- gene multiplex HDR efficiency.
  • each line represents the genotype of one colony at each specified locus.
  • Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele.
  • the percentage of colonies with an edit at the specified locus is indicated below each column.
  • FIG. 8B provides a tally of the number of colonies edited at 0-5 loci.
  • FIGs. 9A and 9B is another five-gene multiplex trial data set that shows colony genotypes.
  • each line represents the genotype of one colony at each specified locus.
  • Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele.
  • the percentage of colonies with an edit at the specified locus is indicated below each column.
  • FIG. 9B provides a tally of the number of colonies edited at 0-5 loci.
  • FIG. 10 depicts a process of making an F0 generation chimera with targeted nucleases that produce a desired gene knockout or choice of alleles.
  • FIG. 11 depicts establishment of an F0 generation animal with a normal phenotype and progeny with a failure to thrive (FTT) phenotype and genotype.
  • FTT failure to thrive
  • FIG. 12 depicts a process for making chimeric animals with gametes having the genetics of the donor embryo.
  • FIGs. 13A-C depict multiplex editing at three targeted loci of NKX2-5, GATA4, and
  • FIG. 13A is a schematic of the experiment
  • FIG. 13B shows the targeting of the genes, with the NKX2-5, GATA4, and MESP1 listed as SEQ ID NOs: 1-3, respectively.
  • FIG. 13C depicts the results of an assay for the experiments. Oligo sequences for each target gene. Novel nucleotides are represented by capital letters. The PTC is represented by light color letters in boxes and the novel Hindlll RFLP site is underlined.
  • FIG. 14 depicts multiplex gene-editing using a combination of TALENs and RGENs; assay of transfected cells evaluated by RFLP revealed HDR at both sites.
  • FIGs. 15A-E are images depicting incorporation of human umbilical cord blood stem cells (hUCBSC) into parthenogenetic porcine blastocyst.
  • FIG. 15 A is a phase contrast image of blastocyst.
  • FIG. 15B is a DAPI image of cells within the blastocyst.
  • FIG. 15C is a human nuclear antigen (HNA) staining.
  • FIG. 15D is a merged DAPI and HuNu image.
  • FIG. 15E is a merged image of FIGs. 15A-15C.
  • FIG. 15F is a graph showing quantification of HuNu cells in the inner cell mass (ICM), trophectoderm (TE), or blastocoel cavity (CA).
  • FIG. 15G is a graph showing the proliferation of HNA cells at days 6, 7, and 8 after activation of oocyte. Injection of hUCBSC was at day 6.
  • FIGs. 16A-16C are images showing chimeric human-porcine fetus.
  • FIG. 16A is an image showing chimeric fetus at 28 days in gestation following injection of hUCBSCs into parthenogenetic porcine blastocysts.
  • FIG. 16B is an immunohistochemistry image showing the staining for human nuclear antigen (HNA) in red and DAPI in blue.
  • FIG. 16C is a control for the immunohistochemistry staining in FIG. 16B, with no primary antibody added in the staining.
  • HNA human nuclear antigen
  • FIGs. 17A and 17B depicts TALEN mediated knockout of porcine genes.
  • FIG 17A is a schematic showing cleavage sites for LMXA1, NURR1, and PITX3.
  • FIG. 17B provides electrophoresis images showing TALEN cleavage products as indicated by double arrows.
  • FIGs. 18A-F are images showing ocular effects of complementation of PITX3 knockout in porcine blastocysts with human umbilical cord blood stem cells. Images of FIGs. 18A-F show the gross morphology of fetal pig eyes at 62 days in gestation. FIGs. 18A and 18B shows the eye of wild type pig. FIGs. 18C and 18D show small eye from PITX3 knockout pigs. FIGs. 18E and 18F show large eye from PITX3 knockout with human umbilical cord blood stem cells (hUCBSC) complementation. Arrows in FIGs. 18A, 18C, and 18E point to the location of the eye for each fetus. FIGs.
  • hUCBSC human umbilical cord blood stem cells
  • FIG. 19A and 19B depict TALEN-mediated knockout ⁇ 2.
  • FIG. 19A is a schematic showing the three-tiered PCR assay utilized to detect gene editing. Amplification from primers a-d indicated a deletion allele was present. To distinguish between heterozygous and homozygous clones, primers a-b and c-d were used to amplify the wild type allele. Only when the a-d product is present and both a-b, c-d products are absent is the clone considered homozygous for the deletion allele.
  • FIG. 19B provides electrophoresis images showing the confirmation of homozygous deletion. Clones fitting the criteria described above are enclosed by a green box.
  • FIGs. 20A-20H depict the loss of porcine ETV2 recapitulated the mouse Etv2 mutant phenotype.
  • FIG. 20 A shows wild-type El 8.0 pig embryo and
  • FIG. 20B shows ETV2 knockout embryo at the same developmental stage.
  • Insets show enlarged views of the allantois. Note an abnormal overall morphology with lack of vascular plexus formation in the mutant (inset).
  • FIGs. 20C-20H are sections through the allantois (FIGs. 20 C and D), the heart level (FIGs.
  • the endocardium, cardinal veins (CV), and dorsal aortae (DA) are clearly visible in the wild- type embryo (E, G).
  • ETV2 null embryos completely lacked these structures although the heart progenitors and gut marked by Gata4 (green) were present (F and H, respectively).
  • Scale bars 1000 ⁇ (FIGs. 20A and B), 200 ⁇ (insets in FIGs. 20A and B), 100 ⁇ (FIGs. 20C-20H).
  • FIGs. 21A-21C are immunohistochemistry images depicting complementation of ETV2 mutant porcine embryos with human induce pluripotent stem cells (hiPSCs). ETV2 mutant blastocysts were generated by SCNT, and injected with ten hiPSCs at the morula stage and subsequently transferred into hormonally synchronized gilts.
  • FIG. 21A shows in situ hybridization using the human specific Alu sequence.
  • FIGs. 2 IB and 21C are
  • FIG. 21B immunohistochemistry images against human CD31 (FIG. 21B), HNA (FIG. 21C, red), and human vWF (FIG. 21C, green). Boxed areas are enlarged in panels below. Arrowheads point to positive cells. Note formation of vessel-like structures. All scale bars indicate 50 microns, nt: neural tube, noto: notochord, som: somite.
  • FIGs. 22A-22D are images showing that Nkx2-5 and Handll (also known as dHand) double knockouts lack both ventricles (rv and lv) and have a single, small primitive atrium (dc).
  • FIG. 22 A shows a wild type animal.
  • FIG. 22B shows a NL ⁇ 2.5 ⁇ ' ⁇ animal.
  • FIG. 22C shows a dHand' ' animal.
  • FIG 22D shows a NKx2.5 ⁇ ' ⁇ dHand' ' double knockout animal.
  • FIGs. 23 A and 23B depict double knockout of NKX2-5 and HANDII in swine fibroblasts.
  • FIG. 23A is a schematics of the coding sequence for each gene shown; alternating colors indicate exon boundaries, the blue region (below) indicates the DNA binding domain of each transcription factor, and the triangles indicate the location TALENs binding sites.
  • FIG. 23B provides electrophoresis images of RFLP analysis of fibroblast colonies for bialleic KO of HANDII and NKX2-5.
  • FIGs. 24A-24C depict that Nkx2-5/HANDII/TBX5 triple knockout porcine embryos have acardia.
  • FIG. 24A provides images of immunohistochemistry staining of Gata4 protein. Wild type embryos (top) stained positively, while the triple knockout porcine embryos (bottom) lacked a heart with essentially no Gata4 immunohistochemically positive cells (marking the heart) at E18.0 (h, heart and fg, foregut).
  • FIG. 24B provides images of DAPI staining for wild type embryos (top) and triple knockout embryos (bottom).
  • FIG. 24C provides merged images of FIG. 24A and FIG. 24B.
  • FIGs. 25A and 25B are images depicting Myod expression.
  • Myf5, Myod and Mrf4 are master regulators of skeletal muscle and are restricted to skeletal muscle in development and in the adult. Shown here is Myod-GFP transgenic expression which is restricted to the somites, diaphragm and established skeletal muscle at El 1.5 (FIG. 25 A).
  • FIG. 25B in situ hybridization of a parasagittal section of an E13.5 (mid-gestation) mouse embryo using a 35S- labeled MyoD riboprobe. Note expression in back, intercostal and limb muscle groups.
  • FIGs. 26A-26C depict the knockout of swine MYOD, MYF5, and MYF6 gene.
  • FIG. 26A is a schematic showing that. TALEN pairs were designed for swine MYOD, MYF5, and MYF6 (aka MRF4) genes. TALEN binding sites (denoted by red arrow heads) were upstream the important basic (+) helix-loop-helix (HLH) domain for each gene. The TALEN binding sites are shown below (denoted by red arrows) and the amino acid that was targeted for a premature STOP codon by homology dependent repair (HDR) are denoted by yellow arrows.
  • FIG. 26B provide electrophoresis images showing the HDR events confirmed by RFLP analysis.
  • HDR templates were designed to introduce the premature STOP codon and a novel restriction enzyme recognition site (Hindlll) to allow facile analysis of HDR events.
  • the region of interest for each gene was amplified by PCR and RFLP was assessed for the population of transfected cells.
  • the closed arrow heads denote the uncut or wild type alleles, while the open arrow heads denote the HDR alleles.
  • the percent of alleles positive for HDR for MYOD, MYF5, and MYF6 were 14%, 31%, and 36%, respectively.
  • FIG. 26C provides electrophoresis images and sequencing graphs to confirm the triple knockout. These populations were plated out for individual colony isolation.
  • FIGs. 27A and 27B depict a phenotype of MYF5/MYOD/MRF4 triple knockout (KO).
  • wild-type (Wt) embryos had well defined somite(s), desmin positive (red) myotomes (m) and developing musculature (FIG. 27A).
  • the developing heart tube demonstrated strong desmin signal (h).
  • MYF5/MYOD/MRF4 KO embryos showed a lack of myotome formation while the heart remained desmin positive (FIG. 27B).
  • FIGs. 28A-28C depict complementation of MYF5/MY OD/MRF4 null embryos complemented with GFP labeled blastomeres.
  • FIG. 28A is an image showing E20 porcine MYF5/MYOD/MRF4 null embryos complemented with GFP labeled blastomeres. Native GFP is observed in the liver and yolk sac of the embryo.
  • FIG. 28B is an image showing section of porcine liver from MYF5/MY OD/MRF4 null embryos (E20) complemented with GFP labeled blastomeres. Native GFP is visible in the sinusoids of the liver.
  • FIG. 28A is an image showing E20 porcine MYF5/MYOD/MRF4 null embryos complemented with GFP labeled blastomeres. Native GFP is visible in the sinusoids of the liver.
  • 28C is a bar graph showing PCR of yolk sac from E20 porcine MYF5/MY OD/MRF4 null embryos complemented with GFP labeled blastomeres (Embryos 1 [shown in FIGs. 28A and 28B], 3, 5). GFP -labeled pig fibroblasts is positive control while WT pig liver is negative control.
  • FIGs. 29A to 29E depict the generation of PDXl-/- pigs.
  • FIG. 29A is a schematic showing TALEN gene editing of the pig PDXl locus.
  • FIG. 29B provides electrophoresis images showing that RFLP analysis identified unmodified, heterozygous knockouts (open arrowhead) or homozygous knockouts (closed arrowhead). 41% of the clones were homozygous knockouts for PDXl.
  • FIGs. 29C and 29D are images showing pancreas ablation ( ⁇ ) in cloned E32 Pdxl-/- pig embryos (FIG. 29D) compared to the pancreas in WT E30 embryos (FIG. 29C).
  • FIG. 29E is an image showing the comparison of nascent ⁇ cells between wild type fetus and PDX ⁇ mutant fetus. P pancreas, S stomach, D duodenum of Wt E30
  • FIGs. 30A-30C depict the generation of ffiffi knockouts (KOs) by gene-editing.
  • FIG. 30A-30C depict the generation of ffiffi knockouts (KOs) by gene-editing.
  • FIG. 30 A is a schematic illustrating the knockout of HHEX gene.
  • the HHEXgms is comprised of 4 exons.
  • the Hindlll KO allele was inserted into exon 2 of the HHEX gene by gene-editing.
  • FIG. 30B provides electrophoresis image showing that the efficiency of gene-editing was measured on the transfected population by a Hindlll RFLP assay.
  • the proportion of chromosomes with the novel Hindlll KO allele is indicated on the gel.
  • FIG. 30C provides electrophoresis images showing that fibroblast clones were also screened using the Hindlll RFLP assay. Homozygous KO clones are indicated with an asterisk.
  • FIGs. 31 A and 3 IB depict liver development in wild-type (FIG. 31 A) and HHEX KO pig embryos (FIG. 3 IB) at 30 days in gestation. Note absence of liver development in HHEX KO specimen in FIG. 3 IB. Wild- type control at the same gestational age is shown in FIG. 31 A.
  • FIGs. 32A-32C are images showing that lungs develop in wild type animals.
  • FIGs. 32D-32F are images showing that lungs fail to develop in NKX2.1 knockout animal.
  • FIG. 33 depicts MR imaging of fetal pig at 16.4T showing internal organs. Pig gestational age is 30 days when crown-rump length is approximately 20 mm.
  • FIG. 34 is a schematic showing the function of PITX3 in dopamine neuron/eye lens development.
  • the inset is the same gel electrophoresis image showing the TALEN cleavage product of PITX3 gene in FIG. 17.
  • FIG. 35 is an image showing the introduction of donor hiPSC or hUCBSC into PITX3 KO porcine morula.
  • FIG. 36 provides a schematic showing the transfer of chimeric PITX3 knockout (KO) morula to surrogate sows.
  • FIG. 37 is a dot plot showing that human stem cell complementation rescues ocular defects in porcine PITX3 knockout.
  • PITX3 knockout essentially eliminated open eyelid.
  • FIG. 37 represents the quantitative analysis of the "eyelids" shown in Figures 18A, 18C, and 18E.
  • Figure 18A the eye in the wild type pig fetus can be observed.
  • the eye is not visible in the PITX3 knockout (FIG. 18C) because the overlying skin or eyelid is not translucent.
  • PITX3 knockout fetuses complemented with stem cells the eye can now been seen through the translucent or "open” eyelid (FIG. 18E).
  • FIGs. 38A and B provide immunohistochemistry images showing the loss of dopamine neurons in fetal substantia nigra (SNc) due to the porcine PITX3 knockout.
  • FIG. 38A shows tyrosine hydroxylase (TH) positive neurons in the SNc of wild-type fetal pig 62 days in gestation.
  • FIG. 38B shows TH positive neurons in the SNc of PITX3 knockout fetal pig. Note loss of dopamine neurons in the knockout pig.
  • VTA ventral tegmental area.
  • FIGs. 39A-39D provides images showing the incorporation of human neurons in chimeric fetuses.
  • FIG. 39A shows HNA staining of five fetal pigs to demonstrate that human neuron cells were present at least in some chimeric fetuses.
  • FIG. 39B shows tyrosine hydroxylase staining and
  • FIG. 39C shows NeuN staining of the same five fetus pigs as in FIG. 39A.
  • FIG. 39D provides merged images of FIGs. 39A-39C.
  • HNA human nuclear antigen
  • TH Tyrosine hydroxylase.
  • FIG. 40 provides provide immunohistochemistry images showing the staining of substantia nigra stained with TH (tyrosine hydroxylase), DAPI and NeuN to substantia nigra dopamine neurons.
  • FIG. 41 provides bar plots showing that hiPSC complementation induced survival of nigral dopamine neurons.
  • FIG. 41 depicts the estimate of number of TH-immunooreactivity (TH-IR) counts in both whole substantia nigral dopamine neurons and medial substantia nigra in wild type pigs (WT), PITX3 knockout pigs (KO), PITX3 KO complemented with hUCBSCs, and PITX3 KO complemented with hiPSCs.
  • WT wild type pigs
  • KO PITX3 knockout pigs
  • hUCBSCs wild type pigs
  • PITX3 KO PITX3 KO complemented with hiPSCs.
  • FIG. 42 provides a schematic and an image showing LMX1A/PITX3 and dopamine neuron development.
  • the inset image is the same as those of the FIG. 17 on LMXA1 and PITX3 gene knockout using TALEN.
  • FIG. 43 provides a schematic showing the generation of chimeric pig with dopamine neuron knockout and complementation with GFP expression porcine blastomeres.
  • LMX1A/PITX3 knockout fibroblasts were generated as described herein. Wild type porcine blastomeres were introduced into the LMX1A/PITX3 blastocysts derived from the knockout fibroblasts to create chimeric blastocysts. The chimeric blastocysts develops into a chimeric pig with immune system organs/cells originated from donor.
  • FIGs. 44A-44F provides images showing that the porcine-porcine complementation in PITX3/LMX1A rescued fetal development.
  • FIGs. 44A-44C exemplify PITX3/LMX1A knockout fetuses with successful complementation based on normal size of development for their gestational age.
  • FIGs. 44D-44F show poor embryonic development indicating poor complementation.
  • FIGs. 45A shows Haemotoxylin and Eosin (H&E) staining of the wild-type fetal porcine lens (L).
  • FIG45B exemplifies lack of lens (L) development in the PITX3 knockout fetus.
  • R retina
  • C cornea.
  • FIGs. 46A-C provide Haemotoxylin and Eosin (H&E) staining showing that porcine- porcine complementation in PITX3/LMX1A knockout (KO) reinstates lens development following stem cell complementation with porcine blastomere cells.
  • FIGs. 47A-47D depicts immunohistochemistry images showing that donor GFP + cells contributed to the fetal pig brain.
  • FIGs. 47B-47D are the enlarged detailed view of the corresponding sections in FIG. 47A.
  • FIGs. 48A-48C provide tyrosine hydroxylase immunohistochemistry images showing that porcine-porcine complementation results in development of DA neurons in primordial ventral mesencephalon (VM) in PITX3/LMX1A knockout pig fetuses.
  • VM shown in FIGs 48A- 48C were derived from fetuses exhibiting normal development based on Crown-Rump length.
  • FIG. 49 provides a schematic showing that RAG2 and IL2Rg are important for T cell, B cell, and NK cell development.
  • FIG. 50 provides a schematic showing that RAG2 and IL2Rg are important for thymus development.
  • FIG. 51 provides a schematic showing the procedure for hematopoietic system knockout and complementation with GFP-expressing porcine blastomeres.
  • IL2Rg/RAG2 knockout fibroblasts were generated as described herein. Wild type porcine blastomeres were introduced into the IL2RG/RAG2 knockout blastocysts to create chimeric blastocysts. The chimeric blastocysts develops into a chimeric pig with immune system organs/cells originated from donor.
  • FIGs. 52A-52C provides images showing that complementation in IL2Rg/RAG2/C-KIT knockout results in generation of thymus.
  • Thymus developed in wild-type pig fetus (FIG. 52A), but failed to do so in IL2Rg/RAG2 knockout pig fetus (FIG. 52B).
  • Complementation with blastomeres from wild type animal resulted in the development of thymus in chimeric
  • FIG. 53 provides graphs showing the generation of GFP + immune cells in chimeric pigs.
  • Cells were collected from cord blood, thymus, spleen, peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN). Live cells were subject to FACS without staining. The percentage of GFP + cells of live cells is shown in each graph.
  • PBMC peripheral blood mononuclear cells
  • MN mesenteric lymph nodes
  • FIGs. 54A-54F provides graphs showing the generation of immune cells in the thymus.
  • FIG. 55 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated T cells (CD 172a " CD2 + CD3 ) in blood.
  • Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count T cells.
  • FIG. 56 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated T cells (CD 172a " CD2 + CD3 ) in spleen.
  • Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count T cells.
  • FIG. 57 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated B cells (CD3 " CD79a + CD21 + or CD21 " ) in blood. Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count B cells.
  • FIG. 58 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated B cells (CD3 " CD79a + CD21 + or CD21 " ) in spleen. Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained.
  • FIG. 59 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated NK cells (CD 172a " CD16 + CD2 ) in blood.
  • Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count NK cells.
  • FIG. 60 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated NK cells (CD 172a " CD16 + CD2 ) in spleen.
  • Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count NK cells.
  • FIG. 61 provides a schematic showing that PAX3 regulates myogenesis.
  • FIGs. 62A and 62B provides images showing PAX3 mutation in mice.
  • FIG. 62A depicts the comparison between wild type mice and PAX3 mutant mice.
  • Splotchdelayed mice carry allele of PAX3 gene mutation.
  • Postnatal Splotchdelayed animals at P0 showed spina bifida (red arrow).
  • FIG. 62B depicts the effect of blastocyst complementation using PAX3 mutant mice. Act-GFP iPSCs were preferentially contributed to limb skeletal muscle in PAX3 mutant chimera, compared to wild- type chimera.
  • FIG. 63 provides a schematic showing that PDX1 regulates pancreas development.
  • FIG. 64 provides a schematic showing that NKX2.1 regulates lung development.
  • FIG. 65 provides a schematic showing that OUG1/OLIG2 are important in
  • FIGs. 66A-66C provide schematics, an electrophoresis image, and graphs showing the generation of OLIG1/OLIG2 knockout fibroblast. TALEN technology is used to generate the knockout as described herein.
  • FIG. 66A illustrates the strategy to generate the OLIG1/OLIG2 knockout animals. ssOLIGl CDS and OLIG2 CDS were used to introduce a stop codon in the open reading frames of OLIG1 and OLIG2, respectively. These two polynucleotides also introduce a Hind III restriction site for the RFLP analysis.
  • FIG. 66B is an electrophoresis image showing the confirmation of introduction of stop codon in OLIG1 and OLIG2 gene using RFLP analysis.
  • FIG. 66C provides sequencing results of wild type (WT) cells and three colonies of OLIG1/OLIG2 knockout cells to confirm the introduction of stop codon in the open reading frame of OLIG1 and OLIG2 genes of the mutants' genome.
  • FIGs. 67A and 67B provides images showing OUG1/OLIG2 knockout pig fetus complemented with GFP -expressing porcine blastomeres. Wild type blastomeres expressing were introduced into OLIG1/OLIG2 knockout blastocytes.
  • FIG. 69A shows a porcine fetus at 30 days in gestation.
  • FIG. 69B shows whole body fluorescent imaging of GFP complemented porcine fetus.
  • FIGs. 68A and 68B provide images showing that GFP -expressing porcine blastomeres contribute to OLIG1/OLIG2 knockout fetal pig brain.
  • FIG. 68A is an image of fetal pig brain.
  • FIG. 68B is an image showing the expression of GFP in fetal chimeric pig brain, indicating the incorporation of wild type donor cells in knockout fetal pig brain.
  • FIGs. 69A and 69B provide images showing the 3-D reconstruction of GFP -labelling in spinal cord of OLIG1/OLIG2 knockout (KO) with GFP-blastomere complementation.
  • FIG. 71A is an image showing coronal reconstruction.
  • FIG. 71 B is an image showing lateral reconstruction.
  • FIG. 70 provides a schematic showing that HHEX regulates liver development.
  • FIG. 71 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2 (RG-KO) pigs showing percent of T cells (CD172a CD2 + CD3")
  • FIG. 72 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2
  • FIG. 73 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2 (RG-KO) pigs showing percent of NK cells (CD172a- CD16 + CD2 + ).
  • FIGs. 74A-74C depict the complete hind limb muscle formation in Pax3 mutant chimeric mouse after blastocyst complementation.
  • FIG. 74A provides a schematic showing the introduction of wild type mouse IPSCs (induced pluripotent stem cells) into PAX3 mutant mouse blastocysts.
  • FIG. 74B is an image showing that the hind limb was present in a PAX3 homozygous chimeric mouse. The dashed line indicates where the cross section was made for the histology study of lower hind limb (FIG. 74).
  • FIG. 74C provides an image showing the histology of cross section of lower hind limb. Legends for FIG.
  • TP Tibialis posterior
  • TA Tibialis anterior
  • EDL Extensor digitorum longus
  • FHL Flexor halluces longus
  • FB/FL Fibularis brevis/longus
  • Sol Soleus
  • M-Ga Medial gastrocnemius aponeurosis
  • L-GA Lateral gastrocnemius aponeurosis.
  • FIGs. 75 A-75F depict that PAX3 mutant pigs recapitulate phenotypes of PAX3 mutant mice.
  • FIG. 75A is a schematic showing the knockout of PAX3 gene using TALENS method as described herein.
  • FIG. 75B provides an electrophoresis image showing the confirmation of the mutant cells using RFLP analysis as described herein.
  • FIGs. 75C-75F provide images showing the comparison of wild type animals and mutant animals.
  • FIGs. 75C and 75D are images of wild type and mutant pigs at E30, respectively.
  • FIGs. 75E and 75F are images of wild type and mutant mice at E12.5, respectively. In wild type animals (pig and mouse, FIGs. 75C and 75E), the hind limbs were present, while in mutant animals (pig and mouse, FIGs. 75D and 75F), the hind limb development were significantly affected.
  • FIGs. 76A-76D depicts PAXs mutant pigs display loss of limb skeletal muscle.
  • FIG. 76A is an image of a wild type pig embryo at E30.
  • FIG. 76B is an image of a mutant pig embryo at E30. In both FIGs. 76A and 76B, the dotted line indicates where the cross section was made for the immunohistochemistry study in FIGs. 76C and 76D.
  • FIG. 76C provides images showing the staining of desmin, MHC and Pax7 in wild type animal hind limb.
  • FIG. 76D provides images showing the staining of desmin, MHC and Pax7 in mutant animal hind limb.
  • FIG. 77 provide electrophoresis images depicting knockout of IL2Rg/RAG2 (RG-KO) in piglets day 100 of gestation. Piglets were harvested two weeks before parturition and subject to genotyping. The expected RFLP banding pattern of IL2Rg/RAG2 bi-allelic knockout were observed. The cells of piglet No. 2 were used to add another mutation to the C-Kit gene.
  • FIGs. 78A-78C depicts the incorporation of V831M into IL2Rg/RAG2 RG-KO cells to produce IL2Rg/RAG2/C-KIT (RGK) cells.
  • FIG. 78A provides a schematic showing the location of the introduced mutation in C-KIT gene and the polynucleotides design of the C-Kit V831M HDR.
  • FIG. 78B provides an alignment of a portion of pig C-KIT protein with that of mouse showing the amino acid residue to be replaced.
  • FIGs. 78A and 78B demonstrate that the correct residue was identified.
  • FIG. 78C provides electrophoresis images showing the confirmation of the introduction of mutation into the genome.
  • Colonies 285 and 286 are examples of homozygous positives for the V-M RFLP.
  • FIG. 79 provides sequencing graphs showing the confirmation of the incorporation of C- Kit gene mutation in the genome of the cells.
  • Clones 162, 285, and 286 contains the V831M mutation. These clones were used for chimera productions.
  • FIG. 80 provides electrophoresis images of genotyping of analysis of the chimera fetuses of IL2Rg/RAG2/C-KIT.
  • the uncleaved band as indicated by the red box indicate that the fetus may be chimeric.
  • the present disclosure provides methods to engineer and to produce viable authentic human organs such as hearts, livers, kidneys, lungs, pancreases, and skeletal muscle; and cells such as neurons and oligodendrocytes, immune cells, and endothelial cells for making blood vessels.
  • the strategy to achieve this goal is to disrupt key genes that are important for the development of specific organs. These genes are evaluated using gene editing technology to knockout specific genes to determine which genes alone or in combination can give rise to specific organs or cell types when knocked out in murine and porcine blastocysts.
  • the gene knockouts in blastocysts can create a niche in which normal syngeneic or xenogeneic stem cells should occupy to contribute to the development of the desired organ or cell (FIG.
  • Novel gene editing and gene modulation technologies using TALENS, CRISPR, and synthetic porcine artificial chromosomes are used to knockout desired target genes and to enhance the function of other genes that can minimize off -target effects.
  • Human stem cells are vetted to determine which type of stem cell gives rise to a robust replication of specific human organs and cells. This issue is addressed by evaluating the contribution of various human stem cells to the inner cell mass of porcine blastocysts and to the developing chimeric fetus. The interactions among these three technical areas are important to the successful achievement of creating authentic human organs and cells.
  • Humanized refers to an organ or tissue harvested from a non-human animal whose protein sequences and genetic complement are more similar to those of humans than the non-human host.
  • Organ refers to a collection of tissues joined in a structural unit to serve a common function.
  • tissue refers to a collection of similar cells that together carry out a specific function.
  • Meganuclease as used herein are another technology useful for gene editing and are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome). Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
  • targeted gene refers to a site of chromosomal DNA that is selected for endonuclease attack by design of the endonuclease system, e.g., a TALENs or CRISPR.
  • Gene editing refers to choosing a gene and altering it.
  • Random insertions, gene trapping, and the like are not gene editing.
  • Examples of gene edits are, at targeted sites, gene knockouts, adding nucleic acids, removing nucleic acids, elimination of all function, introgression of an allele, a hypermorphic alteration, a hypomorphic alteration, and a replacement of one or more alleles.
  • the term "knockout, inactivated, and disrupted" and variants thereof are used interchangeably herein to mean that a gene expression product is eliminated or greatly reduced, by any menas, so that the gene's expression no longer has a significant impact on the animal as a whole. These terms are sometimes used elsewhere to refer to observably reducing the role of a gene without essentially eliminating its role.
  • a gene product is functional only if it fulfills its normal (wild-type) functions.
  • Disruption of the gene prevents expression of a functional factor encoded by the gene and comprises an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal.
  • the disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene.
  • replacement of an allele means the change is made from the native allele to the exogenous allele without indels or other changes except for, in some cases, degenerate substitutions.
  • degenerate substitution means that a base in a codon is changed to another base without changing the amino acid that is coded.
  • the degenerate substitution may be chosen to be in an exon or in an intron.
  • One use for a degenerate substitution is to create a restriction site for easy testing of a presence of the introgressed sequence.
  • the endogenous allele is also referred to herein as the native allele.
  • gene is broad and refers to chromosomal DNA that is expressed to make a functional product.
  • select is used to refer to the ability to identify and isolate the cells for further use; there were no expressible reporter genes anywhere in the process, which is a highly significant advantage that distinguishes this process from many other approaches.
  • blastocyst is used broadly herein to refer to embryos from two cells to about three weeks.
  • embryo is used broadly to refer to animals from zygote to live birth.
  • gametogenesis means the production of haploid sex cells (ova and spermatozoa) that each carry one-half the genetic compliment of the parents from the germ cell line of each parent.
  • the production of spermatozoa is spermatogenesis.
  • the fusion of spermatozoa and ova during fertilization results in a zygote cell that has a diploid genome.
  • gametogenic cell refers to a progenitor to an ovum or sperm, typically a germ cell or a spermatogonial cell.
  • large vertebrate refers to simians, livestock, dogs, and cats.
  • livestock refers to animals customarily raised for food, such as cattle, sheep, goats, avian (chicken, turkey), pigs, buffalo, and fish.
  • cognate refers to two biomolecules that typically interact, for example, a receptor and its ligand.
  • one of the biomolecules may be designed with a sequence to bind with an intended, i.e., cognate, DNA site or protein site.
  • insertion is used broadly to mean either literal insertion into the chromosome or use of the exogenous sequence as a template for repair.
  • exogenous nucleic acid means a nucleic acid that is added to the cell or embryo, regardless of whether the nucleic acid is the same or distinct from nucleic acid sequences naturally in the cell.
  • nucleic acid fragment is broad and includes a chromosome, expression cassette, gene, DNA, RNA, mRNA, or portion thereof.
  • the cell or embryo may be, for instance, chosen from the group consisting non-human vertebrates, non- human primates, cattle, horse, swine, sheep, chicken, avian, rabbit, goats, dog, cat, laboratory animal, and fish.
  • operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid.
  • replacement of an allele refers to a non-meiotic process of copying an exogenous allele over an endogenous allele.
  • Genes have alleles. Genotypes are homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. Alleles are alternative forms of a gene (one member of a pair) that are located at a specific position on a specific chromosome. Alleles determine distinct traits. Alleles have basepair (bp) differences at specific positions in their DNA sequences (distinguishing positions or bp) that give rise to the distinct trait and distinguish them from each another, these distinguishing positions serve as allelic markers.
  • Alleles are commonly described, and are described herein, as being identical if they have the same bases at distinguishing positions; animals naturally have certain variations at other bp in other positions. Artisans routinely accommodate natural variations when comparing alleles.
  • the term exactly identical is used herein to mean absolutely no bp differences or indels in a DNA alignment.
  • progeny that result from this complementation can carry cells of each genotype.
  • genes of the host embryo are edited to produce a knock out or otherwise make a non-functional gene.
  • human stem cells When human stem cells are injected into the gene edited blastocyst, they can rescue or "complement" the defects of the host (edited) genome.
  • the gene or genes that are knocked out support the growth of a particular organ or tissue, the resulting complementation produced tissue can be the result of the growth and differentiation of the non- edited, e.g., stem cell derived genotype.
  • the resulting tissue or organ can be composed of human cells. In this way, fully human organs can be produced, in vivo, using an other animal as a host for the complementation produced organ.
  • multiple genes may be responsible for the growth and differentiation of any particular organ or tissue
  • processes for multiplex gene edits are also described.
  • Multiple genes can be modified or knocked out in a cell or embryo that may be used for research or to make whole chimeric animals.
  • These embodiments include the complementation of cell or organ loss by selective depopulation of host niches.
  • These inventions provide for rapid creation of animals to serve as models, food, and as sources of cellular and a cellular products for industry and medicine.
  • FIG. 1 provides a schematic description of the problem and the proposed approach for providing personalized human organs and tissues to those in need using swine as a host animal.
  • IC induced pluripotent stem cells
  • FIG. 2 A has a timeline that illustrates why it takes several years using single edits to make livestock that have only two edited alleles, with the time being about six years for cattle. Edited, in this context, refers to choosing gene and altering it.
  • a gene of interest has to be edited, for instance knocked out (KO), in cultured somatic cells that are cloned to create a heterozygous calf with a targeted KO.
  • KO knocked out
  • the heterozygotes would be raised to maturity for breeding, about 2 years old for cattle, to generate first-generation (Fl) male and female heterozygous calves, which would be bred with each other to generate a homozygous knockout calf (F2).
  • F2 first-generation
  • F2 homozygous knockout calf
  • Swine for example, have a larger number of offspring per mating and a gestational time that is roughly half that of cattle but the time to make multiple edits can require many years. Moreover, schemes that minimize time with aggressive inbreeding may not be reasonably possible for multiple edits. Also, serial cloning is undesirable from a process and an outcome standpoint, especially if the animals are to be useful as livestock or laboratory models.
  • FIG. 3 shows multiple edits being made in a first-generation animal (F0).
  • F0 first-generation animal
  • Embryos are prepared directly or by cloning with two or more edits independently chosen to be heterozygotes or homozygotes and placed in surrogate females to gestate.
  • the resultant animals are F0 generation founders.
  • a plurality of embryos may be prepared and placed in one or more surrogates to produce progeny of both genders, or well-known techniques of embryo-splitting may be used to make a plurality of clonal embryos.
  • Livestock such as pigs that typically produce a litter with both genders may be crossed and propagated.
  • An embodiment is a method of making genetic edits in a vertebrate cell or embryo at a plurality of target chromosomal DNA sites comprising introducing into a vertebrate cell or embryo: a first targeted endonuclease directed to a first target chromosomal DNA site and a first homology directed repair (HDR) template homologous to the first target site sequence; and a second targeted endonuclease directed to a second target chromosomal DNA site and a second HDR template homologous to the second target site sequence, with the first HDR template sequence replacing the native chromosomal DNA sequence at the first target site and the second HDR template sequence replacing the native chromosomal DNA sequence at the second target site sequence.
  • HDR homology directed repair
  • results herein show that too much or too little endonuclease and/or HDR template can have a negative effect, which may have confounded prior research in this area.
  • targeted endonucleases can be designed and made correctly but nonetheless fail because they are too efficient.
  • the population of successfully modified cells often does not improve over time.
  • Artisans modifying cells normally look for longevity of the cell and modification as an indicator of stability and health for successful cloning or other uses. But that expectation has often not been helpful in the multiplexing processes herein.
  • homologous recombination (HR) introgression efficiencies are variable in the multiplex approach as compared to a single-locus introgression. Some loci were very sensitive but others had large drops in efficiency.
  • HR homologous recombination
  • HDR processes taught herein may be readily distinguished by the edits, and resultant organisms, being made only at the intended target sites.
  • inventive HDR editing embodiments can be performed free of insertion of extra gene copies and/or free of disruption of genes other than those targeted by the endonucleases.
  • specific edits are made at one location because the HDR template sequence is not copied into sites without appropriate homology.
  • Embodiments include organisms and processes wherein an exogenous allele is copied into chromosomal DNA only at the site of its cognate allele.
  • HDR-based editing An advantage of HDR-based editing is that the edits can be chosen. In contrast, other attempts, by non-homologous end joining (NHEJ) processes, can make indels at multiple positions such that the indels cancel each other out without making a frame shift. This problem becomes significant when multiplexing is involved. But successful use of HDR provides that the edits can be made to ensure that, if desired, the target gene has an intended frame shift. Moreover, allelic replacement requires HDR and cannot be accomplished by NHEJ, vector- driven insertion of nucleic acids, transposon insertions, and the like. Moreover, choosing organism that are free of unwanted edits further increases the degree of difficulty.
  • NHEJ non-homologous end joining
  • An embodiment of the invention provides processes for creating multiple targeted gene knockouts or other edits in a single cell or embryo, a process referred to herein as multiplex gene knockouts or editing.
  • allelic identity is to align the chromosomal DNA in the altered organism with the chromosomal DNA of the exogenous allele as it is recognized in nature.
  • the exogenous allele can have one or more allelic markers.
  • the DNA alignment upstream and downstream of the markers can be identical for a certain distance. Depending on the desired test, this distance may be from, e.g., 10 to 4000 bp.
  • an HDR template can be expected to create a sequence that has exactly identical, the bases on either side of the templated area can, of course, have some natural variation. Artisans routinely distinguish alleles despite the presence of natural variations.
  • centimorgan In genetics, a centimorgan (cM, also called a map unit (m.u.)) is a unit that measures genetic linkage. It is defined as the distance between chromosome positions (loci or markers of loci) for which the expected average number of intervening chromosomal crossovers in a single generation is 0.01. Genes that are close to each other have a lower chance of crossing over compared to genes that are distant from each other on the chromosome. Crossing over is a very rare event when two genes are right next to each other on the chromosome. Crossing over of a single allele relative to its two neighboring alleles is so improbable that such an event must be the product of genetic engineering. Even in the case where animals of the same breeds are involved, natural versus engineered allele replacement can be readily determined when the parents are known. And parentage can be determined with a high degree of accuracy by genotyping potential parents. Parent determination is routine in herds and humans.
  • Embodiments include multiplex gene editing methods that are simultaneous.
  • simultaneous is in contrast to a hypothetical process of treating cells multiple times to achieve multiple edits, as in serial knockouts or serial cloning or intervening cycles of animal breeding.
  • Simultaneous means being present at a useful concentration at the same time, for instance multiple targeted endonucleases being present.
  • the processes can be applied to zygotes and embryos to make organisms wherein all cells or essentially all cells have edited alleles or knockouts.
  • Essentially all cells in the context of a knockout for instance, refers to knocking the gene out of so many cells that the gene is, for practical purposes, absent because its gene products are ineffective for the organism's function.
  • the processes modify cells, and cells in embryos, over a minimal number cell divisions, preferably about zero to about two divisions.
  • Embodiments include a quick process or a process that takes place over various times or numbers of cell divisions is contemplated, for instance: from 0 to 20 replications (cell divisions).
  • All values and ranges within the expressly stated limits are contemplated, e.g., about 0 to about 2 replications, about 0 to about 3 replications, no more than about 4 replications, from about 0 to about 10 replications, 10-17; less than about 7 days, less than about 1, about 2, about 3, about 4, about 5, or about 6 days, from about 0.5 to about 18 days, and the like.
  • the term low- passage refers to primary cells that have undergone no more than about 20 replications.
  • Example 1 describes experiments that attempted, successfully, to use HDR editing to knockout two genes at once and, further, to be able to select cells that are homozygous for both knockouts or heterozygous for each knockout.
  • Cells were treated to introduce a first and a second targeted endonuclease (each being a TALENS pair) directed to, respectively, a first gene (Recombination Activating Gene 2, RAG2) and a second gene target (Interleukin Receptor 2, gamma, IL2Rg or ILR2y).
  • the TALENS had to be designed to target intended sites and made in adequate amounts. The treatment of the cells took less than five minutes.
  • Electroporation was used but there are many other suitable protein or DNA introducing-processes described herein.
  • the cells were then cultured so that they formed individual colonies of cells that each descended from a single treated cell. Cells from the various colonies were tested after 3 days or 11 days.
  • the rate of knockout of RAG2 was about six times higher than the rate of knockout of IL2Rg; apparently some genes are more difficult to knockout than others.
  • the efficiency of knocking out both genes was high and cells heterozygous or homozygous for both knockouts were successfully identified.
  • dosage of TALEN mRNA and HDR template had specific and non-specific effects.
  • Example 2 see FIGs. 5A-5D, describes experiments that had the same goal of multiplex HDR editing but for different genes.
  • the first gene target was Adenomatous polyposis coli (APC).
  • the second gene target was p53 (the TP53 gene). Cells homozygous for both knockouts and cells heterozygous for both knockouts were detected and isolated.
  • Example 3 see FIGs. 6-9, describes multiplex HDR editing to knockout 2-5 genes.
  • Cas9/CRISPRs vectors were introduced into cells to express
  • modification levels were below detection (data not shown); however, other data points to RGEN multiplex, e.g., Example 9 below.
  • Four genes were found to be edited in all experiments and five genes in one experiment.
  • APC APC, LDLR, RAG 2, IL2Rg, p53.
  • cells and embryos with multiplex knockouts are embodiments of the invention, as well as animals made thereby.
  • Example 4 describes some detailed processes for making various animals and refers to certain genes by way of example.
  • Example 5 describes examples of CRISPR/Cas9 design and production.
  • Example 6 provides further examples of multiplex gene editing with targeted nucleases driving HDR processes.
  • GATA4 GATA binding protein 4
  • NKX2-5 NKX2-5)
  • MEMP1 Mesoderm Posterior Protein 1
  • the objective was to create biallelic knockouts for each gene for use in complementation studies.
  • the process was about 0.5% efficient as 2 clones had the intended biallelic HDR at each gene.
  • the given genes knocked out singly or in combination genes can cause a failure to thrive genotype and early embryonic lethality without complementation. Artisans can appreciate that knockout of these genes individually and interbreeding of heterozygotes to obtain triple knockouts (about 1/66 chance) for FTT and complementation studies is not feasible in livestock.
  • Example 7 provides data that TALENs and Cas9/CRISPR can be mixed to perform multiplex editing of genes. Some genes/alleles are more readily targeted by a TALEN, or Cas9/CRISPR and that the situation may arise that multiplexing must be done with a combination of these tools.
  • EIF4GI Eukaryotic Translation Initiation Factor 4GI
  • RELA p65
  • TALENs and RGENs may be used together or separately for multiplexing Combinations including, for example, 1, 2, 3 4, 5, 6, 7, 8, 9 or 10 TALENs with 1, 2, 3 4, 5, 6, 7, 8, 9 or 10 RGEN reagents, in any combination.
  • Chimeras can be made by preparing a host blastocyst and adding a donor cell from a donor animal.
  • the resultant animal can be a chimera that has cells that originate from both the host and the donor.
  • Some genes are important for the embryo to create certain kinds of cells and cell lineages. When such a gene is knocked out in the host cells, the introduction of a donor cell that has the missing gene can result in those cells and cell lineages being restored to the host embryo; the restored cells have the donor genotype. Such a process is referred to as a complementation process.
  • Pdxl pancreatic and duodenal homeobox 1
  • PSCs pluripotent stem cells
  • xenogenic pluripotent stem cells including human induced PSCs.
  • xenotransplantation has been considered a potential solution to the organ/tissue shortage for greater than 40 years. The fact that no genes were knocked out to disable the formation of the pancreas is significant.
  • Knocking out even one gene in a large vertebrate is a significant investment of resources using conventional processes.
  • overexpression of a gene product in a cell is readily achieved using the present state of the art, for instance, with a plasmid or a vector that places multiple gene cassette copies into the genome. Adding expression of a gene is easier than targeting a gene and knocking it out.
  • the ability to prevent organogenesis by overexpression of a gene product is believed to be unusual at this time.
  • limitations in the ability to engineer large animal genomes can be significant. Nonetheless, the pig is the preferred donor animal for xenotransplantation due to its similarity in size and physiology to humans as well as its high fecundity and growth rate.
  • FIG 10 depicts a multiplex process used herein to make gene knockouts or other gene edits as applied in the context of chimeras.
  • Low-passage primary somatic cells are made with gene knockouts. Cells with exactly the desired distribution of heterozygosity and homozygosity for the knockouts are isolated. These cells are used in cloning to make an embryo that is allowed to develop as a host blastocyst.
  • a donor embryo is established and used as a source of donor cells that provide genes to populate the niche created by the knockouts.
  • the donor cells are introduced into the host blastocyst and reproduce with the host cells to form a chimera having both host and donor cells.
  • the embryo is transferred to a surrogate female and gestated.
  • the progeny of the chimera have host genotypes when the host cells form the gametes.
  • Chimeras have their gender determined by their host blastocyst.
  • FIG 11 illustrates a failure to thrive phenotype (FTT) complementation process.
  • FTT refers to animals that are not expected to live to an age of sexual maturity.
  • a host embryo is provided with an FTT genotype and phenotype.
  • Multiplex processes are ideal because the FTTs available by knockout of just one gene are limited and are not known for some organs and tissues.
  • the donor cells provide the genes missing in the FTT and provide the missing cell types.
  • the embryo can be a large vertebrate animal and the knockouts can be multiplex, e.g., 2- 25 genes.
  • targeted endonucleases can be used to achieve a knockout.
  • an IL2Rg-/y RAG2-/-knockout is the FTT because the host is essentially missing immune functions. But the donor cells do not have those genes missing and the resultant chimera has an essentially normal phenotype for purposes of being able to raise and maintain the animal. But the progeny has the FTT phenotype. The animals can thus be maintained and FTT animals conveniently produced.
  • the chimeras can be any combination of heterozygous and homozygous for the knockouts. Processes for making chimera are thus described that are F0 generation animals that produce failure to thrive (FTT) phenotypes when other processes require an additional generation, or more. Chimera normally pass on the genetics of the host cells. Disclosed herein, however, are alternative chimeras that pass the donor cell genetics to their progeny and not the host cell genetics. It turns out that switching the genetic inheritance can create some useful opportunities.
  • an embryo labeled as G " host is depicted.
  • the embryo has been prepared with nonfunctional gametes.
  • a donor blastocyst is prepared and used as a source of donor cells.
  • the donor cells provide the genes and cell lineages that are needed to make donor gametes.
  • the resultant chimera has the gametes of the donor cells and creates progeny having donor cell genetics.
  • the host embryo is a male Brahman bull.
  • the donor cells are from a double-muscled bull.
  • the chimera has a Brahman bull phenotype but its progeny are double muscled.
  • the host and donors may be from the same or different breeds or same or different species.
  • the host has been prepared to be sterile, meaning that it cannot sexually reproduce.
  • sterile animals may be used to make gametes that are nonfunctional, e.g., immotile sperm, or not make gametes at all, e.g., with early gametogenesis being disrupted.
  • the donor cells may be, for instance, wild-type cells, cells from animal breeds having desirable traits, or genetically modified cells.
  • Embodiments of the invention include chimeric sterile animals, such as chimeric livestock, that have a genetic modification to a chromosome that prevents gametogenesis or spermatogenesis.
  • the chromosome may be an X chromosome, a Y chromosome, or an autosome.
  • the modification may include a disruption of an existing gene.
  • the disruption may be created by altering an existing chromosomal gene so that it cannot be expressed, or by genetically expressing factors that can inhibit the transcription or translation of a gene.
  • One embodiment is a knockout of spermatogonial stem cells (SSC) in the host.
  • the animal may be made with donor cells that have desirable genetics and supplies SSC cells that make gametes with the donor genotype.
  • SSC spermatogonial stem cells
  • Some genes are disrupted in combination to produce one or more effects that cause infertility, for instance, combinations of: Acr/Hl.1/Smcp, Acr/Tnp2/Smcp, Tnp2/Hl. l/Smcp, Acr/Hlt/Smcp, Tnp2/Hlt/Smcp ( ayernia K; Drabent B; Meinhardt A; Adham IM; Schwandt I; Muller C; Sancken U; Kleene KC; Engel W Triple knockouts reveal gene interactions affecting fertility of male mice. Mol. Reprod. Dev 70(4):406- 5 16, 2005).
  • Embodiments include a first line of animals with a knockout of a first gene or genes and a second line of animals with a knockout of a second gene or genes so that male progeny of the lines are infertile.
  • Embodiments include the production of genetically and genomically sterile animals that can serve as hosts for donated genetic material. Sexual intercourse by the host can lead to reproduction of the donor's genetic material.
  • a group of genetically sterile animals can be used to disseminate identical genes from a single donor by sexual reproduction so that many donor progeny may be rapidly generated.
  • Embodiments include animals that are modified to produce only one gender of animal so that users receiving the animals are not be able to easily breed the animals with the traits.
  • Embodiments include making a genetic modification to cells or embryos to inactivate a gene or plurality of genes selective for gametogenesis or spermatozoa activity.
  • One process of genetic modification involves introduction of a targeted nuclease, e.g., a Cas9/CRISPR or mRNA for a TALEN pair that specifically binds to the gene.
  • An animal is cloned from the cells or the modified embryo is directly raised in a surrogate mother.
  • the animal may be a livestock animal or other animal.
  • Gametogenesis may be blocked at an early stage.
  • spermatozoa activity may be disrupted that is important for fertility but is not otherwise important to the animal. The animal is thus sterile because it cannot sexually reproduce: however, ARTs may be used to create progeny from the modified sperm.
  • a donor animal that has desirable genetic traits (as a result of breeding and/or genetic engineering) is selected.
  • two, three, or more genes (2-25) may be simultaneously knocked out to produce an FO generation with the desired combination of alleles. If homozygosity for all of the knockouts creates an FTT, then one option is to make the founders homozygous for all of the knockouts except for one - or whatever the minimum heterozygosity should be for that situation.
  • the one heterozygote gene can allow for a non-FTT phenotype.
  • the multiplex knockouts can be used in combination with complementation to make fostering chimera that have FTT progeny. This process can eliminate generations in the creation of a multiple knockout animal.
  • One group of embodiments relates to immunodeflcient pigs or other livestock and processes of making them. These embodiments are examples of multiplex edits, e.g., knockouts that take advantage of the opportunity to manage selection of homozygous and heterozygous nockout genotypes. These demonstrate the power of multiplex to rapidly establish founder lines. They also include further aspects of the inventions that involve making chimeras.
  • the pig is the most relevant, non-primate animal model that mimics the size and physiology of humans.
  • fully immunodeflcient pigs are not widely available because (1) single gene knockout (KO) is usually not sufficient, (2) intercrossing to create multi- locus null animals is extremely costly and depending on the number of Kos may be possible, and (3) only small scale germ-free facilities are available for pigs.
  • embodiments include large vertebrate animals with a knockout of both & G2 andIL2Rg (i.e., RG-KO). The genes can be knocked out of somatic cells that are then used for cloning to produce a whole animal.
  • embryos can be treated to knockout the genes, with the animals being derived directly from the embryos.
  • the multiplex gene-targeting platform can simultaneously disrupt of T, B and NK cell development in the pig. Accordingly, animals made without such cells can be made directly with the methods herein, as F0 founders, but the phenotype is FTT.
  • the editing of food animal genomes can be greatly accelerated by editing numerous loci at the same time, saving generations of animal breeding that would be carried out to bring together alleles that are generated instead one at a time.
  • some agricultural traits are complex, meaning that they are manifest as a result of the influence of alleles at more than one gene (from 2 to hundreds). For example, polymorphisms at DGAT, ABCG2, and a
  • Livestock cells or embryos can be subjected to multiplex editing of numerous genes, including various agricultural targets: one or more of AC AN, AMELY, BLG, BMP IB (FecB), DAZL, DGAT, Eif4GI, GDF8, Horn-poll locus, IGF2, CWC15,
  • Some traits like cancer, are caused on the basis of mutations at multiple genes (see APC/p53).
  • numerous disease traits are so-called Complex traits that manifest as a result of the influence of alleles at more than one gene.
  • diabetes, metabolism, heart disease, and neurological diseases are considered complex traits.
  • Embodiments include animal models that are heterozygous and homozygous for individual alleles, or in combination with alleles at other genes, in different combinations.
  • mature onset diabetes of the young loci cause diabetes individually and additively, including; MODY 1 (HNF4a), MODY 2 (GCK), MODY 3 (HNFla), MODY 4 (Pdxl), MODY 5 (HNF- ⁇ ), MODY 6 (eurogenic differentiation 1), MODY 7 (KLF11), MODY 8 (CEL), MODY 9 (PAX4), MODY 10 (INS), MODY 11 (BLK).
  • MODY 1 HNF4a
  • MODY 2 GCK
  • MODY 3 HNFla
  • MODY 4 Pdxl
  • MODY 5 HNF- ⁇
  • MODY 6 eurogenic differentiation 1
  • MODY 7 KLF11
  • MODY 8 CEL
  • MODY 9 PAX4
  • MODY 10 INS
  • MODY 11 BLK
  • Livestock cells or embryos can be subjected to multiplex editing of numerous genes for animal modelling, including various disease modeling targets: APC, ApoE, DMD, GHRHR, HR, HSD11B2, LDLR, NF1, NPPA, NR3C2, p53, PKD1, Rbm20, SCNN1G, tP53, DAZL, FAH, HBB, IL2RG, PDX1, PITX3, Runxl, RAG2, GGTA.
  • Embodiments include cells, embryos, and animals with one or more of the above targets being edited, e.g., KO.
  • spermatogonial stem cells offer a second method genetic modification of livestock. Genetic modification or gene edits can be executed in vitro in spermatogonial stem cells isolated from donor testes. Modified cells are transplanted into germ cell-depleted testes of a recipient. Implanted spermatogonial stem cells produce sperm that carry the genetic modification(s) that can be used for breeding via artificial insemination or in vitro fertilization (IVF) to derive founder animals.
  • IVF in vitro fertilization
  • Complementation of nullomorphic cell or organ loss by selective depopulation of host niches Multiplex editing can be used to purposefully ablate cells or organs from a specific embryonic or animal niche, creating an environment conducive to better donor cell integration, proliferation, and differentiation, enhancing their contribution by complementation of orthologous cells, tissues or organs in the embryo, fetus or animal.
  • the animal with the empty niche is a deficiency carrier because it has been created to have a deficiency that can be filled by donor cells and genes.
  • Specific examples include the recipient-elimination, and donor-rescue of gametogenic cell lineages (DAZL, VASA, MIWI, PIWI, and so forth.).
  • multiplex gene editing can be used to induce congenital alopecia, providing opportunity for donor derived cells to participate in hair folliculogenesis.
  • the genes considered for multiplex gene editing to cause alopecia include those identified in OMIM and thru Human Phenotype Ontology database; DCAF17, VDR, PNPLA1, HRAS, Telomerase-vert, DSP, SNRPE, RPL21, LAM A3, UROD, EDAR, OFD1, PEX7, COL3A1, ALOX12B, HLCS, NIPAL4, CERS3, ANTXR1, B3GALT6, DSG4, UBR1, CTC1, MBTPS2 ,UROS, ABHD5, NOP10, ALMS1, LAMB3, EOGT, SAT1, RBPJ, ARHGAP31, ACVR1, IKBKG, LPAR6, HR, ATR, HTRA1, AIRE, BCS1L, MCCC2, DKC1, PORCN, EBP,
  • the ablation of organs or tissues in pigs or other vertebrates and growth of organs or tissues from human origins is particularly useful as a source of medical organs or tissues.
  • PRKDC PRKDC
  • BCLl la BMI1, CCR5, CXCR4, DKKl, ETV2, FLU, FLKl, GATA2, GATA4, HHEX, C-KIT, LMXIA, MYF5, MYODl, MYOG, NKX2-5, NR4A2, PAX3, PDXl, PITX3, Runxl, RAG2, GGTA, HR, HANDII, TBX5.
  • Embodiments include targeting one, two, or more (2-25) of the above targets in a multiplex approach or by other approaches. Edited Genes
  • Animals may be made that are mono-allelic or bi-allelic for a chromosomal modification, using methods that either leave a genetically expressible marker in place, allow for it to be bred out of an animal, or by methods that do not place such a marker in the animal.
  • methods of homologous dependent recombination have been used to make changes to, or insertion of exogenous genes into, chromosomes of animals.
  • Tools such as TALENs and recombinase fusion proteins, as well as conventional methods, are discussed elsewhere herein.
  • Gestation length and maturation to reproduction age for livestock such as pigs and cattle is a significant barrier to research and to production.
  • generation of a homozygous knockout from heterozygous mutant cells (both sexes) by cloning and breeding would require 16 and 30 months for pigs and cattle respectively.
  • the ability to routinely generate bi-allelic KO cells prior to SCNT is a significant advancement in large animal genetic engineering.
  • Bi-allelic knockout has been achieved in immortal cells lines using other processes such as ZFN and dilution cloning (Liu et al., 2010).
  • Another group recently demonstrated bi-allelic KO of porcine GGTAl using commercial ZFN reagents (Hauschild et al., 2011) where bi-allelic null cells could be enriched by FACS for the absence of a GGTAl- dependent surface epitope. While these studies demonstrate certain useful concepts, they do not show that animals or livestock could be modified because simple clonal dilution is generally not feasible for primary fibroblast isolates (fibroblasts grow poorly at low density) and biological enrichment for null cells is not available for the majority of genes.
  • Targeted nuclease-induced homologous recombination can be used so as to eliminate the need for linked selection markers.
  • TALENs may be used to precisely transfer specific alleles into a livestock genome by homology dependent repair (HDR).
  • HDR homology dependent repair
  • a specific 1 lbp deletion (the Belgian Blue allele) (Grobet et al., 1997; Kambadur et al., 1997) was introduced into the bovine GDF8 locus (see U.S. 2012/0222143).
  • the btGDF8.1 TALEN pair cleaved up to 16% of chromosomes at the target locus.
  • HDR Homology directed repair
  • Homology directed repair is a mechanism in cells to repair ssDNA and double stranded DNA (dsDNA) lesions. This repair mechanism can be used by the cell when there is an HDR template present that has a sequence with significant homology to the lesion site.
  • Specific binding refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like.
  • Specific hybridization is a form of specific binding between nucleic acids that have complementary sequences.
  • Proteins can also specifically bind to DNA, for instance, in TALENs or CRISPR/Cas9 systems or by Gal4 motifs.
  • Introgression of an allele refers to a process of copying an exogenous allele over an endogenous allele with a template-guided process.
  • the endogenous allele might actually be excised and replaced by an exogenous nucleic acid allele in some situations but present theory is that the process is a copying mechanism. Since alleles are gene pairs, there is significant homology between them.
  • the allele might be a gene that encodes a protein, or it could have other functions such as encoding a bioactive RNA chain or providing a site for receiving a regulatory protein or RNA.
  • the HDR template is a nucleic acid that comprises the allele that is being introgressed.
  • the template may be a dsDNA or a single-stranded DNA (ssDNA).
  • ssDNA templates are preferably from about 20 to about 5000 residues although other lengths can be used. Artisans can immediately appreciate that all ranges and values within the explicitly stated range are contemplated; e.g., from 500 to 1500 residues, from 20 to 100 residues, and so forth.
  • the template may further comprise flanking sequences that provide homology to DNA adjacent to the endogenous allele or the DNA that is to be replaced.
  • the template may also comprise a sequence that is bound to a targeted nuclease system, and is thus the cognate binding site for the system's DNA-binding member.
  • Genome editing tools such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have impacted the fields of biotechnology, gene therapy and functional genomic studies in many organisms. More recently, RNA-guided endonucleases (RGENs) are directed to their target sites by a complementary RNA molecule.
  • the Cas9/CRISPR system is a REGEN.
  • tracrRNA is another such tool.
  • These are examples of targeted nuclease systems: these system have a DNA-binding member that localizes the nuclease to a target site. The site is then cut by the nuclease.
  • TALENs and ZFNs have the nuclease fused to the DNA- binding member.
  • Cas9/CRISPR are cognates that find each other on the target DNA.
  • the DNA- binding member has a cognate sequence in the chromosomal DNA.
  • the DNA-binding member is typically designed in light of the intended cognate sequence so as to obtain a nucleolytic action at nor near an intended site. Certain embodiments are applicable to all such systems without limitation; including, embodiments that minimize nuclease re-cleavage, embodiments for making SNPs with precision at an intended residue, and placement of the allele that is being introgressed at the DNA-binding site.
  • TALEN as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN.
  • TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site.
  • TALENs that work together may be referred to as a left- TALEN and a right-TALEN, which references the handedness of DNA or a TALEN-pair.
  • each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence.
  • the residues may be assembled to target a DNA sequence.
  • a target site for binding of a TALEN is determined and a fusion molecule comprising a nuclease and a series of RVDs that recognize the target site is created.
  • the nuclease cleaves the DNA so that cellular repair machinery can operate to make a genetic modification at the cut ends.
  • TALEN means a protein comprising a Transcription Activator-like (TAL) effector binding domain and a nuclease domain and includes monomeric TALENs that are functional per se as well as others that require dimerization with another monomeric TALEN.
  • the dimerization can result in a homodimeric TALEN when both monomeric TALEN are identical or can result in a heterodimeric TALEN when monomeric TALEN are different.
  • TALENs have been shown to induce gene modification in immortalized human cells by means of the two major eukaryotic DNA repair pathways, non-homologous end joining (NHEJ) and homology directed repair. TALENs are often used in pairs but monomeric TALENs are known.
  • NHEJ non-homologous end joining
  • Cells for treatment by TALENs include a cultured cell, an immortalized cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, a blastocyst, or a stem cell.
  • a TAL effector can be used to target other protein domains (e.g., non- nuclease protein domains) to specific nucleotide sequences.
  • a TAL effector can be linked to a protein domain from, without limitation, a DNA 20 interacting enzyme (e.g., a methylase, a topoisomerase, an integrase, a transposase, or a ligase), a transcription activators or repressor, or a protein that interacts with or modifies other proteins such as histones.
  • a DNA 20 interacting enzyme e.g., a methylase, a topoisomerase, an integrase, a transposase, or a ligase
  • a transcription activators or repressor e.g., a protein that interacts with or modifies other proteins such as histones.
  • TAL effector fusions include, for example, creating or modifying epigenetic regulatory elements, making site- specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.
  • nuclease includes exonucleases and endonucleases.
  • endonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule.
  • Non-limiting examples of endonucleases include type II restriction endonucleases such as Fokl, Hhal, Hindlll, Notl, Bbv X, EcoKl, Bglll, and Alwl.
  • Endonucleases comprise also rare- cutting endonucleases when having typically a polynucleotide recognition site of about 12-45 basepairs (bp) in length, more preferably of 14-45 bp.
  • Rare-cutting endonucleases induce DNA double- strand breaks (DSBs) at a defined locus.
  • Rare-cutting endonucleases can for example be a targeted endonuclease, a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl or a chemical endonuclease.
  • ZFN Zinc-Finger nuclease
  • a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence.
  • endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences.
  • TFOs triplex-forming oligonucleotides
  • Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention.
  • endonuclease examples include I-See I, I-Chu L I-Cre I, I-Csm I, Pi-See L PI-Tti L PI- Mtu I, I-Ceu I, I-See I 1- See III, HO, Pi-Civ I, Pl-Ctr I PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I PI-Mav I PI-Meh I, PI-Mfu I PI-Mfl I, PI-Mga I PI-Mgo I, PI-Min I PI-Mka I PI-Mle I, PI- Mma I, PI- 30 Msh I PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I PI-Rma I, PI- Spb I, PI-S
  • a genetic modification made by TALENS or other tools may be, for example, chosen from the list consisting of an insertion, a deletion, insertion of an exogenous nucleic acid fragment, and a substitution.
  • a target DNA site is identified and a TALEN-pair is created that can specifically bind to the site.
  • the TALEN is delivered to the cell or embryo, e.g., as a protein, mRNA or by a vector that encodes the TALEN.
  • the TALEN cleaves the DNA to make a double-strand break that is then repaired, often resulting in the creation of an indel, or incorporating sequences or polymorphisms contained in an accompanying exogenous nucleic acid that is either inserted into the chromosome or serves as a template for repair of the break with a modified sequence.
  • This template-driven repair is a useful process for changing a chromosome, and provides for effective changes to cellular chromosomes.
  • Some embodiments involve a composition or a method of making a genetically modified livestock and/or artiodactyl comprising introducing a TALEN-pair into livestock and/or an artiodactyl cell or embryo that makes a genetic modification to DNA of the cell or embryo at a site that is specifically bound by the TALEN-pair, and producing the livestock
  • TALEN TALEN and/or other factors may be introduced into a cell using any of many known techniques for introduction of proteins, RNA, mRNA, DNA, or vectors.
  • Genetically modified animals may be made from the embryos or cells according to known processes, e.g., implantation of the embryo into a gestational host, or various cloning methods.
  • a genetic modification to DNA of the cell at a site that is specifically bound by the TALEN means that the genetic modification is made at the site cut by the nuclease on the TALEN when the TALEN is specifically bound to its target site.
  • the nuclease does not cut exactly where the TALEN-pair binds, but rather at a defined site between the two binding sites.
  • Some embodiments involve a composition or a treatment of a cell that is used for cloning the animal.
  • the cell may be a livestock and/or artiodactyl cell, a cultured cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, or a stem cell.
  • an embodiment is a composition or a method of creating a genetic modification comprising exposing a plurality of primary cells in a culture to TALEN proteins or a nucleic acid encoding a TALEN or TALENs.
  • the TALENs may be introduced as proteins or as nucleic acid fragments, e.g., encoded by mRNA or a DNA sequence in a vector.
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to alter the genomes of higher organisms. ZFNs may be used in method of inactivating genes.
  • a zinc finger DNA-binding domain has about 30 amino acids and folds into a stable structure. Each finger primarily binds to a triplet within the DNA substrate. Amino acid residues at key positions contribute to most of the sequence-specific interactions with the DNA site. These amino acids can be changed while maintaining the remaining amino acids to preserve the necessary structure. Binding to longer DNA sequences is achieved by linking several domains in tandem. Other functionalities like non-specific Fokl cleavage domain (N), transcription activator domains (A), transcription repressor domains (R) and methylases (M) can be fused to a ZFPs to form ZFNs respectively, zinc finger transcription activators (ZFA), zinc finger transcription repressors (ZFR, and zinc finger methylases (ZFM).
  • N non-specific Fokl cleavage domain
  • A transcription activator domains
  • R transcription repressor domains
  • M methylases
  • nucleic acids may be introduced into cells, for knockout purposes, for inactivation of a gene, to obtain expression of a gene, or for other purposes.
  • nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double- stranded or single-stranded (i.e., a sense or an antisense single strand).
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
  • the target nucleic acid sequence can be operably linked to a regulatory region such as a promoter.
  • Regulatory regions can be porcine regulatory regions or can be from other species.
  • type of promoter can be operably linked to a target nucleic acid sequence.
  • promoters include, without limitation, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters responsive or unresponsive to a particular stimulus.
  • a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used (i.e., a constitutive promoter).
  • a beta-actin promoter such as the chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase (PGK) promoter can be used, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter.
  • HSV-TK herpes simplex virus thymidine kinase
  • CMV cytomegalovirus
  • a fusion of the chicken beta actin gene promoter and the CMV enhancer is used as a promoter. See, for example, Xu et al., Hum. Gene Ther. 12:563, 2001; and Kiwaki et al., Hum. Gene Ther. 7:821, 1996.
  • Additional regulatory regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, can sometimes be obtained without such additional elements.
  • a nucleic acid construct may be used that encodes signal peptides or selectable expressed markers.
  • Signal peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface).
  • selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B- phosphtransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT).
  • selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.
  • a sequence encoding a selectable marker can be flanked by recognition sequences for a recombinase such as, e.g., Cre or Flp.
  • the selectable marker can be flanked by loxP recognition sites (34-bp recognition sites recognized by the Cre recombinase) or FRT recognition sites such that the selectable marker can be excised from the construct.
  • loxP recognition sites 34-bp recognition sites recognized by the Cre recombinase
  • FRT recognition sites such that the selectable marker can be excised from the construct.
  • a transposon containing a Cre- or Flp- activatable transgene interrupted by a selectable marker gene also can be used to obtain transgenic animals with conditional expression of a transgene.
  • a promoter driving expression of the marker/transgene can be either ubiquitous or tissue-specific, which would result in the ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs).
  • Tissue specific activation of the transgene can be accomplished, for example, by crossing a pig that ubiquitously expresses a marker-interrupted transgene to a pig expressing Cre or Flp in a tissue-specific manner, or by crossing a pig that expresses a marker-interrupted transgene in a tissue-specific manner to a pig that ubiquitously expresses Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.
  • the exogenous nucleic acid encodes a polypeptide.
  • a nucleic acid sequence encoding a polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection).
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAGTM tag (Kodak, New Haven, CT).
  • Nucleic acid constructs can be introduced into embryonic, fetal, or adult
  • artiodactyl/livestock cells of any type including, for example, germ cells such as an oocyte or an egg, a progenitor cell, an adult or embryonic stem cell, a primordial germ cell, a kidney cell such as a PK-15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast, using a variety of techniques.
  • germ cells such as an oocyte or an egg
  • a progenitor cell such as an adult or embryonic stem cell
  • a primordial germ cell such as a PK-15 cell
  • an islet cell a beta cell
  • a liver cell or a fibroblast such as a dermal fibroblast
  • transposon systems the transcriptional unit of a nucleic acid construct, i.e., the regulatory region operably linked to an exogenous nucleic acid sequence, is flanked by an inverted repeat of a transposon.
  • transposon systems including, for example, Sleeping Beauty (see, U.S. 6,613,752 and U.S. 2005/0003542); Frog Prince (Miskey et al., Nucleic Acids Res. 31:6873, 2003); Tol2 (Kawakami, Genome Biology 8(Suppl. l):S7, 2007); Minos (Pavlopoulos et al., Genome Biology, 8(Suppl.
  • a transposase can be delivered as a protein, encoded on the same nucleic acid construct as the exogenous nucleic acid, can be introduced on a separate nucleic acid construct, or provided as an mRNA (e.g., an in vitro- transcribed and capped mRNA).
  • Nucleic acids can be incorporated into vectors.
  • a vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA.
  • a vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment.
  • Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non-viral vectors (e.g., transposons) used for gene delivery in animals have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence.
  • Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.
  • Plasmids and viral vectors are known.
  • Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, ⁇ -elements, Tol-2, Frog Prince, piggyBac).
  • plasmids which may also be a carrier of another type of vector
  • adenovirus e.g., adeno-associated virus (AAV)
  • lentivirus e.g., modified HIV-1, SIV or FIV
  • retrovirus e.g., ASV, ALV or MoMLV
  • transposons e.g., Sleeping Beauty, ⁇ -elements, Tol-2, Frog Prince, piggyBac.
  • nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids, e.g., synthetic bases or alternative backbones.
  • a nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand).
  • transgenic is used broadly herein and refers to a genetically modified organism or genetically engineered organism whose genetic material has been altered using genetic engineering techniques. A knockout artiodactyl is thus transgenic regardless of whether or not exogenous genes or nucleic acids are expressed in the animal or its progeny. Genetically modified animals
  • Animals may be modified using TALENs or other genetic engineering tools, including recombinase fusion proteins, or various vectors that are known.
  • a genetic modification made by such tools may comprise disruption of a gene.
  • Materials and methods of genetically modifying animals are further detailed in U.S. 8,518,701; U.S. 2010/0251395; and U.S. 2012/0222143 which are hereby incorporated herein by reference for all purposes; in case of conflict, the instant specification is controlling.
  • the term trans-acting refers to processes acting on a target gene from a different molecule (i.e., intermolecular).
  • a trans-acting element is usually a DNA sequence that contains a gene.
  • trans-acting gene codes for a protein (or microRNA or other diffusible molecule) that is used in the regulation the target gene.
  • the trans-acting gene may be on the same chromosome as the target gene, but the activity is via the intermediary protein or RNA that it encodes.
  • Embodiments of trans-acting gene are, e.g., genes that encode targeting
  • Inactivation of a gene using a dominant negative generally involves a transacting element.
  • cis-regulatory or cis-acting means an action without coding for protein or RNA; in the context of gene inactivation, this generally means inactivation of the coding portion of a gene, or a promoter and/or operator that is necessary for expression of the functional gene.
  • somatic cells such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation (Wilmut et al., Nature, 385:810-813, 1997; and Wakayama et al., Nature, 394:369-374, 1998).
  • Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques.
  • An animal that is genomically modified is an animal wherein all of its cells have the genetic modification, including its germ line cells. When methods are used that produce an animal that is mosaic in its genetic modification, the animals may be inbred and progeny that are genomically modified may be selected.
  • Cloning may be used to make a mosaic animal if its cells are modified at the blastocyst state, or genomic modification can take place when a single-cell is modified. Animals that are modified so they do not sexually mature can be homozygous or heterozygous for the modification, depending on the specific approach that is used. If a particular gene is inactivated by a knock out modification, homozygousity would normally not be sufficient. If a particular gene is inactivated by an RNA interference or dominant negative strategy, then heterozygosity is often adequate.
  • a nucleic acid construct is introduced into a fertilized egg; 1 or 2 cell fertilized eggs are used as the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm.
  • Pronuclear staged fertilized eggs can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of donor animals).
  • In vitro fertilized eggs can be produced as follows. For example, swine ovaries can be collected at an abattoir, and maintained at 22-28°C during transport.
  • Ovaries can be washed and isolated for follicular aspiration, and follicles ranging from 4-8 mm can be aspirated into 50 mL conical centrifuge tubes using 18 gauge needles and under vacuum. Follicular fluid and aspirated oocytes can be rinsed through pre-filters with commercial TL-HEPES (Minitube, Verona, WI).
  • Oocytes surrounded by a compact cumulus mass can be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, WI) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 ⁇ 2- mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for approximately 22 hours in humidified air at 38.7°C and 5% C02.
  • PMSG pregnant mare serum gonadotropin
  • hCG human chorionic gonadotropin
  • the oocytes can be moved to fresh TCM-199 maturation medium, which does not contain cAMP, PMSG or hCG and incubated for an additional 22 hours. Matured oocytes can be stripped of their cumulus cells by vortexing in 0.1%
  • mature oocytes can be fertilized in 500 ⁇ Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, WI) in Minitube 5-well fertilization dishes.
  • IVF in vitro fertilization
  • freshly -collected or frozen boar semen can be washed and resuspended in PORCPRO IVF Medium to 4 x 10 ⁇ sperm.
  • Sperm concentrations can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, WI).
  • Final in vitro insemination can be performed in a ⁇ volume at a final concentration of
  • Linearized nucleic acid constructs can be injected into one of the pronuclei. Then the injected eggs can be transferred to a recipient female (e.g., into the oviducts of a recipient female) and allowed to develop in the recipient female to produce the transgenic animals.
  • a recipient female e.g., into the oviducts of a recipient female
  • in vitro fertilized embryos can be centrifuged at 15,000 X g for 5 minutes to sediment lipids allowing visualization of the pronucleus.
  • the embryos can be injected with using an Eppendorf FEMTOJET injector and can be cultured until blastocyst formation. Rates of embryo cleavage and blastocyst formation and quality can be recorded.
  • Embryos can be surgically transferred into uteri of asynchronous recipients.
  • Embryos can be surgically transferred into uteri of asynchronous recipients.
  • 100-200 (e.g., 150-200) embryos can be deposited into the ampulla-isthmus junction of the
  • a transgenic artiodactyl cell e.g., a transgenic pig cell or bovine cell
  • a transgenic artiodactyl cell such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell that includes a nucleic acid construct described above
  • Oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area.
  • an injection pipette with a sharp beveled tip is used to inject the transgenic cell into an enucleated oocyte arrested at meiosis 2.
  • oocytes arrested at meiosis-2 are termed eggs.
  • the embryo After producing a porcine or bovine embryo (e.g., by fusing and activating the oocyte), the embryo is transferred to the oviducts of a recipient female, about 20 to 24 hours after activation. See, for example, Cibelli et al., Science 280: 1256-1258, 1998, and U.S. 6,548,741.
  • recipient females can be checked for pregnancy approximately 20-21 days after transfer of the embryos.
  • Standard breeding techniques can be used to create animals that are homozygous for the exogenous nucleic acid from the initial heterozygous founder animals. Homozygosity may not be required, however.
  • Transgenic pigs described herein can be bred with other pigs of interest.
  • a nucleic acid of interest and a selectable marker can be provided on separate transposons and provided to either embryos or cells in unequal amount, where the amount of transposon containing the selectable marker far exceeds (5-10 fold excess) the transposon containing the nucleic acid of interest.
  • Transgenic cells or animals expressing the nucleic acid of interest can be isolated based on presence and expression of the selectable marker. Because the transposons can integrate into the genome in a precise and unlinked way (independent transposition events), the nucleic acid of interest and the selectable marker are not genetically linked and can easily be separated by genetic segregation through standard breeding. Thus, transgenic animals can be produced that are not constrained to retain selectable markers in subsequent generations, an issue of some concern from a public safety perspective.
  • transgenic animal Once transgenic animal have been generated, expression of an exogenous nucleic acid can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether or not integration of the construct has taken place. For a description of Southern analysis, see sections 9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; NY., 1989.
  • PCR Polymerase chain reaction
  • Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. See, for example, Lewis, Genetic Engineering News 12: 1, 1992; Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874, 1990; and Weiss, Science 254: 1292, 1991.
  • embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci USA, 99:4495, 2002).
  • telomere length a region of DNA sequence encoding a polypeptide in the tissues of transgenic pigs.
  • techniques that include, for example, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis,
  • RNAi interfering RNA
  • dsRNA Double-stranded RNA
  • RISC RNA-induced silencing complex
  • RISC metabolizes dsRNA to small 21-23 -nucleotide small interfering RNAs (siRNAs).
  • RISC contains a double stranded RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut 2 or Ago2).
  • RISC utilizes antisense strand as a guide to find a cleavable target.
  • siRNAs and microRNAs miRNAs
  • a method of disrupting a gene in a genetically modified animal comprises inducing RNA interference against a target gene and/or nucleic acid such that expression of the target gene and/or nucleic acid is reduced.
  • the exogenous nucleic acid sequence can induce RNA interference against a nucleic acid encoding a polypeptide.
  • double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to a target DNA can be used to reduce expression of that DNA.
  • Constructs for siRNA can be produced as described, for example, in Fire et al., Nature 391:806, 1998; Romano and Masino, Mol. Microbiol. 6:3343, 1992; Cogoni et al., EMBO J. 15:3153, 1996; Cogoni and Masino, Nature, 399: 166, 1999; Misquitta and Paterson Proc. Natl. Acad. Sci.
  • shRNAs are transcribed as a single-stranded RNA molecule containing complementary regions, which can anneal and form short hairpins.
  • the probability of finding a single, individual functional siRNA or miRNA directed to a specific gene is high.
  • the predictability of a specific sequence of siRNA for instance, is about 50% but a number of interfering RNAs may be made with good confidence that at least one of them can be effective.
  • Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that express an RNAi directed against a gene, e.g., a gene selective for a developmental stage.
  • the RNAi may be, for instance, selected from the group consisting of siRNA, shRNA, dsRNA, RISC and miRNA.
  • An inducible system may be used to control expression of a gene.
  • Various inducible systems are known that allow spatiotemporal control of expression of a gene.
  • the term inducible system includes traditional promoters and inducible gene expression elements.
  • An example of an inducible system is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of the nucleic acid.
  • TetR mutated Tet repressor
  • tTA transcriptional activator
  • dox doxycycline
  • Alternative inducible systems include the ecdysone or rapamycin systems.
  • Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor and the product of the ultraspiracle gene (USP).
  • Expression is induced by treatment with ecdysone or an analog of ecdysone such as muristerone A.
  • the agent that is administered to the animal to trigger the inducible system is referred to as an induction agent.
  • the tetracycline-inducible system and the Cre/loxP recombinase system are among the more commonly used inducible systems.
  • the tetracycline-inducible system involves a tetracycline-controlled transactivator (tTA)/ reverse tTA (rtTA).
  • tTA tetracycline-controlled transactivator
  • rtTA reverse tTA
  • a method to use these systems in vivo involves generating two lines of genetically modified animals.
  • One animal line expresses the activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter.
  • Another set of transgenic animals express the acceptor, in which the expression of the gene of interest (or the gene to be modified) is under the control of the target sequence for the tTA/rtTA transactivators (or is flanked by loxP sequences).
  • tetracycline-dependent regulatory systems rely on two components, i.e., a tetracycline-controlled transactivator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner.
  • tTA tetracycline-controlled transactivator
  • tTA/rtTA-dependent promoter that controls expression of a downstream cDNA
  • tet-OFF The tet system that uses tTA is termed tet-OFF, because tetracycline or doxycycline allows transcriptional down- regulation.
  • Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo.
  • rtTA is a variant of tTA that is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation.
  • This tet system is therefore termed tet-ON.
  • the tet systems have been used in vivo for the inducible expression of several transgenes, encoding, e.g., reporter genes, oncogenes, or proteins involved in a signaling cascade.
  • the Cre/lox system uses the Cre recombinase, which catalyzes site-specific
  • Cre-mediated recombination by crossover between two distant Cre recognition sequences, i.e., loxP sites.
  • a DNA sequence introduced between the two loxP sequences (termed floxed DNA) is excised by Cre-mediated recombination.
  • Control of Cre expression in a transgenic animal using either spatial control (with a tissue- or cell-specific promoter) or temporal control (with an inducible system), results in control of DNA excision between the two loxP sites.
  • conditional gene inactivation conditional knockout
  • Another approach is for protein over- expression, wherein a floxed stop codon is inserted between the promoter sequence and the DNA of interest.
  • Inducible Cre recombinases have also been developed.
  • the inducible Cre recombinase is activated only by administration of an exogenous ligand.
  • the inducible Cre recombinases are fusion proteins containing the original Cre recombinase and a specific ligand-binding domain. The functional activity of the Cre recombinase is dependent on an external ligand that is able to bind to this specific domain in the fusion protein.
  • Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that comprise a gene under control of an inducible system.
  • the genetic modification of an animal may be genomic or mosaic.
  • the inducible system may be, for instance, selected from the group consisting of Tet-On, Tet-Off, Cre-lox, and Hiflalpha.
  • An embodiment is a gene set forth herein.
  • Genes may thus be disrupted not only by removal or RNAi suppression but also by creation/expression of a dominant negative variant of a protein which has inhibitory effects on the normal function of that gene product.
  • the expression of a dominant negative (DN) gene can result in an altered phenotype, exerted by a) a titration effect; the DN PASSIVELY competes with an endogenous gene product for either a cooperative factor or the normal target of the endogenous gene without elaborating the same activity, b) a poison pill (or monkey wrench) effect wherein the dominant negative gene product ACTIVELY interferes with a process important for normal gene function, c) a feedback effect, wherein the DN ACTIVELY stimulates a negative regulator of the gene function.
  • DN dominant negative
  • founder animals may be produced by cloning and other methods described herein.
  • the founders can be homozygous for a genetic modification, as in the case where a zygote or a primary cell undergoes a homozygous modification.
  • founders can also be made that are heterozygous.
  • the founders may be genomically modified, meaning that the cells in their genome have undergone modification.
  • Founders can be mosaic for a modification, as may happen when vectors are introduced into one of a plurality of cells in an embryo, typically at a blastocyst stage. Progeny of mosaic animals may be tested to identify progeny that are genomically modified. An animal line is established when a pool of animals has been created that can be reproduced sexually or by assisted reproductive techniques, with heterogeneous or homozygous progeny consistently expressing the modification.
  • An animal line may include a trait chosen from a trait in the group consisting of a production trait, a type trait, a workability trait, a fertility trait, a mothering trait, and a disease resistance trait. Further traits include expression of a recombinant gene product.
  • Embodiments of the invention include administration of a targeted nuclease system with a recombinase (e.g., a RecA protein, a Rad51) or other DNA-binding protein associated with DNA recombination.
  • a recombinase forms a filament with a nucleic acid fragment and, in effect, searches cellular DNA to find a DNA sequence substantially homologous to the sequence.
  • a recombinase may be combined with a nucleic acid sequence that serves as a template for HDR. The recombinase is then combined with the HDR template to form a filament and placed into the cell.
  • the recombinase and/or HDR template that combines with the recombinase may be placed in the cell or embryo as a protein, an mRNA, or with a vector that encodes the recombinase.
  • the disclosure of U.S. 2011/0059160 (U.S. Patent Application No. 12/869,232) is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling.
  • the term recombinase refers to a genetic recombination enzyme that enzymatically catalyzes, in a cell, the joining of relatively short pieces of DNA between two relatively longer DNA strands.
  • Recombinases include Cre recombinase, Hin recombinase, RecA, RAD51, Cre, and FLP. Cre recombinase is a Type I topoisomerase from PI
  • Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria
  • Salmonella. Hin belongs to the serine recombinase family of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination.
  • RAD51 is a human gene. The protein encoded by this gene is a member of the RAD51 protein family which assists in repair of DNA double strand breaks. RAD51 family members are homologous to the bacterial RecA and yeast Rad51.
  • Cre recombinase is an enzyme that is used in experiments to delete specific sequences that are flanked by loxP sites.
  • FLP refers to Flippase recombination enzyme (FLP or Flp) derived from the 2 ⁇ plasmid of the baker's yeast Saccharomyces cerevisiae.
  • RecA or “RecA protein” refers to a family of RecA-like recombination proteins having essentially all or most of the same functions, particularly: (i) the ability to position properly oligonucleotides or polynucleotides on their homologous targets for subsequent extension by DNA polymerases; (ii) the ability topologically to prepare duplex nucleic acid for DNA synthesis; and (iii) the ability of RecA/oligonucleotide or
  • RecA/polynucleotide complexes efficiently to find and bind to complementary sequences.
  • the best characterized RecA protein is from E. coli; in addition to the original allelic form of the protein a number of mutant RecA-like proteins have been identified, for example, RecA803.
  • RecA-like strand-transfer proteins including, for example, yeast, Drosophila, mammals including humans, and plants. These proteins include, for example, Reel, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2 and DMC1.
  • An embodiment of the recombination protein is the RecA protein of E. coli.
  • the RecA protein can be the mutant RecA-803 protein of E. coli, a RecA protein from another bacterial source or a homologous recombination protein from another organism.
  • the present invention also provides compositions and kits containing, for example, nucleic acid molecules encoding site-specific endonucleases, CRISPR, Cas9, ZNFs, TALENs, RecA-gal4 fusions, polypeptides of the same, compositions containing such nucleic acid molecules or polypeptides, or engineered cell lines.
  • An HDR may also be provided that is effective for introgression of an indicated allele. Such items can be used, for example, as research tools, or therapeutically.
  • Pig were maintained at 37 at 5% C02 in DMEM supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin and streptomycin, and 2mM L-Glutamine.
  • DMEM fetal bovine serum
  • penicillin and streptomycin 100 I.U./ml penicillin and streptomycin
  • 2mM L-Glutamine 2mM L-Glutamine.
  • TALENs and HDR templates were delivered through transfection using the NEON Transfection system (Life Technologies). Briefly, low passage Ossabaw, Landrace reaching 100% confluence were split 1 :2 and harvested the next day at 70-80% confluence. Each transfection was comprised of 500,000-600,000 cells resuspended in buffer "R" mixed with TALEN mRNA and oligos and electroporated using the ⁇ tips that provide a 100 ⁇ working volume by the following parameters: input Voltage; 1800V; Pulse Width; 20ms; and Pulse Number; 1.
  • TALEN mRNA and 1 -4 ⁇ of HDR templates single stranded oligonucleotides specific for the gene of interest were included in each transfection. Deviation from those amounts is indicated in he figures and legends.
  • HDR templates single stranded oligonucleotides
  • PCR flanking the intended sites was conducted using PLATINUM Taq DNA polymerase HiFi (Life Technologies) with 1 ⁇ of the cell lysate according to the manufacturer's recommendations.
  • the frequency of mutation in a population was analysed with the SURVEYOR Mutation Detection Kit (Transgenomic) according to the manufacturer's recommendations using 10 ⁇ of the PCR product as described above.
  • RFLP analysis was performed on 10 ⁇ of the above PCR reaction using the indicated restriction enzyme.
  • Surveyor 5 and RFLP reactions were resolved on a 10% TBE poly aery lamide gels and visualized by ethidium bromide staining.
  • Densitometry measurements of the bands were performed using IMAGEJ; and mutation rate of Surveyor reactions was calculated as described in Guschin et al., 2010(1).
  • Percent homology directed repair (HDR) was calculated by dividing the sum intensity of RFLP fragments by the sum intensity of the parental band + RFLP fragments.
  • RFLP analysis0 of colonies was treated similarly except that the PCR products were amplified by IX MYTAQ RED MIX (Bioline) and resolved on 2.5% agarose gels.
  • Transfected cells populations at day 3 and 10 were collected from a well of a 6-well dish and 10-30% were resuspended in 50 ⁇ of IX PCR compatible lysis buffer: 10 mM Tris-Cl pH 8.0, 2 mM EDTA, 0.45% TRYTON X-100(vol/vol), 0.45% TWEEN-20(vol/vol) freshly supplemented with 200 ⁇ g/ml Proteinase K.
  • the lysates were processed in a thermal cycler5 using the following program: 55°C for 60 minutes, 95°C for 15 minutes. Colony samples from dilution cloning were treated as above using 20-30 ⁇ of lysis buffer.
  • TALEN mRNA and HDR templates directed to pig RAG2 and IL2Ry were co-transfected into pig fibroblasts.
  • a fixed quantity of RAG2 mRNA and template were used for each transfection whereas the quantity of IL2Rg TALEN mRNA and HDR template is altered for each condition as indicated.
  • the dosage of TALEN mRNA and HDR template has both on and off target effects.
  • An increase in TALEN mRNA for IL2Ry led to an increase in both NHEJ and HDR for IL2Ry while NHEJ levels for RAG2 were unchanged.
  • FIG. 4A-4D Multiplex gene editing of swine RAG2 and IL2Ry .
  • FIG. 4A-4D Multiplex gene editing of swine RAG2 and IL2Ry .
  • FIG. 4A SURVEYOR and RFLP analysis to determine the efficiency of non- homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection.
  • FIG. 4B RFLP analysis for homology dependent repair on cell populations 11 days post transfection.
  • FIG. 4C Percentage of colonies positive for HDR at IL2Ry, RAG2 or both. Cells were plated from the population indicated by a "C" in FIG. 4A. Distribution of colony genotypes is shown below.
  • FIG. 4D Colony analysis from cells transfected with TALEN mRNA quantities of 2 and 1 ⁇ g for IL2Ry and RAG2 and HDR template at 1 ⁇ for each. Distribution of colony genotypes is shown below.
  • FIGs. 5A-5D Multiplex gene editing of swine APC and p53.
  • FIG. 5A Surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection.
  • FIG. 5B RFLP analysis for homology dependent repair on cell populations 11 days post transfection.
  • FIGs. 5C and 5D Percentage of colonies positive derived from the indicated cell population (indicated in FIG. 5 A, "C" and "D") for HDR at APC, p53 or both. Colonies with 3 or more HDR alleles are listed below.
  • Example 3 Multiplex with at least three genes
  • Example 1 a non-specific reduction in HDR was observed at high concentration of
  • HDR oligo thus it was unknown whether 2+ HDR oligos could be effective without nonspecific inhibition of HDR.
  • Two concentrations were tested, 1 uM and 2 uM for each target site. While TALEN activity was not significantly altered between the two conditions, HDR was blunted significantly at 2 uM concentration for each template.
  • Clones derived from the 1 uM condition had a variety of genotypes, some of those with edits in each gene and up to 7 alleles (FIGs. 7A and 7B). If treated as independent events, the expected frequency of the genotype denoted by an "a", with 7 alleles edited, is 0.001 percent.
  • Binomial distribution predicts the likelihood of identifying 2+ colonies of such a genotype in a sample size of 72, as was done here, is less than 0.000026 percent. This high rate of success could not be predicted and is unexpected and surprising. This result was replicated with two addition combinations of
  • FIGs. 6A and 6B Effect of Oligonucleotide HDR template concentration on 5-gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Rg, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 uM (FIG. 6A) or 1 uM (FIG. 6B) of each cognate HDR template. Percent NHEJ and HDR were measured by Surveyor and RFLP assay. Referring to FIGs. 7A and 7B: Colony genotypes from 5-gene multiplex HDR. Colony genotypes were evaluated by RFLP analysis. In FIG.
  • each line represents the genotype of one colony at each specified locus.
  • Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele.
  • the percentage of colonies with an edit at the specified locus is indicated below each column.
  • FIG. 7B provides a tally of the number of colonies edited at 0-5 loci.
  • FIGs. 8A-8B Colony genotypes of a second 5-gene multiplex trial.
  • FIG. 8A Each line represents the genotype of one colony at each specified locus.
  • FIG. 8B A tally of the number of colonies edited at 0-5 loci.
  • FIGs. 9A and 9B Colony genotypes a third 5-gene multiplex trial.
  • FIG. 9A Each line represents the genotype of one colony at each specified locus.
  • FIG. 9B A tally of the number of colonies edited at 0-5 loci.
  • Example 4A Develop RAG2/IL2Rg null (RG-KO) pig fibroblasts by multiplex gene editing.
  • Male pig fetal fibroblasts are transfected with TALENs and oligonucleotide templates for disruption of RAG2 and IL2Rg using previously defined methods (Tan, W., et al., Efficient nonmeiotic allele introgression in livestock using custom endonucleases.
  • PNAS
  • RG-KO candidates are identified by, e.g., RFLP, as confirmed by sequencing. At least about 5 validated RG-KO colonies arepooled as a resource for cloning and chimera production.
  • Example 4B Production of chimeric embryos using RG-KO host blastocysts.
  • RG-KO embryos and female EGFP-labeled donor cells are produced using chromatin transfer technology followed by in vitro culture to the blastocyst stage.
  • RG-KO cells from Example 1 may be used.
  • Day-7 inter cell mass clumps from EGFP blastocysts are injected into day-6 RG-KO embryos prior to embryo transfer to a synchronized sow.
  • Nagashima and colleagues observed chimerism in >50 percent of liveborn piglets
  • Example 4C Determine if non-chime ric offspring are deficient for T, B and NK cells.
  • Non-chimeric offspring is tested to determine if they deficient for T, B and NK cells.
  • C-section derivation is conducted on each sow carrying presumptive chimeras and one bred sow carrying wild-type piglets.
  • Umbilical cord blood is isolated from each piglet immediately after C-section derivation.
  • Cord blood leukocytes are evaluated by fluorescence-activated cell sorting (FACS) for T, B and NK cell populations as well as donor derived EGFP expression.
  • FACS fluorescence-activated cell sorting
  • chimerism is evaluated by PCR from cord blood, ear and tail biopsy. This initial analysis is completed within 6 hours of birth, such that non-chimeric piglets can be monitored closely and humanely euthanized with signs of infection. A portion of non-chimeric animals, or those lacking immune cells, is euthanized for necropsy.
  • Example 4D Identify chimeric pigs and determine origin of T, B and NK cells.
  • Chimeric pigs are tested to determine origin of T, B and NK cells. The following process is one technique for the same. Chimeric piglets are identified using the methods above. Weekly evaluation of circulating lymphocytes and serum immunoglobulin is compared between chimeric, non-chimeric and wild-type piglets over a 2 month period. Populations of sorted T, B and NK cells are evaluated for EGFP expression and microsatellite analysis to confirm donor origin. The maintenance of samples and semen collections from chimeric pigs are supported by RCI until Phase II funding is available.
  • lymphocytes are cryopreserved for potential allograft treatments while the remainder is used for FACS analysis of lymphocytes.
  • Peripheral blood samples are collected at 2, 4, 6 and 8 weeks of age by standard methods. RBCs are removed and approximately 1-2E+5 cells are distributed into tubes.
  • T cells CD4 and CD8
  • B cells CD45RA ad CD3
  • NK cells CD 16 and CD3
  • myeloid cells CD3
  • Antigen expression is quantified on the LS RII Flow Cytometer (BD Biosciences). Fluorophores are carefully selected to enable multiplex evaluation of donor derived EGFP cells along with surface antigens. Single cell suspensions from the spleen are analyzed by the same methods.
  • All major organs and tissues are grossly examined for appropriate anatomic development and appropriate samples from all major organs and tissues including pancreas, liver, heart, kidneys, lungs, gastrointestinal, immune system (peripheral and mucosal lymph nodes and spleen), and CNS are collected for DNA isolation.
  • Single cell suspensions are prepared from the spleen for FACS analysis.
  • Tissues are prepared for histological examination to further assess chimerism and for any alterations that may be associated with the chimeric state and for the presence of any underlying illness.
  • Quantitative PCR is conducted on cord blood, ear, and tail biopsy using primers specific to the EGFP transgene and compared to a standard curve with known ratios of EGFP to wild type-cells. Specimens are also evaluated for RG-KO alleles via the RFLP assay previously described. Engraftment of EGFP+ cells are evaluated macroscopically on whole animals and organs during necropsy. Tissues from the major organs are sectioned for EGFP
  • DAPI 6-diamidino-2-phenylindole
  • Animals are screened for informative microsatellites for host and donor genetics from those routinely used in the lab. Samples from tissues and blood (sorted lymphocytes or myeloid lineages, EGFP positive and negative) are evaluated. Relative quantities of donor versus host cells are evaluated by multiplexed amplicon sequencing on the MISEQ instrument (Illumina).
  • Non-chimeric pigs are made having an absence of T, B and NK cells in cord and peripheral blood. Chimeric pigs have levels substantially similar to nearly wild-type levels. Moreover, T, B and NK cell positive chimeras have substantially normal immune functions and remain healthy when reared in standard conditions.
  • Example 5 CRISPR/Cas9 design and production.
  • Gene specific gRNA sequences were cloned into the Church lab gRNA vector (Addgene ID: 41824) according their methods.
  • the Cas9 nuclease was provided either by co-transfection of the hCas9 plasmid (Addgene ID: 41815) or mRNA synthesized from RCIScript-hCas9.
  • This RCIScript-hCas9 was constructed by sub-cloning the Xbal-Agel fragment from the hCas9 plasmid (encompassing the hCas9 cDNA) into the RCIScript plasmid. Synthesis of mRNA was conducted as above except that linearization was performed using Kpnl.
  • Example 6 Multiplex gene editing with targeted endonucleases and HDR.
  • FIG. 13 A is a schematic of each gene in the multiplex experiment (depicted as a cDNA- exons denoted by alternating shades) and the site targeted by TALENS is indicated. The sequence coding the DNA binding domain for each gene is indicated below. Swine fibroblasts were co- transfected with 1 ug of each TALEN mRNA and 0.1 nMol of each HDR oligo (FIG. 13B), targeting each gene, designed to insert a premature termination codon as well as a novel Hindlll RFLP site for genotyping. A total of 384 colonies were isolated for genotyping. The GATA4 and Nkx2-5 RFLP assays were performed (FIG. 13C) and MESP1 was evaluated by sequencing (not shown).
  • FIG. 13C Two colonies (2/384, 0.52%) were homozygous HDR knockouts for all three genes. The triple knockouts are labeled with asterisks (FIG. 13C). Additional genotypes can be observed in FIG. 13C, example colony 49 with no HDR edits; colony 52 and 63 with heterozygous edits to NKX2-5; colony 59 with heterozygous edits to both NKX2-5 and GATA4 and so on.
  • Example 7 Multiplex gene-editing using a combination of TALENs and RGENs.
  • Swine fibroblasts were co-transfected with TALENS (1 ug EIF4G 14.1 mRNA) + Cas9/CRISPR components (2 ug Cas9 mRNA + 2 ug p65 Gls guide RNA) and 02 nMol of HDR oligo for each gene. Transfected cells were evaluated by RFLP assay revealing HDR at both sites. Cells from this population are plated for colony isolation and isolates with edits in both genes are identified.
  • Example 8 Human-porcine chimeric blastocysts.
  • An important first step in creating human organs/cells in the pig using blastocyst complementation is to determine whether human stem cells can be incorporated into the inner cell mass as opposed to the trophectoderm and blastocoele cavity.
  • an assay system was developed using parthenogenetic blastocysts. The parthenotes are created by the electrical activation of pig oocytes resulting in the formation of a diploid cell from the combination of DNA from the maternal pronucleus and the polar body. The single diploid cell then divides and the 6th day after activation becomes a well-formed blastocyst suitable for injection of human stem cells.
  • Human -porcine chimeric fetus derived from complemented PITX3 knockout blastocysts Porcine nigral dopamine neurons in pig-pig chimeras are also created and characterized; and human nigral dopamine neurons in human-pig chimeras.
  • NURR1, LMX1A, and PITX3 knockout blastocysts are generated using TALEN technology in fibroblasts and cloning. It is determined whether the knockout blastocysts are capable of generating complementation based nigral dopamine neurons by using labeled porcine blastomeres as a source of stem cells.
  • TALEN-knockout of LMX1A, PITX3, and NURR1 in pig fibroblasts were developed to cleave in exons 1, 2 and 3 respectively for LMXA1, PITX3, and also NURRl, another gene that plays a major role in dopamine neuron development (see FIG. 17A, black triangle).
  • TALENs were co-transfected with a homology dependent repair template designed to introduce a novel stop codon, Hindlll site, and a frame-shift after the novel stop codon to ensure disruption of the targeted allele.
  • Populations of transfected cells were analyzed for Hindlll dependent cleavage produced by a PCR-restriction fragment polymorphism assay (FIG. 17B).
  • hUCBSCs were injected into PITX3 knockout porcine blastocysts and transferred blastocysts to hormonally synchronized gilts.
  • Chimeric fetuses were harvested at 62 days in gestation and examined for the status of the eyelids (FIGs. 18A, 18C, and 18E).
  • a portion of the chimeric fetuses displayed open eyelids similar to wild-type pig fetuses while others exhibited closed eyelids.
  • Example 11 ETV2 knockout pig embryos
  • Etv2 is a master regulatory gene for vascular and hematopoietic lineages, and is an ideal candidate for gene editing studies.
  • the Etv2 gene locus was mutated to generate vascular and hematopoietic deficient pig embryos for several reasons.
  • Etv2 is a master regulatory gene for vascular and hematopoietic development in mice (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012, Rasmussen 2012, Chan 2013, Rasmussen 2013, Behrens 2014, Shi 2014).
  • Example 12 ETV2 knockout pig embryos lack vascular and hematopoietic lineages.
  • Etv2 is important for vasculogenesis and hematopoiesis in the mouse as embryos lacking Etv2 are lethal at approximately E9.5 with an absence of vasculature and blood (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012).
  • ETV2 is the key regulator of the vasculature and blood in mammals, and thus, the ETV2 knockout in the pig phenocopies the mouse.
  • the entire ETV2 coding sequence was removed using two TALEN pairs flanking the gene in porcine fibroblasts (FIGs. 19 A and 19B). The process was 15% efficient at complete gene removal; 79/528 of the genotyped clones were homozygous for the deletion of the ETV2 gene.
  • ETV2 homozygous knockout fibroblast clones were used for nuclear cloning (Somatic Cell Nuclear Transfer; SCNT) to generate ETV2 null embryos which were transferred to surrogate sows.
  • SCNT Somatic Cell Nuclear Transfer
  • E18.0 Embryos were harvested and analyzed at E18.0 (FIGs. 20A-20H).
  • E18.0 wild-type (Wt) embryos were vascularized with a well-developed vascular plexus in the allantois (FIG. 20 A) and had evidence of blood development (FIG. 20C).
  • ETV2 KO embryos showed clear developmental defects. Growth was retarded in ETV2 KOs relative to the Wt embryo, though both embryos were at the 24-somite stage (FIG 20B), and lacked both blood and vascular lineages (FIGs. 20 C-20H).
  • ETV2 KO embryos lacked cardinal veins, dorsal aortae, and the endocardium, that are clearly developed in the Wt embryos (FIGs. 20 E-20H). These results reflect a similar phenotype and suggest that the function of ETV2 is conserved between mice and pigs. Further, these data strongly support the hypothesis that multiple mutations can be directed into the porcine genome to support growth of chimeric organs that are humanized in more than one cell type.
  • Example 13 Complementation of ETV2 knockout porcine blastocysts with human iPSCs.
  • hiPSCs were injected into ETV2 knockout porcine blastocysts and transferred these blastocysts to hormonally synchronized gilts.
  • Chimeric fetuses were harvested at 18 days of gestational age and immunohistochemically examined for the status of the hiPSCs (FIGs. 21A-21C).
  • Human cells were identified by genomic in situ hybridization using the probe to Alu repetitive sequence, as well as staining against human nuclear antigen (HNA).
  • Example 14 Nkx2-5 and Handll as important regulators of cardiogenesis.
  • Cardiac development is a complex highly-orchestrated event that includes the specification, proliferation, migration and differentiation of cardiac progenitors that become electrically coupled and ultimately form a functional syncytium. These stages of cardiogenesis are governed by transcriptional networks, which have been shown, using gene disruption technology, to be important for heart formation and viability (Lyons 1995, Srivastava 1997, Tanaka 1999, Bruneau 2001, Yamagishi 2001, Garry 2006, Ferdous 2009, Caprioli 2011) (Table 1).
  • Nkx2-5 is the vertebrate homolog of the Drosophila homeodomain protein, Tinman (Csx). The Tinman mutation results in the absence of heart formation in the fly (Bodmer 1993).
  • Nkx2-5 is one of the earliest transcription factors expressed in the cardiac lineage. Targeted disruption of NL ⁇ 2-5 results in perturbed heart morphogenesis, severe growth retardation and embryonic lethality at approximately E9.5 (Lyons 1995, Tanaka 1999).
  • Handll (dHand) is a bHLH transcription factor that has also been shown to be important for cardiac morphogenesis. Handll mutant embryos are lethal during early embryogenesis and have severe right ventricular hypoplasia and aortic arch defects (Srivastava 1997). Moreover, mice lacking both ⁇ 3 ⁇ 4 ⁇ 2-5 and Handll demonstrate ventricular agenesis and have only a single atrial chamber (FIGs. 22A-22D) (Y amagishi 2001). These gene disruption studies in the mouse model illustrate the effectiveness of using a gene editing strategy in the pig model.
  • Example 15 Multiplex knockout of porcine NKX2-5 and HANDII genes.
  • NKX2-5/HANDII mutant porcine fibroblasts were used to generate NKX2-5/HANDII mutant porcine fibroblasts. Each gene was targeted either within or immediately prior to their conserved transcription factor/DNA binding domains (FIG. 23 A). This strategy was favored over targeting the gene near the transcription start site to reduce the chance of producing a functional peptide by initiation at a downstream AUG.
  • NKX2-5 a homology template was provided to generate a novel in-frame stop codon, restriction site for RFLP screening, and an additional five base insertion after the stop codon to prevent a functional read-through protein. Double mutants were identified (FIG. 23 B). The ability to reliably produce double null pig fibroblast cell lines in a single shot is unique and a transformative technology important for complementation.
  • Example 16 Perturbed cardiogenesis in triple knockout pig embryos.
  • Embryos were harvested and analyzed at E18, which is equivalent to El 1 of the mouse.
  • the triple knockout porcine embryos have vasculature, skeletal muscle and blood but essentially lack a heart (minimal GATA4 immunohisto-chemically positive cardiomyo-cytes) (FIGs. 24A-24C) compared to the wildtype control porcine embryo.
  • FIGs. 24A-24C minimal GATA4 immunohisto-chemically positive cardiomyo-cytes
  • NKX2-5/HANDII double knockout porcine model to limit the involvement of other lineages (i.e. neuronal lineage in the TBX5 KO) and be more reflective of congenital heart disease models (i.e. hypoplastic right and left heart defects). This approach results in the engineering of humanized biventricular hearts in the porcine model.
  • Example 17 Myf5, Myod and Mrf4 as important regulators of myogenesis.
  • Myod family members are master myogenic regulators as they transactivate a broad spectrum of gene families, including muscle specific genes, transcription factors, cell cycle genes, etc. to promote a myogenic cell fate .
  • Previous gene disruption studies have demonstrated that mice lacking Myf5/Myod/MRF4 lack skeletal muscle and are lethal early following birth presumably due to their inability for respiration (due to the absence of a diaphragm). These gene disruption studies in the mouse illustrate the effectiveness of using gene editing strategies in the pig.
  • MYF5/MYOD/MRF4 knockout pig embryos lack skeletal muscle lineages. Embryos were harvested and analyzed at E18.0 (FIGs. 27A and 27B). The results in the mouse and pig reflect a similar phenotype and support the notion that the function of MYF5/MY OD/MRF4 are conserved between mice and pigs as mutant embryos lack skeletal muscle. Further, these data strongly support the hypothesis that direct multiple mutations into the porcine genome to support growth of chimeric organs that are humanized in more than one cell type.
  • Example 18 Complementation of MYF5/MYOD/MRF4 knockout phenotype with GFP WT pig blastomeres.
  • Porcine MYF5/MYOD/MRF4 null blastocysts were generated using SCNT, and injected with GFP -labeled porcine blastomeres (since no validated porcine ES cells are available, blastomeres were utilized for this experiment).
  • the resulting chimeras were implanted in pseudopregnant sows and examined at E20. The feasibility of complementation was demonstrated as liver and yolk sac were GFP positive. Additionally, it was estimated that approximately 10% of porcine MYF5/MYOD/MRF4 null blastocysts were GFP labeled (FIGs. 28A-28C).
  • porcine MYF5/MYOD/MRF4 null blastocysts were GFP labeled (FIGs. 28A-28C).
  • Example 19 PDXl knockout results in apancreatic fetal pigs.
  • Pdxl ⁇ l ⁇ mice are apancreatic and die shortly after birth due to the inability of the pancreatic bud to develop into the mature organ (Offield et al, 1996). Rescue of the mouse
  • Pdxl " ⁇ " phenotype by blastocyst complementation has been demonstrated by injecting wild-type mouse or rat iPSCs into Pdxl ⁇ ' mouse blastocysts, producing mice that had normal functioning pancreases, derived from the donor cells (Kobayashi et al, 2010). Blastocyst complementation of Pdxl deficiency was also recently described in the pig where a functional pancreas was produced in a trans-genic apancreatic pig following the injection of labeled WT blastomere cells into pig blastocysts expressing the dominant Pdxl:hesl transgene (Matsunaria et al., 2013).
  • Pdxl knockout pigs are not susceptible to the unpredictable nature of position effects or expression levels seen when using transgenes and offer a more consistent platform for the production of pancreas ablated pigs.
  • TALEN technology has been used extensively to biallelically knockout the PDXl gene in pig fibroblasts (FIG. 29A) using a TALEN pair that targets the important homeobox domain of the PDXl gene, and an HDR construct to introduce a STOP codon, frameshift, and novel restriction enzyme site. Homozygous PDXl knockouts were obtained at a rate of 41% (76/184 clones) (FIG. 29B).
  • HHEX KO clones were generated to test the efficiency of this gene-editing method. Constructs were developed to cleave exon 2 of HHEX gene (see FIG. 3 OA black triangle) within the N-terminus of the homeo-domain-like region important for DNA binding. Fibroblasts were transfected with vector constructs and a homology dependent repair template designed to introduce a novel stop codon, a Hindlll site, and a frame- shift mutation after the novel stop codon to ensure disruption of the targeted allele.
  • FIG. 30B Over 50% the transfected population was positive for the Hindlll KO allele by PCR-restriction fragment polymorphism assay (FIG. 30B) and several individual clones derived from the population were either heterozygous or homozygous for the KO allele (FIG. 30C). In total, 22 clones with sequence validated KO alleles were cryopreserved. The same vector constructs are used to generate both HHEX and Ubc KO blastocysts.
  • HHEXKO embryonic lethal in pigs._To determine the effect of HHEXKO in pigs, HHEX-/- fibroblasts were cloned SCNT and transferred to a synchronized recipient. At 30-32 days in gestation the embryos were harvested and assessed for the development of the liver. All embryos were genotyped and confirmed for knockout of HHEX. All specimens exhibited delayed development with a clear absence of the liver (FIG. 3 IB). Samples were taken from each specimen to grow fibroblasts as a source of HHEX knockout cells for future experiments to combine this knockout with editing of other targeted genes such as ETV2 to create human liver with human vasculature.
  • Example 21 Summary of preliminary studies on porcine gene knockouts and
  • Example 22 MR imaging of fetal porcine organs at 16.4T.
  • FIG. 33 shows a fetal pig 30 days in gestation (20 mm crown-rump length) where all of the internal organs are quite visible in great detail.
  • the pulse sequence used in this figure was optimized for visualizing the liver.
  • Other pulse sequences are developed to optimize contrast for other organs for quantitation of parameters such as organ volume in addition to 3D morphology to provide important information regarding the anatomical features of complemented organs. This provides a rapid quantitative approach for determine the success of complementation following the knockout of target genes to generate specific organs.
  • Parkinson's disease is a movement disorder that is caused by the progressive loss of dopamine neurons in the substantial nigra area of the brain.
  • Current treatments include medications such as L-DOPA, a precursor for the synthesis of dopamine.
  • L-DOPA therapy is effective for a period of time, but ultimately medication-induced dykinesias occur making this therapy undesirable.
  • Clinical trials of human fetal dopamine cell transplantation for treating Parkinson disease demonstrate the amelioration of motor deficits (Lindvall et al., 1989; Freed et al., 2001; Olanow et al., 2003).
  • Blastocysts are generated from each type of gene knockout mouse.
  • GFP -labeled mouse iPSC cells are injected into blastocysts and transferred to surrogate dams. Offspring are analyzed for histological evidence of GFP -positive cells in the brain.
  • porcine blastocysts are generated from each type of gene knockout.
  • GFP -labeled pig blastomere cells are injected into blastocysts and transferred to surrogate sows/gilts. Fetuses are analyzed at day 60 in gestation for histological evidence of GFP -positive cells in the brain.
  • Human stem cells are injected into blastocysts to assess human-porcine complementation.
  • FIG. 34 shows the procedure used to introduce donor hiPSC or human umbilical cord blood stem cell into PITX3 KO porcine morula. Chimeric fetuses were generated as shown in FIG. 36. Complementation of PITX3 knockout in porcine blastocysts with human stem cells rescued the ocular development defects (FIG. 18 and FIG. 37). Porcine PITX3 knockout also resulted in loss of dopamine neurons in fetal substantia nigra (FIGs.
  • LMX1A and PITX3 gene are important in dopamine neuron development (FIG. 42).
  • wild-type blastomeres expressing GFP were introduced into LMX1A ⁇ /PITX3 ⁇ ⁇ blastocysts (FIG. 43).
  • lens was ablated (FIGs. 45A and 45B).
  • FIGs. 46A-46C Porcine- porcine complementation in PITX3/LMX1A knockout animal reinstated lens development.
  • FIGs. 46A-46C Cells of donor origin (GFP positive cells) were found in chimeric fetal pig brain (FIGs. 47A-47D).
  • DA neurons Dopamine Neurons
  • Table E provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • OPCs oligodendrocyte progenitor cells
  • a potential therapeutic strategy is the manufacture of oligodendrocyte progenitor cells (OPCs).
  • Current iPSC-based approaches result in poor yield and incomplete reprogramming of OPCs. Genes involved in the development of OPCs are identified and can be targeted for knockout for large-scale production of authentic human OPCs in the pig brain.
  • mice The combined knockout of OLIG1 and OLIG2 in mice is investigated to determine if knockout blastocysts can give rise to murine OPCs following murine-murine complementation, i) OLIG1 and OLIG2 knockout mice are created and interrogated for OPC deficiency, ii) mouse oligodendrocyte progenitor cells (OPCs) are created in young mice via complementation of OLIG1/OLIG2 knockout blastocysts (or NKX2.1, NKX2.2 and/or SOX10 as choosen), iii) functionality of the OPCs are confirmed by transplanting them into a congenitally dysmyelinated mouse and demonstrating myelination.
  • OPCs mouse oligodendrocyte progenitor cells
  • OLIG1/OLIG2 OLIG1/OLIG2 knockout fibroblast were generated (FIGs. 66A-66C). Wild type blastomeres expressing GFP were introduced into OLIG1/OLIG2 knockout blastocysts to generate chimeric pig. GFP expression was observed in whole body fluorescent imaging of the porcine fetus complemented with wild type cells (FIGs. 67A and 67B ). The GFP expressing cells were also incorporated into central nervous system of the chimeric pig (FIGs. 68A and 68B, and 69A and 69B). Specifically, GFP expressing cells were present in both chimeric pig brain (FIGs. 68A and 68B) and spinal cord (FIGs. 69A and 69B).
  • Table F provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • Blastocysts with knockouts of RAG2, IL2rg, C-KIT, and ETV2 alone or in combination can be used to generate murine hematopoiesis via complementation using GFP -labeled murine iPSCs.
  • Immune cells can be identified through a series of antibody panels designed to distinguish between murine and human cells. The T-cell panel use CD3 to isolate the T cell population, CD4 to stain for helper T cells, CD8a for cytotoxic T cells, and CD25 is used as an activation marker for each of these T cell subtypes. In a separate panel, FoxP3 can be used to identify regulatory T cells, and CD25 is used to identify activated regulatory T cells.
  • Microglial cells are analyzed by staining for CD45 and CD1 lb. Also in this panel is CD200, which is a marker of microglial quiescence; and CD163, which is a marker of the alternately activated M2- like microglia. Young mice that exhibit robust complementation are used as the young parabiotic partner for circulatory coupling. Both parabiosis and blood plasma infusion approaches are employed to provide young blood/plasma to old mice and Alzheimer's mice. Young mice engineered to produce human blood are jointed to aged mice or Alzheimer's mice by parabiotic surgery. In parallel, blood plasma is pooled from young mice and intravenously infused to aged or Alzheimer's mice.
  • mice Three to six months after the surgery /plasma infusion, the mice (the parabiotic pair separated) are subjected to a battery of behavioral tests for the assessment of cognitive function, which include an open field, an elevated plus maze test, a novel object recognition test, and the Morris water maze test. Non-parabiosed and untreated age-matched mice are used as controls. Porcine studies evaluate RAG2, IL2rg, C-KIT, and ETV2 knockouts alone or in combination to generate porcine hematopoietic cells using labeled pig blastomeres. See, FIGs. 71-73.
  • a panel of antibodies are used to determine the cell phenotype in pigs, including (CD45, CD3, ⁇ -chain TCR, CD8, CD4 to identify ⁇ and ⁇ T cells: CD3, CD21 and CD79a for B cells; CD16. CD172a, CD2, and CD8 for NK cells and macrophages; and FoxP3 for Tregs).
  • the results from the pig-pig chimeras provide information as to which knockouts are suitable for generating human hematopoiesis in the pig.
  • candidate human stem cells from Technical Aim III are identified for starting studies to engineer human blood in pigs.
  • a panel of antibodies that specifically recognize human blood cells are used to analyze the phenotype the cells using multicolor flowcytometry and hierarchical analysis where species specific antibodies are not available.
  • the genotype of host cells (carriers) used are: RAG2 "/ 7IL2rg “/ 7C-KIT “/” /ETV2 "/” ;RAG2 "/"
  • RAG2/IL2Rg are important for T cell, B cell, and NK cell development and thymus development (FIGs. 49 and 50). It is also hypothesized that C-KIT gene is important for T cell, B cell, and NK cell development and thymus development.
  • IL2Rg/RAG2 knockout animals chimeric animals were generated by introducing wild type blastomeres expressing GFP into IL2Rg/RAG2 knockout blastocysts (FIG. 51). Further, IL2Rg/RAG2/C-KIT animal were generated (FIGs. 77-79). A IL2Rg/RAG2/C-KIT piglet complemented with wild type cell (Recip ID 6035) was harvested and subject to further study. Phenotyping analysis indicated that the complemented animal may have both wild type genome and mutant genome (FIG. 80). Complementation in IL2Rg/RAG2/C-KIT knockout rescued the gene disruptions and resulted in generation of thymus (FIGs.
  • GFP + immune cells were observed in cells collected from cord blood, thymus, spleen, peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN) (FIG. 53). Immune cells were generated in the thymus in chimeric animals (FIGs. 54A-54F). In blood and spleen, chimeric pig embryos contained T cells in an amount similar to wild type pig embryos, while the T cells were significantly reduced in IL2Rg/RAG2/C-KIT knockout pig embryos (FIGs. 55 & 56). Similar phenotypes were observed with respect to B cells and NK cells (FIGs. 57-60).
  • chimeric pig embryos have similar immune cells profiles as wild type pig embryos, while immune cells are significantly reduced in knockout pig embryos.
  • Table G provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Green the TALENs are made and validated for function.
  • Carrier (host) genotypes used include: RUNXl ⁇ /C-KIT ⁇ /FLKl “7” ; RUNX 1 _/ 7FLK 1 _/” ; RUNXl ⁇ /C-KIT “7” ; C-KIT ⁇ /FLKl “7 ; RUNX1 "7” ; C-KIT "7” ; FLK1 "7” .
  • Blastocysts are generated from each type of knockout mouse. GFP -labeled mouse iPSC cells are injected into blastocysts and transferred to surrogate dams. Offspring are analyzed for histological evidence of GFP -positive cells in blood, bone marrow, thymus, lymph nodes and skin.
  • Histological demonstration of cells in the murine blood, bone marrow, thymus, lymph nodes and skin that are labeled with GFP and express mesodermal and hematopoietic markers CD45, CD3, and PDGFR1 alfa are indications of successful complementation.
  • FACS plots and photomicrographs showing histological evidence of cells in blood, bone marrow, thymus, lymph nodes and skin of the mouse that are labeled with GFP, CD45, CD3, and PDGFR1 alfa indicate successful complementation.
  • Blastocysts are generated from each type of knockout pig.
  • GFP-labeled pig blastomere cells are injected into blastocysts and transferred to surrogate sows/gilts.
  • Fetuses are analyzed at day 60 in gestation for histological evidence of GFP -positive cells in blood, bone marrow, thymus, lymph nodes and skin. Histological demonstration of cells in blood, bone marrow, thymus, lymph nodes and skin of the pig fetus that are labeled with GFP and express mesodermal and hematopoietic markers CD45, CD3, and PDGFR1 alfa.
  • Table H provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Vascular disease is extremely common as peripheral artery disease affects more than 10M Americans resulting in more than 150,000 limb amputations each year in the U.S (Hirsch 2013). In addition, more than 300,000 patients have coronary artery bypass grafting (surgical revascularization). These diseases collectively are amplified by the rising incidence of diabetes, obesity and cardiovascular disease. Importantly, these complications result in considerable morbidity and mortality. Current medical therapies for vascular disease include limb amputation, vascular bypass grafting (using the patient's diseased vasculature) or vascular grafts-all these therapeutic interventions have significant limitations.
  • Etv2 was previously discovered as a master regulator of development of the vascular and the hematopoietic lineages, and that Etv2 null embryos were lethal and lacked hematoendothelial lineages (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012, Rasmussen 2012, Rasmussen 2013, Behrens 2014, Shi 2014).
  • ETV2 mutant porcine embryos also lacked hemato-endothelial lineages. Humanized vasculature in the ETV2 mutant porcine model is generated.
  • ETV2 mutant porcine embryos are generated by SCNT and comprehensively analyzed.
  • ETV2 mutant morulae are injected with GFP -labeled porcine blastomeres. These GFP -labeled blastomeres are also generated using SCNT. Injected morulae are transferred to sows, and embryos are collected at El 8, E24 and neonates are collected at postnatal day 7 (P7).
  • ETV2 mutant morulae are injected with human stem cells (four separate lines are analyzed. Injected morulae are transferred to sows, and embryos are collected at E18 and E24. Porcine- or human stem cell- complemented ETV2 null pig embryo is generated and examined and the cardiac and vascular functions by MRI and transthoracic echocardiography, respectively. The pig-pig chimeras are analyzed at E18, E24, and P7; and human-pig chimeras are analyzed at E18 and E24.
  • Host genotypes include: ETV2 _/" .
  • Table I provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Congenital cardiovascular malformation is the most common birth defect and it contributes to advanced or endstage heart failure in the pediatric and adult population.
  • Congenital heart disease afflicts approximately 1% of all live births and has considerable morbidity and mortality (Garry 2006, Hoffman 1995, Kang 2000, Kramarow 2012, Rasmussen 2011).
  • the only curative therapy for congenital heart disease-induced heart failure is orthotopic heart transplantation. Due to the shortage of organs for transplantation, less than 2% receive such lifesaving therapy. Those that receive a heart transplant require lifelong immunosuppression, which also has deleterious side effects and limits survival. Therefore, there is a need to develop an alternative source of human heart tissues.
  • Humanized biventricular hearts which serve as an unlimited source of organs for transplantation and provide a paradigm shifting platform for the treatment of congenital heart disease and endstage heart failure, are generated.
  • a NKX2-5/HANDII double knockout porcine embryo can be engineered, using gene editing technologies, which lack ventricular myocardium.
  • Human stem cells, SCNT and blastocyst complementation are used to produce a humanized biventricular heart.
  • the humanized pigs also serve as a large animal model to study regeneration of human lineages or response(s) to pharmacological agents and lead to improved therapies for cardiovascular diseases including hypoplastic right and left heart congenital and heart failure diseases.
  • NKX2-5/HandII mutant morulae are generated using SCNT and comprehensively characterized at E18 and E24.
  • NKX2-5/HandII mutant morulae are injected with GFP -labeled porcine blastomeres. These GFP-labeled blastomeres are also generated using SCNT.
  • Injected morulae are transferred to sows, and embryos are collected at E18, E24, and neonates are collected at postnatal day 7 (P7). Contribution of GFP positive cells to embryos are examined.
  • NKX2-5/HandII mutant morulae are injected with human stem cells (four separate lines are analyzed and they are selected.
  • Injected morulae are transferred to sows, and embryos are collected at E18 and E24.
  • porcine- or human stem cell-complemented NKX2- 5/HandII double knockout pig embryos are generated and the cardiac and vascular functions are examined by MRI and transthoracic echocardiography.
  • the pig-pig chimeras are analyzed at E18, E24 and P7; and human-pig chimeras are analyzed at E18 and E24.
  • Genotypes of host and donor cells used include: NKX2-5-'-/HANDir'-/TBX5- / -; NKX2-5 ' '- /HANDir'-; HANDir l ⁇ /TBX5 ⁇ l ⁇ ; NKX2-5 ⁇ ITBX5-'-; HANDir 1' ; TBX5 ⁇ ' ⁇ ; NKX2-5 ⁇ .
  • Table J provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Green the TALENs arc made and ⁇ alidatcd for function.
  • Myopathic diseases such as muscular dystrophies and aging are common and deadly. While skeletal muscle has a tremendous capacity for regeneration, this potential ultimately fails with disease and aging. No treatments are currently available for terminal muscle diseases and fifty percent of the falls in the elderly lead to their demise. Additionally, U.S. Army soldiers, even light infantry troops, lose substantial training hours due to musculoskeletal injuries. The incidence of these injuries accounts for 56% of all sick-call diagnoses. Prolonged recovery is typically due to the need for long term physical therapies (Smith and Cashman, 2002).
  • MYF5/MYOD/MRF4 (aka Myf6) knockout pigs.
  • These humanized pigs serve as a large animal model to study the regeneration of the human skeletal muscle, and/or response(s) to
  • MYF5/MYOD/MRF4 triple mutant porcine embryos are generated by SCNT and comprehensively analyzed.
  • MYF5/MYOD/MRF4 triple mutant morulae are injected with GFP- labeled porcine blastomeres. These GFP-labeled blastomeres are also generated using SCNT.
  • Injected morulae are transferred to sows, and embryos are collected at E24, E50 and neonates are collected at postnatal day 7 (P7). Contribution of GFP positive cells to the embryos are examined.
  • MYF5/MYOD/MRF4 triple mutant morulae can be injected with human stem cells (four separate lines can be analyzed and they are selected as outlined in Technical Area III).
  • Injected morulae are transferred to sows, and embryos are collected at E24 and E50.
  • porcine- or human stem cell-complemented MYF5/MYOD/MRF4 null pig embryo is generated and the skeletal muscle functions are examined by MRI, contractility assays and lameness testing.
  • the pig-pig chimeras are analyzed at E24, E50, and P7; and human-pig chimeras are analyzed at E24 and E50.
  • Genotypes of host and donor cells used include: MYF5 “/ 7MYOD “/ 7MRF4 “/” ; MYF5 “7” /MYOD “7” ; MYF5 _/ 7 MRF4 “7” ; MYOD “/ 7MRF4 “/” ; MYF5 “7” ; MYOD “7” ; MRF4 “7” ; PAX3 ⁇ .
  • Green the TALENs are made and validated for function.
  • HHEX and Ubc knockout blastocysts are the homeobox gene HHEX and the polyubiquitin gene Ubc.
  • HHEX and Ubc the homeobox gene HHEX and the polyubiquitin gene Ubc.
  • knockout either of these genes in mouse zygotes using the TALEN and CRISPR gene editing technologies are sequence the genome of KO zygotes to determine the absence of full-length HHEX and Ubc genes; and
  • HHEX-KO and Ubc-KO murine blastocysts are produced by injecting hLDPCs into HHEX-KO and Ubc-KO murine blastocysts, and implant them into C57BL/6 female mice.
  • the engraftment of LDPCs are studied after injection into mouse embryos. The livers of pups born to female recipients of blastocysts are analyzed.
  • HHEX and Ubc mice are each embryonic lethal, and embryos of these mice survive beyond the E14.5 stage only if the hepatocyte and liver epithelial progenitor cell proliferation is complemented by the implanted hLPDCs.
  • Genotypes of host and donor cells used include: HHEX '1' ! Ubc '1' ; HHEX 1' ; Ubc '1' .
  • Table L provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Green the TALENs are made and validated for function.
  • Example 31 Engineer Pancreas for Transplant and to Treat Diabetes
  • pancreatic diseases and disorders including diabetes
  • pancreatic diseases and disorders including diabetes
  • bladderstocyst complementation of apancreatic phenotypes has been demonstrated with exogenic rat iPSCs in PdxV' ' mice and syngenic cells in Pdxl .Hesl pigs (Kobayashi et al., 2010, Matsunaria et al, 2013).
  • the pancreas formed in the complemented animals was derived entirely from progeny of the donor cells.
  • Chimeric embryos at mid-gestation, early postnatal neonates and adult complemented pigs are generated to provide human pancreas tissue and islets for testing in vitro and in vivo including testing the ability of the human islets generated in pigs to restore blood glucose homeostasis in rodent and non human primate models of diabetes.
  • Genotypes of host and donor cells used include: Pdxr / 7Etv2 "/” ;Pdxl “/” ; Etv2 _/” .
  • Table M provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
  • Lung disease accounts for 400,000 deaths per year in the US and transplantation is the only "cure" for many end-stage lung diseases, such as chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, primary pulmonary hypertension and cystic fibrosis.
  • COPD chronic obstructive pulmonary disease
  • emphysema emphysema
  • idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis
  • Major obstacles to lung transplantation include the paucity of donor lungs available, chronic rejection since lungs are typically not HLA-matched, the requirement for lifelong immunosuppression and high mortality. A new source of transplantable lung tissue that is not subject to rejection is therefore warranted. Genes involved in lung development are tested to determine which can be targeted for knockout in blastocyst complementation studies that ultimately generate human lungs.
  • KO pigs are cloned using parthenogenetic porcine blastocysts to evaluate generation of lung-deficient phenotypes.
  • GFP-expressing porcine blastomeres are injected into KO blastocyst embryos.
  • Donor cell engraftment and complementation of the lung phenotype are determined in the chimeric fetal pigs at 30 and 60 days gestation to check for complementation ability of the KO.
  • human pluripotent stem cells can complement lung- defective pig blastocysts
  • GFP-expressing human iPS cells, naive iPS cells, UCBSCs or MAPCs are injected into KO pig blastocysts.
  • the lungs are evaluated for mosaicism and
  • Genotypes of host and donor cells used include: Nkx2.1 "/ 7 Sox2 "/ 7Id2 "/ 7Tbx4 “/” ;Nkx2. V 1' /Sox2 "/ 7Id2- / -; Nkx2. l “/ 7Sox2 "/ 7Tbx4 "/” ; Nkx2. l “/ 7Id2 "/ 7 Tbx4 _/” ; Sox2 "/ 7Id2 "/ 7 Tbx4 "A ; Nkx2.1 "7” /Sox2 _/” ; Nkx2. l “/ 7Id2 "/” ; Nkx2.
  • NKX2.1 regulates lung development (FIG. 64).
  • NKX2.1 knockout pigs were generated. Pig fibroblasts were knocked out (TALEN gene editing) for the transcription factor Wx2.1, a gene important for lung development. Pig embryos at day 30 were shown to have blunted lung growth at the early pseudoglandular stage (FIGs. 32A-32F). Therefore complementation at the blastocyst stage with human stem cells may be a source of generating human lungs for transplantation or lung disease studies.
  • the NKX2.1 knockout pigs can be used to resolve the issues of shortage of donor lungs for transplantation, and lack of suitable in vivo models of human lung disease.
  • Table N provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.

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Abstract

Human or humanized tissues and organs suitable for transplant are disclosed herein. Gene editing of a host animal provides a niche for complementation of the missing genetic information by donor stem cells. Editing of a host genome to knock out or disrupt genes responsible for the growth and/or differentiation of a target organ and injecting that animal at an embryo stage with donor stem cells to complement the missing genetic information for the growth and development of the organ. The result is a chimeric animal in which the complemented tissue (human/humanized organ) matches the genotype and phenotype of the donor. Such organs may be made in a single generation and the stem cell may be taken or generated from the patient's own body. As disclosed herein, it is possible to do so by simultaneously editing multiple genes in a cell or embryo creating a "niche" for the complemented tissue. Multiple genes can be targeted for editing using targeted nucleases and homology directed repair (HDR) templates in vertebrate cells or embryos.

Description

COMPOSITIONS AND METHODS FOR CHIMERIC EMBRYO-ASSISTED ORGAN
PRODUCTION
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional patent applications Ser. Nos.
62/246,926, 62/246,927, 62/246,929, 62/246,947, 62/247,092, 62/947,096, 62/247,115, 62/247,117, 62/247,118 and 62/247,122, each of which was filed on October 27, 2015. The entire contents of each of the aforementioned provisional applications are incorporated herein by reference.
The subject matter of this application may be related to that disclosed in international patent application publication Nos. WO2015/168125A1, published November 5, 2015, WO 2016/141234, published September 9, 2016 in international application Nos.
PCT/US2016/040378, filed June 30, 2016, and PCT/US2016/040431, filed June 30, 2016. The entire contents of each of the aforementioned international applications are incorporated herein by reference.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
This invention was made with government support under Grant No. W81XWH-15-1- 0393 awarded by the Department of Defense and Grant Nos. 1R43HL124781-01A1 and
1R43GM113525-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
In the past 100 years scientists and physicians have been spectacularly effective in keeping people alive and healthy, at least until the last decades of their lives when a panoply of old-age diseases and disorders set in. Over $1 trillion dollars are spent annually in the United States for treatment of these diseases. Organ transplant can be effective but there are far too few organs available and in many cases immunological mismatches lead to problems. For example, over 7,000 Americans have died while awaiting an organ transplant since 2003.
Genetic complementation of animal somatic cells by various stem cells allows for the engineering and production of humanized tissues and organs for use in therapy, transplant and regenerative medicine. Currently the source of organs for transplantation are either mechanical or biological, coming from: human donors; cadavers; and, in limited cases, xenotransplants from other species of mammals, most particularly swine. Unfortunately, all are subject to rejection by the host body and/or may elicit other side effects. SUMMARY OF THE INVENTION
The following paragraphs enumerated consecutively from 1 through 92 describe various aspects and associated embodiments of the invention.
1. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue.
2. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, excluding chimeric embryos wherein:
the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells; and
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
3. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes; the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
4. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RU X1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells; the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
5. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
6. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ and/or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
7. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons. 8. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes.
9. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
10. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
11. The chimeric embryo any one of paragraphs 1 or 3, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
12. The chimeric embryo any one of paragraphs 1 or 3, wherein the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
13. The chimeric embryo any one of paragraphs 1 or 3, wherein the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
14. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
15. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise HHEX or Ubc and the human organ or tissue comprises human liver cells.
16. The chimeric embryo according to any one of paragraphs 1-3, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
17. The chimeric embryo according to paragraph 1 or paragraph 2, wherein both alleles of the endogenous gene have been disrupted.
18. The chimeric embryo according to any one of paragraphs 1-17, wherein the non- human embryo is a non-human vertebrate embryo.
19. The chimeric embryo of paragraph 18, wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
20. The chimeric embryo according to paragraph 18, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, crustacean, and fish. 21. The chimeric embryo of paragraph 20, wherein the vertebrate non-human embryo is a cow, pig, sheep, goat, chicken or rabbit embryo.
22. The chimeric embryo of any one of paragraphs 1-21, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS) , Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNs), molecules encoding site- specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi, CRISPRi or combinations thereof.
23. The chimeric embryo of paragraph 22, wherein the one or more endogenous genes have been disrupted by Cas9.
24. The chimeric embryo according to any one of paragraphs 1-23, wherein the human cells are derived from at least one donor cell and the at least one donor cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
25. The chimeric embryo according to any of paragraphs 1-24, wherein the disruption comprises a gene edit, a knockout, an insertion of one or more DNA residues, a deletion of one or more bases, or both an insertion and a deletion of one or more DNA residues.
26. The chimeric embryo according to any of paragraphs 1-24, wherein the disruption comprises a substitution of one or more DNA residues.
27. The chimeric embryo of paragraph 26, wherein the disruption consists of a substitution of one or more DNA residues.
28. An animal that has developed from the chimeric embryo according to any one of paragraphs 1-27.
29. A human tissue or organ harvested from an animal that has developed from the chimeric embryo according to any one of paragraphs 1-27.
30. A method of producing a chimeric embryo comprising:
a) disrupting one or more endogenous genes responsible for the development of one or more endogenous organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo.
31. A method of producing a chimeric embryo comprising: a) disrupting one or more endogenous genes responsible for the development of one or more endogenous organs or tissues in at least one non-human cell or non-human embryo; b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues;
thereby producing a chimeric embryo, excluding chimeric embryos wherein:
the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells; and
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
32. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
33. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
34. A method of producing a chimeric embryo comprising a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
35. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
36. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons.
37. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes. 38. The method according to any one of paragraphs 30-32 wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
39. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
40. The method according to paragraphs 30 or 32, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
41. The method according to paragraphs 30 or 32, wherein the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
42. The method according to paragraphs 30 or 32, wherein the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
43. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
44. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells.
45. The method according to any one of paragraphs 30-32, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
46. The method of paragraph 30 or paragraph 31, wherein both alleles of the one or more endogenous genes are disrupted.
47. The method according to any one of paragraphs 30-46, wherein the non-human embryo is a non-human vertebrate embryo.
48. The method of paragraph 47, wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
49. The method according to paragraph 47, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
50. The method of any one of paragraphs 30-49, wherein the at least one human donor cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
51. The method of any one of paragraphs 30-50, further comprising implanting the chimeric embryo into a uterus of an animal wherein the chimeric embryo develops into a chimeric animal comprising human cells. 52. The method of paragraph 51, further comprising harvesting the human cells from the chimeric animal.
53. The method of paragraph 52, further comprising transplanting the human cells into a human patient in need thereof.
54. The method of paragraph 53, wherein the at least one human cell is donated by the human patient.
55. The method of any one of paragraphs 30-54, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNS), molecules encoding site-specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi,CRISPRi or combinations thereof.
56. The method of paragraph 55, wherein the method is Cas9.
57. The method of any one of paragraphs 55-56, further comprising introducing a homology directed repair (HDR) template having a template sequence with homology to one of the endogenous genes, with the template sequence replacing at least a portion of the endogenous gene sequence to disrupt the endogenous gene.
58. The method of paragraph 57, further comprising introducing a plurality of homology directed repair (HDR) template, each having a template sequence with homology to one of the endogenous genes, with each the template sequences replacing at least a portion of one of the endogenous gene sequences to disrupt the endogenous gene.
59. The method of paragraph 57 or 58, wherein the disruption comprises a substitution of one or more DNA residues of the endogenous gene.
60. The method of paragraph 57 or 58, wherein the disruption consists of a substitution of one or more DNA residues of the endogenous gene.
61. A chimeric embryo or chimeric animal created using the method of any one of paragraphs 30-60.
62. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
wherein the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal.
63. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting one or more endogenous genes responsible for the development of one or more endogenous organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, excluding chimeric embryos, wherein:
the endogenous genes comprise MYF5, MYOD, and/or MRF4 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells; and
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
64. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and
c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
wherein the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, and wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons; the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
65. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and
c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
wherein the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood; the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
66. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and
c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue; wherein:
the endogenous genes are RAG2 and IL2rg or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
67. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein: the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
68. The method according to any one of paragraphs 62-64 , wherein the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons.
69. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes.
70. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
71. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
72. The method according to paragraphs 62 or 64, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
73. The method according to paragraphs 62 or 64, wherein the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
74. The method according to paragraphs 62 or 64, wherein the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
75. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
76. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells.
77. The method according to any one of paragraphs 62-64, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
78. The method of paragraph 62 or paragraph 63, wherein both alleles of the one or more endogenous genes are disrupted.
79. The method according to any one of paragraphs 62-78, wherein the non-human embryo is a non-human vertebrate embryo. 80. The method of paragraph 79 , wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
81. The method according to paragraph 79, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
82. The method of any one of paragraphs 62-81, wherein the at least one human cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
83. The method of paragraph 62-82, further comprising harvesting the human cells from the chimeric animal.
84. The method of paragraph 83, further comprising transplanting the human cells into a human patient in need thereof.
85. The method of paragraph 84, wherein the at least one human cell is donated by the human patient.
86. The method of any one of paragraphs 62-85 further comprising introducing a homology directed repair (HDR) template having a template sequence with homology to one of the endogenous genes, with the template sequence replacing at least a portion of the endogenous gene sequence to disrupt the endogenous gene.
87. The method of paragraph 86 further comprising introducing a plurality of homology directed repair (HDR) template, each having a template sequence with homology to one of the endogenous genes, with each the template sequences replacing at least a portion of one of the endogenous gene sequences to disrupt the endogenous gene.
88. The method of paragraph 86 or 87 wherein the disruption comprises a substitution of one or more DNA residues of the endogenous gene.
89. The method of paragraph 86 or 87 wherein the disruption consists of a substitution of one or more DNA residues of the endogenous gene.
90. The method of any one of paragraphs 62-89, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS) , Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNS), molecules encoding site-specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi, CRISPRi or combinations thereof.
91. The method of paragraph 90, wherein the one or more endogenous genes of the non- human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Cas9. 92. A chimeric animal created using the method of any one of paragraphs 62-91.
Specific elements of any of the foregoing aspects and embodiments of the invention be combined or substituted for elements in other aspects and embodiments of the invention. Furthermore, although advantages associated with certain aspects and embodiments of the disclosure are described in the context of these aspects and embodiments, other aspects and embodiments can also exhibit such advantages, and not all aspects and embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram illustrating the problem of tissue/organ transplantation and the solution provided by genome engineering of Human Cells and Animals for Organ Transplant.
FIG. 2A depicts a process for making animals homozygous for two knockouts using single edits.
FIG. 2B depicts a hypothetical process of making animals with multiple edits by making of a single edit at a time.
FIG. 3 depicts multiplex gene edits used to establish founders at generation F0.
FIGs. 4A-4D depict multiplex gene editing of swine RAG2 and IL2Ry (or IL2Rg). FIG. 4A is a graph showing surveyor and restriction fragment length polymorphism (RFLP) analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection. FIG. 4B is a graph showing RFLP analysis for homology dependent repair on cell populations 11 days post transfection. FIG. 4C is a graph showing percentage of colonies positive for HDR at IL2Ry, RAG2 or both. Cells were plated from the population indicated by a "C" in FIG. 4A. FIG. 4D is a graph showing colony analysis from cells transfected with Transcription Activator-Like Effector Nucleases (TALENS) mRNA 30 quantities of 2 and 1 μg for IL2Ry and RAG2 and HDR template at 1 μΜ for each. Distribution of colony genotypes is shown below. In present application, IL2Ry and IL2Rg are used interchangeably.
FIGs. 5A - 5D depict Multiplex gene editing of swine APC and p53. FIG. 5A is a graph showing surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair (HDR) on cell populations 3 days post transfection. FIG. 5B is a graph showing RFLP analysis for homology dependent repair on cell populations 11 days post transfection. FIGs. 5C and 5D are graphs showing percentage of colonies positive derived from the indicated cell population (indicated in FIG. 5 A, "C" and "D") for HDR at APC, p53 or both. Colonies with 3 or more HDR alleles are listed below. FIGs. 6A and 6B depict effect of oligonucleotide HDR template concentration on Five- gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Ry, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 μΜ (FIG. 6A) or 1 μΜ (FIG. 6B) of each cognate HDR template. Percent NHEJ and HDR were measured by Surveyor and RFLP assay.
FIGs. 7A and 7B are a five-gene multiplex data set that shows plots of experimental data for the effect of oligonucleotide HDR template concentration on 5 -gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Ry, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 μΜ or 1 uM of each cognate HDR template. Percent NHEJ and HDR were measured by Surveyor and RFLP assay. Colony genotypes from 5-gene multiplex HDR: Colony genotypes were evaluated by RFLP analysis. In FIG. 7A, each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 7B provides a tally of the number of colonies edited at 0-5 loci.
FIGs. 8A and 8B are another five-gene multiplex data set that shows plots of experimental data for a second experiment involving the effect of oligonucleotide HDR template concentration on Five- gene multiplex HDR efficiency. Colony genotypes of a second 5-gene multiplex trial. In FIG. 8A, each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 8B provides a tally of the number of colonies edited at 0-5 loci.
FIGs. 9A and 9B is another five-gene multiplex trial data set that shows colony genotypes. In FIG. 9A, each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 9B provides a tally of the number of colonies edited at 0-5 loci.
FIG. 10 depicts a process of making an F0 generation chimera with targeted nucleases that produce a desired gene knockout or choice of alleles. FIG. 11 depicts establishment of an F0 generation animal with a normal phenotype and progeny with a failure to thrive (FTT) phenotype and genotype.
FIG. 12 depicts a process for making chimeric animals with gametes having the genetics of the donor embryo.
FIGs. 13A-C depict multiplex editing at three targeted loci of NKX2-5, GATA4, and
MESP1. FIG. 13A is a schematic of the experiment, FIG. 13B shows the targeting of the genes, with the NKX2-5, GATA4, and MESP1 listed as SEQ ID NOs: 1-3, respectively. FIG. 13C depicts the results of an assay for the experiments. Oligo sequences for each target gene. Novel nucleotides are represented by capital letters. The PTC is represented by light color letters in boxes and the novel Hindlll RFLP site is underlined.
FIG. 14 depicts multiplex gene-editing using a combination of TALENs and RGENs; assay of transfected cells evaluated by RFLP revealed HDR at both sites.
FIGs. 15A-E are images depicting incorporation of human umbilical cord blood stem cells (hUCBSC) into parthenogenetic porcine blastocyst. FIG. 15 A is a phase contrast image of blastocyst. FIG. 15B is a DAPI image of cells within the blastocyst. FIG. 15C is a human nuclear antigen (HNA) staining. FIG. 15D is a merged DAPI and HuNu image. FIG. 15E is a merged image of FIGs. 15A-15C. FIG. 15F is a graph showing quantification of HuNu cells in the inner cell mass (ICM), trophectoderm (TE), or blastocoel cavity (CA). FIG. 15G is a graph showing the proliferation of HNA cells at days 6, 7, and 8 after activation of oocyte. Injection of hUCBSC was at day 6.
FIGs. 16A-16C are images showing chimeric human-porcine fetus. FIG. 16A is an image showing chimeric fetus at 28 days in gestation following injection of hUCBSCs into parthenogenetic porcine blastocysts. FIG. 16B is an immunohistochemistry image showing the staining for human nuclear antigen (HNA) in red and DAPI in blue. FIG. 16C is a control for the immunohistochemistry staining in FIG. 16B, with no primary antibody added in the staining.
FIGs. 17A and 17B depicts TALEN mediated knockout of porcine genes. FIG 17A is a schematic showing cleavage sites for LMXA1, NURR1, and PITX3. FIG. 17B provides electrophoresis images showing TALEN cleavage products as indicated by double arrows.
FIGs. 18A-F are images showing ocular effects of complementation of PITX3 knockout in porcine blastocysts with human umbilical cord blood stem cells. Images of FIGs. 18A-F show the gross morphology of fetal pig eyes at 62 days in gestation. FIGs. 18A and 18B shows the eye of wild type pig. FIGs. 18C and 18D show small eye from PITX3 knockout pigs. FIGs. 18E and 18F show large eye from PITX3 knockout with human umbilical cord blood stem cells (hUCBSC) complementation. Arrows in FIGs. 18A, 18C, and 18E point to the location of the eye for each fetus. FIGs. 19A and 19B depict TALEN-mediated knockout ΟΪΕΊΥ2. FIG. 19A is a schematic showing the three-tiered PCR assay utilized to detect gene editing. Amplification from primers a-d indicated a deletion allele was present. To distinguish between heterozygous and homozygous clones, primers a-b and c-d were used to amplify the wild type allele. Only when the a-d product is present and both a-b, c-d products are absent is the clone considered homozygous for the deletion allele. FIG. 19B provides electrophoresis images showing the confirmation of homozygous deletion. Clones fitting the criteria described above are enclosed by a green box.
FIGs. 20A-20H depict the loss of porcine ETV2 recapitulated the mouse Etv2 mutant phenotype. FIG. 20 A shows wild-type El 8.0 pig embryo and FIG. 20B shows ETV2 knockout embryo at the same developmental stage. Insets show enlarged views of the allantois. Note an abnormal overall morphology with lack of vascular plexus formation in the mutant (inset). FIGs. 20C-20H are sections through the allantois (FIGs. 20 C and D), the heart level (FIGs. 20 E and F) and the trunk level (FIGs 20 G and H) of the embryos shown in A and B, respectively, were stained for Tie2, an endothelial marker; Gata4, a cardiac lineage marker; and 4',6- diamidino-2-phenylindole (DAPI), a nuclear counterstain. The wild-type allantois was highly vascularized with Tie2 positive endothelial lining and contained blood (FIG. 20C, arrows), whereas, the mutant lacked these populations (FIG. 20D). The endocardium, cardinal veins (CV), and dorsal aortae (DA) are clearly visible in the wild- type embryo (E, G). In contrast, ETV2 null embryos completely lacked these structures although the heart progenitors and gut marked by Gata4 (green) were present (F and H, respectively). Scale bars: 1000 μιη (FIGs. 20A and B), 200 μιη (insets in FIGs. 20A and B), 100 μιη (FIGs. 20C-20H).
FIGs. 21A-21C are immunohistochemistry images depicting complementation of ETV2 mutant porcine embryos with human induce pluripotent stem cells (hiPSCs). ETV2 mutant blastocysts were generated by SCNT, and injected with ten hiPSCs at the morula stage and subsequently transferred into hormonally synchronized gilts. FIG. 21A shows in situ hybridization using the human specific Alu sequence. FIGs. 2 IB and 21C are
immunohistochemistry images against human CD31 (FIG. 21B), HNA (FIG. 21C, red), and human vWF (FIG. 21C, green). Boxed areas are enlarged in panels below. Arrowheads point to positive cells. Note formation of vessel-like structures. All scale bars indicate 50 microns, nt: neural tube, noto: notochord, som: somite.
FIGs. 22A-22D are images showing that Nkx2-5 and Handll (also known as dHand) double knockouts lack both ventricles (rv and lv) and have a single, small primitive atrium (dc). FIG. 22 A shows a wild type animal. FIG. 22B shows a NL· 2.5~'~ animal. FIG. 22C shows a dHand'' animal. FIG 22D shows a NKx2.5~'~ dHand'' double knockout animal. FIGs. 23 A and 23B depict double knockout of NKX2-5 and HANDII in swine fibroblasts. FIG. 23A is a schematics of the coding sequence for each gene shown; alternating colors indicate exon boundaries, the blue region (below) indicates the DNA binding domain of each transcription factor, and the triangles indicate the location TALENs binding sites. FIG. 23B provides electrophoresis images of RFLP analysis of fibroblast colonies for bialleic KO of HANDII and NKX2-5.
FIGs. 24A-24C depict that Nkx2-5/HANDII/TBX5 triple knockout porcine embryos have acardia. FIG. 24A provides images of immunohistochemistry staining of Gata4 protein. Wild type embryos (top) stained positively, while the triple knockout porcine embryos (bottom) lacked a heart with essentially no Gata4 immunohistochemically positive cells (marking the heart) at E18.0 (h, heart and fg, foregut). FIG. 24B provides images of DAPI staining for wild type embryos (top) and triple knockout embryos (bottom). FIG. 24C provides merged images of FIG. 24A and FIG. 24B.
FIGs. 25A and 25B are images depicting Myod expression. Myf5, Myod and Mrf4 are master regulators of skeletal muscle and are restricted to skeletal muscle in development and in the adult. Shown here is Myod-GFP transgenic expression which is restricted to the somites, diaphragm and established skeletal muscle at El 1.5 (FIG. 25 A). In FIG. 25B, in situ hybridization of a parasagittal section of an E13.5 (mid-gestation) mouse embryo using a 35S- labeled MyoD riboprobe. Note expression in back, intercostal and limb muscle groups.
FIGs. 26A-26C depict the knockout of swine MYOD, MYF5, and MYF6 gene. FIG.
26A is a schematic showing that. TALEN pairs were designed for swine MYOD, MYF5, and MYF6 (aka MRF4) genes. TALEN binding sites (denoted by red arrow heads) were upstream the important basic (+) helix-loop-helix (HLH) domain for each gene. The TALEN binding sites are shown below (denoted by red arrows) and the amino acid that was targeted for a premature STOP codon by homology dependent repair (HDR) are denoted by yellow arrows. FIG. 26B provide electrophoresis images showing the HDR events confirmed by RFLP analysis. HDR templates were designed to introduce the premature STOP codon and a novel restriction enzyme recognition site (Hindlll) to allow facile analysis of HDR events. The region of interest for each gene was amplified by PCR and RFLP was assessed for the population of transfected cells. The closed arrow heads denote the uncut or wild type alleles, while the open arrow heads denote the HDR alleles. The percent of alleles positive for HDR for MYOD, MYF5, and MYF6 were 14%, 31%, and 36%, respectively. FIG. 26C provides electrophoresis images and sequencing graphs to confirm the triple knockout. These populations were plated out for individual colony isolation. 38 out of 768 (4.9%) colonies demonstrated 4 or more RFLP events and were further analyzed by sequencing. 5 clones were identified to be homozygous knockout for all three genes by either HDR incorporating the premature STOP codon or in/dels that would result in a frameshift and subsequent premature STOP codon. An example of the RFLP analysis and sequencing of a clone that is a triple knockout for MYOD/MYF5/MYF6 is shown.
FIGs. 27A and 27B depict a phenotype of MYF5/MYOD/MRF4 triple knockout (KO). At E18.0, wild-type (Wt) embryos had well defined somite(s), desmin positive (red) myotomes (m) and developing musculature (FIG. 27A). In addition, the developing heart tube demonstrated strong desmin signal (h). In contrast, MYF5/MYOD/MRF4 KO embryos showed a lack of myotome formation while the heart remained desmin positive (FIG. 27B).
FIGs. 28A-28C depict complementation of MYF5/MY OD/MRF4 null embryos complemented with GFP labeled blastomeres. FIG. 28A is an image showing E20 porcine MYF5/MYOD/MRF4 null embryos complemented with GFP labeled blastomeres. Native GFP is observed in the liver and yolk sac of the embryo. FIG. 28B is an image showing section of porcine liver from MYF5/MY OD/MRF4 null embryos (E20) complemented with GFP labeled blastomeres. Native GFP is visible in the sinusoids of the liver. FIG. 28C is a bar graph showing PCR of yolk sac from E20 porcine MYF5/MY OD/MRF4 null embryos complemented with GFP labeled blastomeres (Embryos 1 [shown in FIGs. 28A and 28B], 3, 5). GFP -labeled pig fibroblasts is positive control while WT pig liver is negative control.
FIGs. 29A to 29E depict the generation of PDXl-/- pigs. FIG. 29A is a schematic showing TALEN gene editing of the pig PDXl locus. FIG. 29B provides electrophoresis images showing that RFLP analysis identified unmodified, heterozygous knockouts (open arrowhead) or homozygous knockouts (closed arrowhead). 41% of the clones were homozygous knockouts for PDXl. FIGs. 29C and 29D are images showing pancreas ablation (Δ) in cloned E32 Pdxl-/- pig embryos (FIG. 29D) compared to the pancreas in WT E30 embryos (FIG. 29C). FIG. 29E is an image showing the comparison of nascent β cells between wild type fetus and PDX ~ mutant fetus. P pancreas, S stomach, D duodenum of Wt E30 fetus.
FIGs. 30A-30C depict the generation of ffiffi knockouts (KOs) by gene-editing. FIG.
30 A is a schematic illustrating the knockout of HHEX gene. The HHEXgms is comprised of 4 exons. The Hindlll KO allele was inserted into exon 2 of the HHEX gene by gene-editing. FIG. 30B provides electrophoresis image showing that the efficiency of gene-editing was measured on the transfected population by a Hindlll RFLP assay. The proportion of chromosomes with the novel Hindlll KO allele (indicated by cleavage products, open triangles) is indicated on the gel. FIG. 30C provides electrophoresis images showing that fibroblast clones were also screened using the Hindlll RFLP assay. Homozygous KO clones are indicated with an asterisk.
FIGs. 31 A and 3 IB depict liver development in wild-type (FIG. 31 A) and HHEX KO pig embryos (FIG. 3 IB) at 30 days in gestation. Note absence of liver development in HHEX KO specimen in FIG. 3 IB. Wild- type control at the same gestational age is shown in FIG. 31 A. FIGs. 32A-32F depict that knockout of NKX2.1 results in loss of fetal lung. FIGs. 32A-32C are images showing that lungs develop in wild type animals. FIGs. 32D-32F are images showing that lungs fail to develop in NKX2.1 knockout animal.
FIG. 33 depicts MR imaging of fetal pig at 16.4T showing internal organs. Pig gestational age is 30 days when crown-rump length is approximately 20 mm.
FIG. 34 is a schematic showing the function of PITX3 in dopamine neuron/eye lens development. The inset is the same gel electrophoresis image showing the TALEN cleavage product of PITX3 gene in FIG. 17.
FIG. 35 is an image showing the introduction of donor hiPSC or hUCBSC into PITX3 KO porcine morula.
FIG. 36 provides a schematic showing the transfer of chimeric PITX3 knockout (KO) morula to surrogate sows.
FIG. 37 is a dot plot showing that human stem cell complementation rescues ocular defects in porcine PITX3 knockout. PITX3 knockout essentially eliminated open eyelid.
Complementation with wild type stem cell (hiPSC or hUCBSC) rescued, at least partially, the elimination of open eyelid caused by knockout of PITX2. The graph in FIG. 37 represents the quantitative analysis of the "eyelids" shown in Figures 18A, 18C, and 18E. In Figure 18A, the eye in the wild type pig fetus can be observed. In contrast, the eye is not visible in the PITX3 knockout (FIG. 18C) because the overlying skin or eyelid is not translucent. In some PITX3 knockout fetuses complemented with stem cells, the eye can now been seen through the translucent or "open" eyelid (FIG. 18E). These qualitative findings were quantified by a blinded analysis of 9 people rating each eye as either "open" or "closed". Each data point represents the percentage of individuals who rated each eye as "open" to generate a "Open Eyelid Index".
FIGs. 38A and B provide immunohistochemistry images showing the loss of dopamine neurons in fetal substantia nigra (SNc) due to the porcine PITX3 knockout. FIG. 38A shows tyrosine hydroxylase (TH) positive neurons in the SNc of wild-type fetal pig 62 days in gestation. FIG. 38B shows TH positive neurons in the SNc of PITX3 knockout fetal pig. Note loss of dopamine neurons in the knockout pig. VTA = ventral tegmental area.
FIGs. 39A-39D provides images showing the incorporation of human neurons in chimeric fetuses. FIG. 39A shows HNA staining of five fetal pigs to demonstrate that human neuron cells were present at least in some chimeric fetuses. FIG. 39B shows tyrosine hydroxylase staining and FIG. 39C shows NeuN staining of the same five fetus pigs as in FIG. 39A. FIG. 39D provides merged images of FIGs. 39A-39C. HNA: human nuclear antigen; TH: Tyrosine hydroxylase. FIG. 40 provides provide immunohistochemistry images showing the staining of substantia nigra stained with TH (tyrosine hydroxylase), DAPI and NeuN to substantia nigra dopamine neurons.
FIG. 41 provides bar plots showing that hiPSC complementation induced survival of nigral dopamine neurons. FIG. 41 depicts the estimate of number of TH-immunooreactivity (TH-IR) counts in both whole substantia nigral dopamine neurons and medial substantia nigra in wild type pigs (WT), PITX3 knockout pigs (KO), PITX3 KO complemented with hUCBSCs, and PITX3 KO complemented with hiPSCs.
FIG. 42 provides a schematic and an image showing LMX1A/PITX3 and dopamine neuron development. The inset image is the same as those of the FIG. 17 on LMXA1 and PITX3 gene knockout using TALEN.
FIG. 43 provides a schematic showing the generation of chimeric pig with dopamine neuron knockout and complementation with GFP expression porcine blastomeres.
LMX1A/PITX3 knockout fibroblasts were generated as described herein. Wild type porcine blastomeres were introduced into the LMX1A/PITX3 blastocysts derived from the knockout fibroblasts to create chimeric blastocysts. The chimeric blastocysts develops into a chimeric pig with immune system organs/cells originated from donor.
FIGs. 44A-44F provides images showing that the porcine-porcine complementation in PITX3/LMX1A rescued fetal development. FIGs. 44A-44C exemplify PITX3/LMX1A knockout fetuses with successful complementation based on normal size of development for their gestational age. FIGs. 44D-44F show poor embryonic development indicating poor complementation.
FIGs. 45A shows Haemotoxylin and Eosin (H&E) staining of the wild-type fetal porcine lens (L). FIG45B exemplifies lack of lens (L) development in the PITX3 knockout fetus. R = retina, C = cornea.
FIGs. 46A-C provide Haemotoxylin and Eosin (H&E) staining showing that porcine- porcine complementation in PITX3/LMX1A knockout (KO) reinstates lens development following stem cell complementation with porcine blastomere cells.
FIGs. 47A-47D depicts immunohistochemistry images showing that donor GFP+ cells contributed to the fetal pig brain. FIGs. 47B-47D are the enlarged detailed view of the corresponding sections in FIG. 47A.
FIGs. 48A-48C provide tyrosine hydroxylase immunohistochemistry images showing that porcine-porcine complementation results in development of DA neurons in primordial ventral mesencephalon (VM) in PITX3/LMX1A knockout pig fetuses. VM shown in FIGs 48A- 48C were derived from fetuses exhibiting normal development based on Crown-Rump length. FIG. 49 provides a schematic showing that RAG2 and IL2Rg are important for T cell, B cell, and NK cell development.
FIG. 50 provides a schematic showing that RAG2 and IL2Rg are important for thymus development.
FIG. 51 provides a schematic showing the procedure for hematopoietic system knockout and complementation with GFP-expressing porcine blastomeres. IL2Rg/RAG2 knockout fibroblasts were generated as described herein. Wild type porcine blastomeres were introduced into the IL2RG/RAG2 knockout blastocysts to create chimeric blastocysts. The chimeric blastocysts develops into a chimeric pig with immune system organs/cells originated from donor.
FIGs. 52A-52C provides images showing that complementation in IL2Rg/RAG2/C-KIT knockout results in generation of thymus. Thymus developed in wild-type pig fetus (FIG. 52A), but failed to do so in IL2Rg/RAG2 knockout pig fetus (FIG. 52B). Complementation with blastomeres from wild type animal resulted in the development of thymus in chimeric
IL2Rg/RAG2/C-KIT knockout pig (FIG. 52C).
FIG. 53 provides graphs showing the generation of GFP+ immune cells in chimeric pigs. Cells were collected from cord blood, thymus, spleen, peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN). Live cells were subject to FACS without staining. The percentage of GFP+ cells of live cells is shown in each graph.
FIGs. 54A-54F provides graphs showing the generation of immune cells in the thymus.
Cells were collected from thymus of wild type pig embryo (FIGs. 54A-54C) and chimeric pigs (FIGs. 54D-54F) and stained for T cells, B cells and NK cells. The cells are subject to FACS. Chimeric pigs showed similar immune cell composition in thymus to wild type pigs.
FIG. 55 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated T cells (CD 172a" CD2+ CD3 ) in blood. Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count T cells.
FIG. 56 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated T cells (CD 172a" CD2+ CD3 ) in spleen. Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count T cells.
FIG. 57 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated B cells (CD3" CD79a+ CD21+ or CD21") in blood. Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count B cells. FIG. 58 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated B cells (CD3" CD79a+ CD21+ or CD21") in spleen. Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained.
Collected cells were subject to FACS to identify and count B cells.
FIG. 59 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated NK cells (CD 172a" CD16+ CD2 ) in blood. Cells were collected from blood of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count NK cells.
FIG. 60 provides graphs showing that complementation in IL2Rg/RAG2/C-KIT knockout generated NK cells (CD 172a" CD16+ CD2 ) in spleen. Cells were collected from spleen of wild type pig embryos, chimeric pig embryos and knockout pig embryos and stained. Collected cells were subject to FACS to identify and count NK cells.
FIG. 61 provides a schematic showing that PAX3 regulates myogenesis.
FIGs. 62A and 62B provides images showing PAX3 mutation in mice. FIG. 62A depicts the comparison between wild type mice and PAX3 mutant mice. Splotchdelayed mice carry allele of PAX3 gene mutation. Postnatal Splotchdelayed animals at P0 showed spina bifida (red arrow). No myogenesis occurred in limb muscle level (white arrow). FIG. 62B depicts the effect of blastocyst complementation using PAX3 mutant mice. Act-GFP iPSCs were preferentially contributed to limb skeletal muscle in PAX3 mutant chimera, compared to wild- type chimera.
FIG. 63 provides a schematic showing that PDX1 regulates pancreas development. FIG. 64 provides a schematic showing that NKX2.1 regulates lung development.
FIG. 65 provides a schematic showing that OUG1/OLIG2 are important in
oligodendrocyte development.
FIGs. 66A-66C provide schematics, an electrophoresis image, and graphs showing the generation of OLIG1/OLIG2 knockout fibroblast. TALEN technology is used to generate the knockout as described herein. FIG. 66A illustrates the strategy to generate the OLIG1/OLIG2 knockout animals. ssOLIGl CDS and OLIG2 CDS were used to introduce a stop codon in the open reading frames of OLIG1 and OLIG2, respectively. These two polynucleotides also introduce a Hind III restriction site for the RFLP analysis. FIG. 66B is an electrophoresis image showing the confirmation of introduction of stop codon in OLIG1 and OLIG2 gene using RFLP analysis. FIG. 66C provides sequencing results of wild type (WT) cells and three colonies of OLIG1/OLIG2 knockout cells to confirm the introduction of stop codon in the open reading frame of OLIG1 and OLIG2 genes of the mutants' genome.
FIGs. 67A and 67B provides images showing OUG1/OLIG2 knockout pig fetus complemented with GFP -expressing porcine blastomeres. Wild type blastomeres expressing were introduced into OLIG1/OLIG2 knockout blastocytes. FIG. 69A shows a porcine fetus at 30 days in gestation. FIG. 69B shows whole body fluorescent imaging of GFP complemented porcine fetus.
FIGs. 68A and 68B provide images showing that GFP -expressing porcine blastomeres contribute to OLIG1/OLIG2 knockout fetal pig brain. FIG. 68A is an image of fetal pig brain. FIG. 68B is an image showing the expression of GFP in fetal chimeric pig brain, indicating the incorporation of wild type donor cells in knockout fetal pig brain.
FIGs. 69A and 69B provide images showing the 3-D reconstruction of GFP -labelling in spinal cord of OLIG1/OLIG2 knockout (KO) with GFP-blastomere complementation. FIG. 71A is an image showing coronal reconstruction. FIG. 71 B is an image showing lateral reconstruction.
FIG. 70 provides a schematic showing that HHEX regulates liver development.
FIG. 71 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2 (RG-KO) pigs showing percent of T cells (CD172a CD2+ CD3")
FIG. 72 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2
(RG-KO) pigs showing percent of B cells (CD3" CD79a+CD21+)
FIG. 73 provides a plot showing FACS data from the day 100 (gestation) I12rg/Rag2 (RG-KO) pigs showing percent of NK cells (CD172a- CD16+CD2+).
FIGs. 74A-74C depict the complete hind limb muscle formation in Pax3 mutant chimeric mouse after blastocyst complementation. FIG. 74A provides a schematic showing the introduction of wild type mouse IPSCs (induced pluripotent stem cells) into PAX3 mutant mouse blastocysts. FIG. 74B is an image showing that the hind limb was present in a PAX3 homozygous chimeric mouse. The dashed line indicates where the cross section was made for the histology study of lower hind limb (FIG. 74). FIG. 74C provides an image showing the histology of cross section of lower hind limb. Legends for FIG. 74C: TP: Tibialis posterior; TA: Tibialis anterior; EDL: Extensor digitorum longus; FHL: Flexor halluces longus; FB/FL: Fibularis brevis/longus; Sol: Soleus; M-Ga: Medial gastrocnemius aponeurosis; L-GA: Lateral gastrocnemius aponeurosis.
FIGs. 75 A-75F depict that PAX3 mutant pigs recapitulate phenotypes of PAX3 mutant mice. FIG. 75A is a schematic showing the knockout of PAX3 gene using TALENS method as described herein. FIG. 75B provides an electrophoresis image showing the confirmation of the mutant cells using RFLP analysis as described herein. FIGs. 75C-75F provide images showing the comparison of wild type animals and mutant animals. FIGs. 75C and 75D are images of wild type and mutant pigs at E30, respectively. FIGs. 75E and 75F are images of wild type and mutant mice at E12.5, respectively. In wild type animals (pig and mouse, FIGs. 75C and 75E), the hind limbs were present, while in mutant animals (pig and mouse, FIGs. 75D and 75F), the hind limb development were significantly affected.
FIGs. 76A-76D depicts PAXs mutant pigs display loss of limb skeletal muscle. FIG. 76A is an image of a wild type pig embryo at E30. FIG. 76B is an image of a mutant pig embryo at E30. In both FIGs. 76A and 76B, the dotted line indicates where the cross section was made for the immunohistochemistry study in FIGs. 76C and 76D. FIG. 76C provides images showing the staining of desmin, MHC and Pax7 in wild type animal hind limb. FIG. 76D provides images showing the staining of desmin, MHC and Pax7 in mutant animal hind limb.
FIG. 77 provide electrophoresis images depicting knockout of IL2Rg/RAG2 (RG-KO) in piglets day 100 of gestation. Piglets were harvested two weeks before parturition and subject to genotyping. The expected RFLP banding pattern of IL2Rg/RAG2 bi-allelic knockout were observed. The cells of piglet No. 2 were used to add another mutation to the C-Kit gene.
FIGs. 78A-78C depicts the incorporation of V831M into IL2Rg/RAG2 RG-KO cells to produce IL2Rg/RAG2/C-KIT (RGK) cells. FIG. 78A provides a schematic showing the location of the introduced mutation in C-KIT gene and the polynucleotides design of the C-Kit V831M HDR. FIG. 78B provides an alignment of a portion of pig C-KIT protein with that of mouse showing the amino acid residue to be replaced. FIGs. 78A and 78B demonstrate that the correct residue was identified. FIG. 78C provides electrophoresis images showing the confirmation of the introduction of mutation into the genome. Colonies 285 and 286 are examples of homozygous positives for the V-M RFLP.
FIG. 79 provides sequencing graphs showing the confirmation of the incorporation of C- Kit gene mutation in the genome of the cells. Clones 162, 285, and 286 contains the V831M mutation. These clones were used for chimera productions.
FIG. 80 provides electrophoresis images of genotyping of analysis of the chimera fetuses of IL2Rg/RAG2/C-KIT. The uncleaved band as indicated by the red box indicate that the fetus may be chimeric.
DETAILED DESCRIPTION
The present disclosure provides methods to engineer and to produce viable authentic human organs such as hearts, livers, kidneys, lungs, pancreases, and skeletal muscle; and cells such as neurons and oligodendrocytes, immune cells, and endothelial cells for making blood vessels. The strategy to achieve this goal is to disrupt key genes that are important for the development of specific organs. These genes are evaluated using gene editing technology to knockout specific genes to determine which genes alone or in combination can give rise to specific organs or cell types when knocked out in murine and porcine blastocysts. The gene knockouts in blastocysts can create a niche in which normal syngeneic or xenogeneic stem cells should occupy to contribute to the development of the desired organ or cell (FIG. 1). Novel gene editing and gene modulation technologies using TALENS, CRISPR, and synthetic porcine artificial chromosomes are used to knockout desired target genes and to enhance the function of other genes that can minimize off -target effects. Human stem cells are vetted to determine which type of stem cell gives rise to a robust replication of specific human organs and cells. This issue is addressed by evaluating the contribution of various human stem cells to the inner cell mass of porcine blastocysts and to the developing chimeric fetus. The interactions among these three technical areas are important to the successful achievement of creating authentic human organs and cells.
Definitions
Before further description of the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here.
"Humanized" as used herein refers to an organ or tissue harvested from a non-human animal whose protein sequences and genetic complement are more similar to those of humans than the non-human host.
"Organ" as used herein refers to a collection of tissues joined in a structural unit to serve a common function. "Tissue" as used herein refers to a collection of similar cells that together carry out a specific function.
"Meganuclease" as used herein are another technology useful for gene editing and are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome). Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
The term "targeted gene" refers to a site of chromosomal DNA that is selected for endonuclease attack by design of the endonuclease system, e.g., a TALENs or CRISPR.
Gene editing, as that term is used herein, refers to choosing a gene and altering it.
Random insertions, gene trapping, and the like are not gene editing. Examples of gene edits are, at targeted sites, gene knockouts, adding nucleic acids, removing nucleic acids, elimination of all function, introgression of an allele, a hypermorphic alteration, a hypomorphic alteration, and a replacement of one or more alleles. The term "knockout, inactivated, and disrupted" and variants thereof are used interchangeably herein to mean that a gene expression product is eliminated or greatly reduced, by any menas, so that the gene's expression no longer has a significant impact on the animal as a whole. These terms are sometimes used elsewhere to refer to observably reducing the role of a gene without essentially eliminating its role. These terms generally refer to preventing the formation of a functional gene product. A gene product is functional only if it fulfills its normal (wild-type) functions. Disruption of the gene prevents expression of a functional factor encoded by the gene and comprises an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal. The disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene.
The term "replacement" of an allele means the change is made from the native allele to the exogenous allele without indels or other changes except for, in some cases, degenerate substitutions.
The term "degenerate substitution" means that a base in a codon is changed to another base without changing the amino acid that is coded. The degenerate substitution may be chosen to be in an exon or in an intron. One use for a degenerate substitution is to create a restriction site for easy testing of a presence of the introgressed sequence. The endogenous allele is also referred to herein as the native allele.
The term "gene" is broad and refers to chromosomal DNA that is expressed to make a functional product.
The term "select" is used to refer to the ability to identify and isolate the cells for further use; there were no expressible reporter genes anywhere in the process, which is a highly significant advantage that distinguishes this process from many other approaches.
The term "blastocyst" is used broadly herein to refer to embryos from two cells to about three weeks.
The term "embryo" is used broadly to refer to animals from zygote to live birth.
The term "gametogenesis" means the production of haploid sex cells (ova and spermatozoa) that each carry one-half the genetic compliment of the parents from the germ cell line of each parent. The production of spermatozoa is spermatogenesis. The fusion of spermatozoa and ova during fertilization results in a zygote cell that has a diploid genome.
The term "gametogenic cell" refers to a progenitor to an ovum or sperm, typically a germ cell or a spermatogonial cell.
The term "large vertebrate" refers to simians, livestock, dogs, and cats. The term "livestock" refers to animals customarily raised for food, such as cattle, sheep, goats, avian (chicken, turkey), pigs, buffalo, and fish.
The term "cognate" refers to two biomolecules that typically interact, for example, a receptor and its ligand. In the context of HDR processes, one of the biomolecules may be designed with a sequence to bind with an intended, i.e., cognate, DNA site or protein site.
The term "insertion" is used broadly to mean either literal insertion into the chromosome or use of the exogenous sequence as a template for repair.
The term "exogenous nucleic acid" means a nucleic acid that is added to the cell or embryo, regardless of whether the nucleic acid is the same or distinct from nucleic acid sequences naturally in the cell. The term "nucleic acid fragment" is broad and includes a chromosome, expression cassette, gene, DNA, RNA, mRNA, or portion thereof. The cell or embryo may be, for instance, chosen from the group consisting non-human vertebrates, non- human primates, cattle, horse, swine, sheep, chicken, avian, rabbit, goats, dog, cat, laboratory animal, and fish.
As used herein, "operably linked" refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid.
As used herein, "replacement of an allele" refers to a non-meiotic process of copying an exogenous allele over an endogenous allele. Genes have alleles. Genotypes are homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. Alleles are alternative forms of a gene (one member of a pair) that are located at a specific position on a specific chromosome. Alleles determine distinct traits. Alleles have basepair (bp) differences at specific positions in their DNA sequences (distinguishing positions or bp) that give rise to the distinct trait and distinguish them from each another, these distinguishing positions serve as allelic markers. Alleles are commonly described, and are described herein, as being identical if they have the same bases at distinguishing positions; animals naturally have certain variations at other bp in other positions. Artisans routinely accommodate natural variations when comparing alleles. The term exactly identical is used herein to mean absolutely no bp differences or indels in a DNA alignment.
Genetic complementation
Classically, genetic complementation, refers to the production of a wild-type phenotype when two different mutations are combined in a diploid or a heterokaryon. However, modern techniques of chimera production can now rely on stem cell complementation, whereby cells of more than one embryonic origin are combined to make one genetically mixed animal. In this case, complementation does not involve any change in the genotypes of individual chromosomes; rather it represents the mixing of gene products. Complementation occurs during the time that two cell types are in the same embryo and can each supply a function. Afterward, each respective chromosome remains unaltered. In the case of chimeras, complementation occurs when two different sets of chromosomes, are active in the same embryo. However, progeny that result from this complementation can carry cells of each genotype. In embryonic complementation, genes of the host embryo are edited to produce a knock out or otherwise make a non-functional gene. When human stem cells are injected into the gene edited blastocyst, they can rescue or "complement" the defects of the host (edited) genome. When the gene or genes that are knocked out support the growth of a particular organ or tissue, the resulting complementation produced tissue can be the result of the growth and differentiation of the non- edited, e.g., stem cell derived genotype. When human stem cells are used to complement the host-edited genome, the resulting tissue or organ can be composed of human cells. In this way, fully human organs can be produced, in vivo, using an other animal as a host for the complementation produced organ.
Because multiple genes may be responsible for the growth and differentiation of any particular organ or tissue, processes for multiplex gene edits are also described. Multiple genes can be modified or knocked out in a cell or embryo that may be used for research or to make whole chimeric animals. These embodiments include the complementation of cell or organ loss by selective depopulation of host niches. These inventions provide for rapid creation of animals to serve as models, food, and as sources of cellular and a cellular products for industry and medicine.
FIG. 1 provides a schematic description of the problem and the proposed approach for providing personalized human organs and tissues to those in need using swine as a host animal. Those of skill in the art can appreciate that the technology which allows for the production of induced pluripotent stem cells (IPSC) allows for a patient to provide her or his own stem cells for complementation of the edited genes and production of human or humanized "self organs or tissues.
The use of multiplex gene editing is important for producing a host animal with multiple edited genes in need of complementation. FIG. 2 A has a timeline that illustrates why it takes several years using single edits to make livestock that have only two edited alleles, with the time being about six years for cattle. Edited, in this context, refers to choosing gene and altering it. First, a gene of interest has to be edited, for instance knocked out (KO), in cultured somatic cells that are cloned to create a heterozygous calf with a targeted KO. The heterozygotes would be raised to maturity for breeding, about 2 years old for cattle, to generate first-generation (Fl) male and female heterozygous calves, which would be bred with each other to generate a homozygous knockout calf (F2). Generating homozygotes with respect to multiple targeted mutations using a conventional approach in cattle would be impractical. The number of years and the number of animals used to make further edits increases in an approximately exponential fashion, depending on the particular scheme that is used, as illustrated in FIG. 2B. Among the vertebrates, even those animals that have larger numbers of offspring per generation and have shorter gestational times than cattle nonetheless would require overly long times to achieve multiple edits. Swine, for example, have a larger number of offspring per mating and a gestational time that is roughly half that of cattle but the time to make multiple edits can require many years. Moreover, schemes that minimize time with aggressive inbreeding may not be reasonably possible for multiple edits. Also, serial cloning is undesirable from a process and an outcome standpoint, especially if the animals are to be useful as livestock or laboratory models.
An opportunity presented by the invention is illustrated in FIG. 3, which shows multiple edits being made in a first-generation animal (F0). Embryos are prepared directly or by cloning with two or more edits independently chosen to be heterozygotes or homozygotes and placed in surrogate females to gestate. The resultant animals are F0 generation founders. A plurality of embryos may be prepared and placed in one or more surrogates to produce progeny of both genders, or well-known techniques of embryo-splitting may be used to make a plurality of clonal embryos. Livestock such as pigs that typically produce a litter with both genders may be crossed and propagated.
Multiple alleles can be disrupted or otherwise edited as described herein in a cell or embryo using targeted endonucleases and homology directed repair (HDR). An embodiment is a method of making genetic edits in a vertebrate cell or embryo at a plurality of target chromosomal DNA sites comprising introducing into a vertebrate cell or embryo: a first targeted endonuclease directed to a first target chromosomal DNA site and a first homology directed repair (HDR) template homologous to the first target site sequence; and a second targeted endonuclease directed to a second target chromosomal DNA site and a second HDR template homologous to the second target site sequence, with the first HDR template sequence replacing the native chromosomal DNA sequence at the first target site and the second HDR template sequence replacing the native chromosomal DNA sequence at the second target site sequence.
It was an unexpected and surprising, and not predictable, result to learn that multiple edits such as knockouts or replacements could be obtained. One theorized mechanism is that there are a minority of cells that are receptive to multiple edits because they are at a particular stage in the cell cycle. When exposed to endonucleases and HDR templates, they respond readily. A related theory of operation is that the HDR templating process lends itself to multiple substitutions because activation of cellular repair machinery for one targeted site favors repair, or HDR templating, at other sites as well. HDR has historically been a low efficiency process so that multiple HDR edits were apparently not attempted, observed, or recognized. Heretofore, previous experiments with xenogeneic complementation have only been done on single edit genomes. However, the disclosed platform for multiplex gene editing now provides for a host blastocyst having multiple edited genes allowing for the complementation of those edits by human stem cells and the production of those organs and tissues arising therefrom.
Results herein show that too much or too little endonuclease and/or HDR template can have a negative effect, which may have confounded prior research in this area. In fact, it has been observed that targeted endonucleases can be designed and made correctly but nonetheless fail because they are too efficient. Further, the population of successfully modified cells often does not improve over time. Artisans modifying cells normally look for longevity of the cell and modification as an indicator of stability and health for successful cloning or other uses. But that expectation has often not been helpful in the multiplexing processes herein. Moreover, it has been observed that homologous recombination (HR) introgression efficiencies are variable in the multiplex approach as compared to a single-locus introgression. Some loci were very sensitive but others had large drops in efficiency. There is apparently interference between the endonucleases but the net effect cannot be explained simply, for instance by positing that the endonucleases are competing for common resource.
There are various well-known techniques to insert many genes randomly or imprecisely into a plurality of locations in chromosomal DNA, or to make many random edits that disrupt a plurality of genes. As is evident, random or imprecise processes are not going to assist the scientist that needs to edit a plurality of specifically targeted genes to achieve an effect.
Accordingly, HDR processes taught herein may be readily distinguished by the edits, and resultant organisms, being made only at the intended target sites. One difference is that the inventive HDR editing embodiments can be performed free of insertion of extra gene copies and/or free of disruption of genes other than those targeted by the endonucleases. And the specific edits are made at one location because the HDR template sequence is not copied into sites without appropriate homology. Embodiments include organisms and processes wherein an exogenous allele is copied into chromosomal DNA only at the site of its cognate allele.
An advantage of HDR-based editing is that the edits can be chosen. In contrast, other attempts, by non-homologous end joining (NHEJ) processes, can make indels at multiple positions such that the indels cancel each other out without making a frame shift. This problem becomes significant when multiplexing is involved. But successful use of HDR provides that the edits can be made to ensure that, if desired, the target gene has an intended frame shift. Moreover, allelic replacement requires HDR and cannot be accomplished by NHEJ, vector- driven insertion of nucleic acids, transposon insertions, and the like. Moreover, choosing organism that are free of unwanted edits further increases the degree of difficulty. It is generally believed, however, that multiplex edits as described herein have not been previously achieved at targeted sites in cells or animals relevant to livestock or large vertebrates. It is well known that cloning animals from high-passage cells creates animals with so much genetic damage that they are not useful as F0 founders of laboratory models or livestock.
And gene editing is a stochastic process; as a result, the field has traditionally emphasized various screening techniques to identify the few percent of cells that have successfully been edited. Since it is a stochastic process, the difficulty of making a plurality of edits can be expected by the artisan to increase in an exponential fashion as the number of intended edits increases.
An embodiment of the invention provides processes for creating multiple targeted gene knockouts or other edits in a single cell or embryo, a process referred to herein as multiplex gene knockouts or editing.
A similar test for allelic identity is to align the chromosomal DNA in the altered organism with the chromosomal DNA of the exogenous allele as it is recognized in nature. The exogenous allele can have one or more allelic markers. The DNA alignment upstream and downstream of the markers can be identical for a certain distance. Depending on the desired test, this distance may be from, e.g., 10 to 4000 bp. While an HDR template can be expected to create a sequence that has exactly identical, the bases on either side of the templated area can, of course, have some natural variation. Artisans routinely distinguish alleles despite the presence of natural variations. Artisans can immediately appreciate that all ranges and values between the explicitly stated bounds are contemplated, with any of the following distances being available as an upper or lower limit: 15, 25, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 4000.
Artisans are also able to distinguish gene edits to an allele that are a result of gene editing as opposed to sexual reproduction. It is trivial when the allele is from another species that cannot sexually reproduce to mix alleles. And many edits are simply not found in nature. Edits can be also be readily distinguished when alleles are migrated from one breed to the next, even when a replacement is made that exactly duplicates an allele naturally found in another breed. Alleles are stably located on DNA most of the time. But meiosis during gamete formation causes male and female DNAs to occasionally swap alleles, an event called a crossover. Crossover frequencies and genetic maps have been extensively studied and developed. In the case of livestock, the pedigree of an animal can be traced in great detail for many generations. In genetics, a centimorgan (cM, also called a map unit (m.u.)) is a unit that measures genetic linkage. It is defined as the distance between chromosome positions (loci or markers of loci) for which the expected average number of intervening chromosomal crossovers in a single generation is 0.01. Genes that are close to each other have a lower chance of crossing over compared to genes that are distant from each other on the chromosome. Crossing over is a very rare event when two genes are right next to each other on the chromosome. Crossing over of a single allele relative to its two neighboring alleles is so improbable that such an event must be the product of genetic engineering. Even in the case where animals of the same breeds are involved, natural versus engineered allele replacement can be readily determined when the parents are known. And parentage can be determined with a high degree of accuracy by genotyping potential parents. Parent determination is routine in herds and humans.
Embodiments include multiplex gene editing methods that are simultaneous. The term simultaneous is in contrast to a hypothetical process of treating cells multiple times to achieve multiple edits, as in serial knockouts or serial cloning or intervening cycles of animal breeding. Simultaneous means being present at a useful concentration at the same time, for instance multiple targeted endonucleases being present. The processes can be applied to zygotes and embryos to make organisms wherein all cells or essentially all cells have edited alleles or knockouts. Essentially all cells, in the context of a knockout for instance, refers to knocking the gene out of so many cells that the gene is, for practical purposes, absent because its gene products are ineffective for the organism's function. The processes modify cells, and cells in embryos, over a minimal number cell divisions, preferably about zero to about two divisions. Embodiments include a quick process or a process that takes place over various times or numbers of cell divisions is contemplated, for instance: from 0 to 20 replications (cell divisions). Artisans can immediately appreciate that all values and ranges within the expressly stated limits are contemplated, e.g., about 0 to about 2 replications, about 0 to about 3 replications, no more than about 4 replications, from about 0 to about 10 replications, 10-17; less than about 7 days, less than about 1, about 2, about 3, about 4, about 5, or about 6 days, from about 0.5 to about 18 days, and the like. The term low- passage refers to primary cells that have undergone no more than about 20 replications.
Elsewhere, it has been shown that, in a single embryo, maternal, paternal or both alleles can be edited in bovine and porcine embryos, and that template editing of both alleles can therefore occur using HDR in the embryo. These edits were made at the same locus.
Specifically introgression from sister chromatids was detected. Carlson et al., PNAS
43(109): 17382-17387, 2012.
Example 1, see FIGs. 4A-4D, describes experiments that attempted, successfully, to use HDR editing to knockout two genes at once and, further, to be able to select cells that are homozygous for both knockouts or heterozygous for each knockout. Cells were treated to introduce a first and a second targeted endonuclease (each being a TALENS pair) directed to, respectively, a first gene (Recombination Activating Gene 2, RAG2) and a second gene target (Interleukin Receptor 2, gamma, IL2Rg or ILR2y). The TALENS had to be designed to target intended sites and made in adequate amounts. The treatment of the cells took less than five minutes. Electroporation was used but there are many other suitable protein or DNA introducing-processes described herein. The cells were then cultured so that they formed individual colonies of cells that each descended from a single treated cell. Cells from the various colonies were tested after 3 days or 11 days. The rate of knockout of RAG2 was about six times higher than the rate of knockout of IL2Rg; apparently some genes are more difficult to knockout than others. The efficiency of knocking out both genes was high and cells heterozygous or homozygous for both knockouts were successfully identified. Significantly, dosage of TALEN mRNA and HDR template had specific and non-specific effects. An increase in TALEN mRNA for IL2Rg led to an increase in both NHEJ and HDR for IL2Rg while NHEJ levels for RAG2 were unchanged. An increase in IL2Rg HDR template reduced HDR at the RAG2 locus suggesting a nonspecific inhibition of homology directed repair by escalation of the concentration of oligonucleotide. This dose sensitivity, particularly at these low doses, has possibly lead others away from pursuit of multiplex processes. Cells from Example 1 have been cloned and, at the time of filing, two animals are pregnant with embryos derived from the same.
Example 2, see FIGs. 5A-5D, describes experiments that had the same goal of multiplex HDR editing but for different genes. The first gene target was Adenomatous polyposis coli (APC). The second gene target was p53 (the TP53 gene). Cells homozygous for both knockouts and cells heterozygous for both knockouts were detected and isolated.
Example 3, see FIGs. 6-9, describes multiplex HDR editing to knockout 2-5 genes.
There were three experiments, with the number of cell colonies tested for genotype ranging from 72-192 for each experiment. Cells were treated for multiplex knockout of various combinations the genes APC, p53, RAG2, Low Density Lipoprotein Receptor (LDLR), IL2Rg, Kisspeptin Receptor (KISSR or GPR54), and Eukaryotic Translation Initiation Factor 4GI (EIF4GI). The gene LDLR was consistently less amenable to modification than the other genes. As is evident from the results, multiple alleles can be disrupted simultaneously using the TALEN-specified, homology directed repair (HDR). Five TALEN pairs that each resulted in more than 20% HDR/site and their cognate HDR templates were simultaneously co-transfected in three combinations (Table A). A proportion of colonies from each replicate were positive for HDR events in at least four genes and two colonies from replicate-A had HDR events in all five genes. Although simultaneous indel formation in five genes has been demonstrated by Cas9/CRISPR- stimulated NHEJ in mouse embryonic stem cellss (ES or ESC, used interchangeably), the precise modification of 5 genes (up to 7 alleles) by targeted nuclease-stimulated HDR is unexpected, surprising, and unrivaled. When the TALENS of replicate were replaced
Cas9/CRISPRs (vectors were introduced into cells to express), modification levels were below detection (data not shown); however, other data points to RGEN multiplex, e.g., Example 9 below. Four genes were found to be edited in all experiments and five genes in one experiment.
The speed and efficiency of this process lends itself to scaling-up such that the multiplex knockout of more than 5 genes is achievable without changing the nature of the process.
Referring to Table A, about 72 to 192 cells were tested; now that this process has been established it is not unreasonable to increase the number of tests to a very much larger number of cells such that multiplex of larger numbers of genes/alleles can be expected. A number of multiplex genes or alleles may be from 2-25; artisans can immediately appreciate that all ranges and values between the explicitly stated bounds are contemplated, with any of the following being available as an upper or lower limit in combination with each other: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 25.
Table A: Multiplex HDR in pig fibroblasts
Figure imgf000039_0002
Figure imgf000039_0001
Figure imgf000039_0003
Genes targeted in each replicate:
A. APC, LDLR, RAG 2, IL2Rg, p53.
B. APC, LDLR, RAG2, KISSR, ELF4G1
C. APC, LDLR, RAG2, KISSR, DMD
As is evident, cells and embryos with multiplex knockouts are embodiments of the invention, as well as animals made thereby.
Example 4 describes some detailed processes for making various animals and refers to certain genes by way of example. Example 5 describes examples of CRISPR/Cas9 design and production.
Example 6 provides further examples of multiplex gene editing with targeted nucleases driving HDR processes. GATA binding protein 4 (GATA4); homeobox protein NKX2-5 (NKX2-5) and Mesoderm Posterior Protein 1 (MESP1) were simultaneously targeted with TALENs and HDR templates to direct frame-shift mutations and premature stop-codons into each gene. The objective was to create biallelic knockouts for each gene for use in complementation studies. The process was about 0.5% efficient as 2 clones had the intended biallelic HDR at each gene. The given genes knocked out singly or in combination genes can cause a failure to thrive genotype and early embryonic lethality without complementation. Artisans can appreciate that knockout of these genes individually and interbreeding of heterozygotes to obtain triple knockouts (about 1/66 chance) for FTT and complementation studies is not feasible in livestock.
Example 7 provides data that TALENs and Cas9/CRISPR can be mixed to perform multiplex editing of genes. Some genes/alleles are more readily targeted by a TALEN, or Cas9/CRISPR and that the situation may arise that multiplexing must be done with a combination of these tools. In this example, the Eukaryotic Translation Initiation Factor 4GI (EIF4GI) was targeted by TALENs and the p65 (RELA) gene was targeted by Cas9/CRISPR. The cells were analyzed by RFLP assay, indicative of HDR events, and HDR was evident at both sites. Accordingly, TALENs and RGENs may be used together or separately for multiplexing Combinations including, for example, 1, 2, 3 4, 5, 6, 7, 8, 9 or 10 TALENs with 1, 2, 3 4, 5, 6, 7, 8, 9 or 10 RGEN reagents, in any combination.
Chimeras
Chimeras can be made by preparing a host blastocyst and adding a donor cell from a donor animal. The resultant animal can be a chimera that has cells that originate from both the host and the donor. Some genes are important for the embryo to create certain kinds of cells and cell lineages. When such a gene is knocked out in the host cells, the introduction of a donor cell that has the missing gene can result in those cells and cell lineages being restored to the host embryo; the restored cells have the donor genotype. Such a process is referred to as a complementation process.
Matsunari et al., PNAS 110:4557-4562, 2013, described a complementation process for making a donor-derived pig pancreas. They made a host pig blastocyst that was altered to prevent formation of a functional pancreas. They made the host blastocyst by somatic cell cloning. The somatic cell had been modified to overexpress Hesl under the promoter of Pdxl (pancreatic and duodenal homeobox 1), which was known to inhibit pancreatic development. The added donor cells to the host blastocyst that did not have this modification; the donor cells supplied the cell lineages needed to make the pancreas. They had already demonstrated elsewhere that functional organs can be generated from pluripotent stem cells (PSCs) in vivo by blastocyst complementation in organogenesis-disabled mouse embryos. They proposed future research using xenogenic pluripotent stem cells (PSCs), including human induced PSCs. Indeed, xenotransplantation has been considered a potential solution to the organ/tissue shortage for greater than 40 years. The fact that no genes were knocked out to disable the formation of the pancreas is significant.
Knocking out even one gene in a large vertebrate is a significant investment of resources using conventional processes. In contrast, overexpression of a gene product in a cell is readily achieved using the present state of the art, for instance, with a plasmid or a vector that places multiple gene cassette copies into the genome. Adding expression of a gene is easier than targeting a gene and knocking it out. The ability to prevent organogenesis by overexpression of a gene product is believed to be unusual at this time. In fact, limitations in the ability to engineer large animal genomes can be significant. Nonetheless, the pig is the preferred donor animal for xenotransplantation due to its similarity in size and physiology to humans as well as its high fecundity and growth rate.
FIG 10 depicts a multiplex process used herein to make gene knockouts or other gene edits as applied in the context of chimeras. Low-passage primary somatic cells are made with gene knockouts. Cells with exactly the desired distribution of heterozygosity and homozygosity for the knockouts are isolated. These cells are used in cloning to make an embryo that is allowed to develop as a host blastocyst. A donor embryo is established and used as a source of donor cells that provide genes to populate the niche created by the knockouts. The donor cells are introduced into the host blastocyst and reproduce with the host cells to form a chimera having both host and donor cells. The embryo is transferred to a surrogate female and gestated. The progeny of the chimera have host genotypes when the host cells form the gametes.
Chimeras have their gender determined by their host blastocyst.
FIG 11 illustrates a failure to thrive phenotype (FTT) complementation process. FTT refers to animals that are not expected to live to an age of sexual maturity. A host embryo is provided with an FTT genotype and phenotype. Multiplex processes are ideal because the FTTs available by knockout of just one gene are limited and are not known for some organs and tissues. The donor cells provide the genes missing in the FTT and provide the missing cell types. The embryo can be a large vertebrate animal and the knockouts can be multiplex, e.g., 2- 25 genes. Moreover, targeted endonucleases can be used to achieve a knockout. In an immunodeficiency embodiment, an IL2Rg-/y RAG2-/-knockout is the FTT because the host is essentially missing immune functions. But the donor cells do not have those genes missing and the resultant chimera has an essentially normal phenotype for purposes of being able to raise and maintain the animal. But the progeny has the FTT phenotype. The animals can thus be maintained and FTT animals conveniently produced. The chimeras can be any combination of heterozygous and homozygous for the knockouts. Processes for making chimera are thus described that are F0 generation animals that produce failure to thrive (FTT) phenotypes when other processes require an additional generation, or more. Chimera normally pass on the genetics of the host cells. Disclosed herein, however, are alternative chimeras that pass the donor cell genetics to their progeny and not the host cell genetics. It turns out that switching the genetic inheritance can create some useful opportunities.
Referring to FIG. 12, an embryo labeled as G" host is depicted. The embryo has been prepared with nonfunctional gametes. A donor blastocyst is prepared and used as a source of donor cells. The donor cells provide the genes and cell lineages that are needed to make donor gametes. The resultant chimera has the gametes of the donor cells and creates progeny having donor cell genetics. In the illustration, the host embryo is a male Brahman bull. The donor cells are from a double-muscled bull. The chimera has a Brahman bull phenotype but its progeny are double muscled. The host and donors may be from the same or different breeds or same or different species. The host has been prepared to be sterile, meaning that it cannot sexually reproduce. Some sterile animals may be used to make gametes that are nonfunctional, e.g., immotile sperm, or not make gametes at all, e.g., with early gametogenesis being disrupted. The donor cells may be, for instance, wild-type cells, cells from animal breeds having desirable traits, or genetically modified cells.
Embodiments of the invention include chimeric sterile animals, such as chimeric livestock, that have a genetic modification to a chromosome that prevents gametogenesis or spermatogenesis. The chromosome may be an X chromosome, a Y chromosome, or an autosome. The modification may include a disruption of an existing gene. The disruption may be created by altering an existing chromosomal gene so that it cannot be expressed, or by genetically expressing factors that can inhibit the transcription or translation of a gene. One embodiment is a knockout of spermatogonial stem cells (SSC) in the host. The animal may be made with donor cells that have desirable genetics and supplies SSC cells that make gametes with the donor genotype. Some genes are disrupted in combination to produce one or more effects that cause infertility, for instance, combinations of: Acr/Hl.1/Smcp, Acr/Tnp2/Smcp, Tnp2/Hl. l/Smcp, Acr/Hlt/Smcp, Tnp2/Hlt/Smcp ( ayernia K; Drabent B; Meinhardt A; Adham IM; Schwandt I; Muller C; Sancken U; Kleene KC; Engel W Triple knockouts reveal gene interactions affecting fertility of male mice. Mol. Reprod. Dev 70(4):406- 5 16, 2005). Embodiments include a first line of animals with a knockout of a first gene or genes and a second line of animals with a knockout of a second gene or genes so that male progeny of the lines are infertile.
The use of genetic engineering to create genetically modified large vertebrates can accelerate the creation of animals with desirable traits. Traditional livestock breeding is an expensive and time consuming process that involves careful selection of genetic traits and lengthy waits for generational reproduction. Even with careful trait selection, the variations of sexual reproduction present a considerable challenge in cultivating and passing on desirable trait combinations. But creation of chimeras that pass on donor traits creates methods of animal reproduction that allow for rapid dissemination of desirable genetic traits, as well as for protection of the proprietary control of the traits. Embodiments include the production of genetically and genomically sterile animals that can serve as hosts for donated genetic material. Sexual intercourse by the host can lead to reproduction of the donor's genetic material. A group of genetically sterile animals can be used to disseminate identical genes from a single donor by sexual reproduction so that many donor progeny may be rapidly generated. Embodiments include animals that are modified to produce only one gender of animal so that users receiving the animals are not be able to easily breed the animals with the traits.
Embodiments include making a genetic modification to cells or embryos to inactivate a gene or plurality of genes selective for gametogenesis or spermatozoa activity. One process of genetic modification involves introduction of a targeted nuclease, e.g., a Cas9/CRISPR or mRNA for a TALEN pair that specifically binds to the gene. An animal is cloned from the cells or the modified embryo is directly raised in a surrogate mother. The animal may be a livestock animal or other animal. Gametogenesis may be blocked at an early stage. Or spermatozoa activity may be disrupted that is important for fertility but is not otherwise important to the animal. The animal is thus sterile because it cannot sexually reproduce: however, ARTs may be used to create progeny from the modified sperm. A donor animal that has desirable genetic traits (as a result of breeding and/or genetic engineering) is selected.
Rapid establishment of FO generation founder animal lines with two or more knockouts
With multiplex, two, three, or more genes (2-25) may be simultaneously knocked out to produce an FO generation with the desired combination of alleles. If homozygosity for all of the knockouts creates an FTT, then one option is to make the founders homozygous for all of the knockouts except for one - or whatever the minimum heterozygosity should be for that situation. The one heterozygote gene can allow for a non-FTT phenotype. Alternatively, the multiplex knockouts can be used in combination with complementation to make thriving chimera that have FTT progeny. This process can eliminate generations in the creation of a multiple knockout animal.
In either case, the advantages are large and move many processes into the realm of actually being achievable. Producing animals with knockouts of two loci by conventional breeding is cost prohibitive as only ~6% of offspring would have the desired phenotype in the F2 generation (Table B). In contrast, the multiplex approach enables production of the desired genotype in the F0 generation, a large advantage over conventional knockouts and breeding. It should be stressed that the saving of time and animals is not theoretical: it is an advance that makes some kinds of modifications possible because success is expected instead of failure. Furthermore, to continue the example, breeding between one or two chimeric RG-KO parents would significantly increase the production rate of RG-KO offspring to 25 and 100 percent respectively (Table B).
Figure imgf000044_0001
Immunodeflcient animals
One group of embodiments relates to immunodeflcient pigs or other livestock and processes of making them. These embodiments are examples of multiplex edits, e.g., knockouts that take advantage of the opportunity to manage selection of homozygous and heterozygous nockout genotypes. These demonstrate the power of multiplex to rapidly establish founder lines. They also include further aspects of the inventions that involve making chimeras.
The pig is the most relevant, non-primate animal model that mimics the size and physiology of humans. Unfortunately, fully immunodeflcient pigs are not widely available because (1) single gene knockout (KO) is usually not sufficient, (2) intercrossing to create multi- locus null animals is extremely costly and depending on the number of Kos may be possible, and (3) only small scale germ-free facilities are available for pigs. Herein, embodiments include large vertebrate animals with a knockout of both & G2 andIL2Rg (i.e., RG-KO). The genes can be knocked out of somatic cells that are then used for cloning to produce a whole animal.
Alternatively, embryos can be treated to knockout the genes, with the animals being derived directly from the embryos. The multiplex gene-targeting platform can simultaneously disrupt of T, B and NK cell development in the pig. Accordingly, animals made without such cells can be made directly with the methods herein, as F0 founders, but the phenotype is FTT.
Agricultural Targets for Multiplex Edits
The editing of food animal genomes can be greatly accelerated by editing numerous loci at the same time, saving generations of animal breeding that would be carried out to bring together alleles that are generated instead one at a time. In addition, some agricultural traits are complex, meaning that they are manifest as a result of the influence of alleles at more than one gene (from 2 to hundreds). For example, polymorphisms at DGAT, ABCG2, and a
polymorphism on chromosome 18 together account for a large portion of the variation in Net Dairy Merit in dairy cattle. Livestock cells or embryos can be subjected to multiplex editing of numerous genes, including various agricultural targets: one or more of AC AN, AMELY, BLG, BMP IB (FecB), DAZL, DGAT, Eif4GI, GDF8, Horn-poll locus, IGF2, CWC15,
KissR/GRP54, OFD1Y, p65, PRLR, Prmdl4, PRNP, Rosa, Socs2, SRY, ZFY, β-lactoglobulin, CLPG
Disease Modeling Targets For Multiplexing
Some traits, like cancer, are caused on the basis of mutations at multiple genes (see APC/p53). In addition numerous disease traits are so-called Complex traits that manifest as a result of the influence of alleles at more than one gene. For example, diabetes, metabolism, heart disease, and neurological diseases are considered complex traits. Embodiments include animal models that are heterozygous and homozygous for individual alleles, or in combination with alleles at other genes, in different combinations. For example mature onset diabetes of the young (MODY) loci cause diabetes individually and additively, including; MODY 1 (HNF4a), MODY 2 (GCK), MODY 3 (HNFla), MODY 4 (Pdxl), MODY 5 (HNF-Ιβ), MODY 6 (eurogenic differentiation 1), MODY 7 (KLF11), MODY 8 (CEL), MODY 9 (PAX4), MODY 10 (INS), MODY 11 (BLK). Livestock cells or embryos can be subjected to multiplex editing of numerous genes for animal modelling, including various disease modeling targets: APC, ApoE, DMD, GHRHR, HR, HSD11B2, LDLR, NF1, NPPA, NR3C2, p53, PKD1, Rbm20, SCNN1G, tP53, DAZL, FAH, HBB, IL2RG, PDX1, PITX3, Runxl, RAG2, GGTA. Embodiments include cells, embryos, and animals with one or more of the above targets being edited, e.g., KO.
Genes in one species consistently have orthologs in other species. Humans and mice genes consistently have orthologs in livestock, particularly among cows, pigs, sheep, goats, chicken, and rabbits. Genetic orthologs between these species and fish is often consistent, depending upon the gene's function. Biologists are familiar with processes for finding gene orthologs so genes may be described herein in terms of one of the species without listing orthologs of the other species. Embodiments describing the disruption of a gene thus include disruption of orthologs that have the same or different names in other species. There are general genetic databases as well as databases that are specialized to identification of genetic orthologs. Moreover, artisans are familiar with the commonly used abbreviations for genes and using the context to identify which gene is being referred to in case there is more than one abbreviation for a gene or two genes are referred to by the same abbreviation.
Spermatogonial stem cells offer a second method genetic modification of livestock. Genetic modification or gene edits can be executed in vitro in spermatogonial stem cells isolated from donor testes. Modified cells are transplanted into germ cell-depleted testes of a recipient. Implanted spermatogonial stem cells produce sperm that carry the genetic modification(s) that can be used for breeding via artificial insemination or in vitro fertilization (IVF) to derive founder animals.
Complementation of nullomorphic cell or organ loss by selective depopulation of host niches Multiplex editing can be used to purposefully ablate cells or organs from a specific embryonic or animal niche, creating an environment conducive to better donor cell integration, proliferation, and differentiation, enhancing their contribution by complementation of orthologous cells, tissues or organs in the embryo, fetus or animal. The animal with the empty niche is a deficiency carrier because it has been created to have a deficiency that can be filled by donor cells and genes. Specific examples include the recipient-elimination, and donor-rescue of gametogenic cell lineages (DAZL, VASA, MIWI, PIWI, and so forth.).
In another embodiment multiplex gene editing can be used to induce congenital alopecia, providing opportunity for donor derived cells to participate in hair folliculogenesis. The genes considered for multiplex gene editing to cause alopecia include those identified in OMIM and thru Human Phenotype Ontology database; DCAF17, VDR, PNPLA1, HRAS, Telomerase-vert, DSP, SNRPE, RPL21, LAM A3, UROD, EDAR, OFD1, PEX7, COL3A1, ALOX12B, HLCS, NIPAL4, CERS3, ANTXR1, B3GALT6, DSG4, UBR1, CTC1, MBTPS2 ,UROS, ABHD5, NOP10, ALMS1, LAMB3, EOGT, SAT1, RBPJ, ARHGAP31, ACVR1, IKBKG, LPAR6, HR, ATR, HTRA1, AIRE, BCS1L, MCCC2, DKC1, PORCN, EBP,
SLITRK1, BTK, DOCK6, APCDD1, ZIP4, CASR, TERT, EDARADD, ATP6V0A2, PVRL1, MGP, KRT85, RAG2, RAG-1, ROR2, CLAUDIN1, ABCA12, SLA-DRA1, B4GALT7, COL7A1, NHP2, GNA11, WNT5A, USB1, LMNA, EPS8L3, NSDHL, TRPV3, KRAS, TINF2, TGM1, DCLRE1C, PKP1, WRAP53, KDM5C, ECM1, TP63, KRT14, RIPK4. Chimerism with donor cells that have folliculogenic potential may be used to grow human hair follicles. The ablation of organs or tissues in pigs or other vertebrates and growth of organs or tissues from human origins is particularly useful as a source of medical organs or tissues.
Further complementation targets for multiplexing are: PRKDC, BCLl la, BMI1, CCR5, CXCR4, DKKl, ETV2, FLU, FLKl, GATA2, GATA4, HHEX, C-KIT, LMXIA, MYF5, MYODl, MYOG, NKX2-5, NR4A2, PAX3, PDXl, PITX3, Runxl, RAG2, GGTA, HR, HANDII, TBX5.
Embodiments include targeting one, two, or more (2-25) of the above targets in a multiplex approach or by other approaches. Edited Genes
The methods and inventions described herein with respect to particular targets and targeted endonucleases are broadly applicable, primary livestock cells suitable for cloning with edits with all of the following genes have been prepared.
Figure imgf000047_0001
DKK1 Dickkopf -related protein 1 S
Nuclear receptor subfamily 4, group A,
NR4A2/NURR1 member 2/Nuclear receptor related 1 protein S
FLK1 Fetal Liver Kinase 1 S
HHEX1 Hematopoietically-expressed homeobox S
protein
BCL11A B-cell lymphoma/leukemia 11 A S
RAG2 Recombination activating gene 2 S
RAG1 Recombination activating gene 1 S
IL2RG Interleukin 2 receptor, gamma S
c-KIT/SCFR Mast/stem cell growth factor receptor S
BMI1 poly comb ring finger oncogene S
HANDII Heart- and neural crest derivatives- expressed S
protein 2
TBX5 T-box transcription factor 5 S
GATA2 GATA binding protein 2 S
DAZL Deleted in Azoospermia like S, B
OLIG1 oligodendrocyte transcription factor 1 S
OLIG2 oligodendrocyte transcription factor 2 S
Genetically Modified Animals
Animals may be made that are mono-allelic or bi-allelic for a chromosomal modification, using methods that either leave a genetically expressible marker in place, allow for it to be bred out of an animal, or by methods that do not place such a marker in the animal. For instance, methods of homologous dependent recombination (HDR) have been used to make changes to, or insertion of exogenous genes into, chromosomes of animals. Tools such as TALENs and recombinase fusion proteins, as well as conventional methods, are discussed elsewhere herein. Some of the experimental data supporting genetic modifications disclosed herein is summarized as follows, exceptional cloning efficiency has been demonstrated when cloning from polygenic populations of modified cells, and advocated for this approach to avoid variation in cloning efficiency by somatic cell nuclear transfer (SCNT) for isolated colonies (Carlson et al., 2011). Additionally, however, TALEN-mediated genome modification, as well as modification by recombinase fusion molecules, provides for a bi-allelic alteration to be accomplished in a single generation. For example, an animal homozygous for a knocked-out gene may be made by SCNT and without inbreeding to produce homozygosity. Gestation length and maturation to reproduction age for livestock such as pigs and cattle is a significant barrier to research and to production. For example, generation of a homozygous knockout from heterozygous mutant cells (both sexes) by cloning and breeding would require 16 and 30 months for pigs and cattle respectively. Some have allegedly reduced this burden with sequential cycles of genetic modification and SCNT (Kuroiwa et al., 2004) however, this is both technically challenging and cost prohibitive, moreover, there are many reasons for avoiding serial cloning for making F0 animals that are to be actually useful for large vertebrate laboratory models or livestock. The ability to routinely generate bi-allelic KO cells prior to SCNT is a significant advancement in large animal genetic engineering. Bi-allelic knockout has been achieved in immortal cells lines using other processes such as ZFN and dilution cloning (Liu et al., 2010). Another group recently demonstrated bi-allelic KO of porcine GGTAl using commercial ZFN reagents (Hauschild et al., 2011) where bi-allelic null cells could be enriched by FACS for the absence of a GGTAl- dependent surface epitope. While these studies demonstrate certain useful concepts, they do not show that animals or livestock could be modified because simple clonal dilution is generally not feasible for primary fibroblast isolates (fibroblasts grow poorly at low density) and biological enrichment for null cells is not available for the majority of genes.
Targeted nuclease-induced homologous recombination can be used so as to eliminate the need for linked selection markers. TALENs may be used to precisely transfer specific alleles into a livestock genome by homology dependent repair (HDR). In a pilot study, a specific 1 lbp deletion (the Belgian Blue allele) (Grobet et al., 1997; Kambadur et al., 1997) was introduced into the bovine GDF8 locus (see U.S. 2012/0222143). When transfected alone, the btGDF8.1 TALEN pair cleaved up to 16% of chromosomes at the target locus. Co-transfection with a supercoiled homologous DNA repair template harboring the 1 lbp deletion resulted in a gene conversion frequency (HDR) of up to 5% at day 3 without selection for the desired event. Gene conversion was identified in 1.4 % of isolated colonies that were screened. These results demonstrated that TALENs can be used to effectively induce HDR without the aid of a linked selection marker.
Homology directed repair (HDR)
Homology directed repair (HDR) is a mechanism in cells to repair ssDNA and double stranded DNA (dsDNA) lesions. This repair mechanism can be used by the cell when there is an HDR template present that has a sequence with significant homology to the lesion site. Specific binding, as that term is commonly used in the biological arts, refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like. Specific hybridization is a form of specific binding between nucleic acids that have complementary sequences. Proteins can also specifically bind to DNA, for instance, in TALENs or CRISPR/Cas9 systems or by Gal4 motifs. Introgression of an allele refers to a process of copying an exogenous allele over an endogenous allele with a template-guided process. The endogenous allele might actually be excised and replaced by an exogenous nucleic acid allele in some situations but present theory is that the process is a copying mechanism. Since alleles are gene pairs, there is significant homology between them. The allele might be a gene that encodes a protein, or it could have other functions such as encoding a bioactive RNA chain or providing a site for receiving a regulatory protein or RNA.
The HDR template is a nucleic acid that comprises the allele that is being introgressed. The template may be a dsDNA or a single-stranded DNA (ssDNA). ssDNA templates are preferably from about 20 to about 5000 residues although other lengths can be used. Artisans can immediately appreciate that all ranges and values within the explicitly stated range are contemplated; e.g., from 500 to 1500 residues, from 20 to 100 residues, and so forth. The template may further comprise flanking sequences that provide homology to DNA adjacent to the endogenous allele or the DNA that is to be replaced. The template may also comprise a sequence that is bound to a targeted nuclease system, and is thus the cognate binding site for the system's DNA-binding member.
Targeted Endonuc lease Systems
Genome editing tools such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have impacted the fields of biotechnology, gene therapy and functional genomic studies in many organisms. More recently, RNA-guided endonucleases (RGENs) are directed to their target sites by a complementary RNA molecule. The Cas9/CRISPR system is a REGEN. tracrRNA is another such tool. These are examples of targeted nuclease systems: these system have a DNA-binding member that localizes the nuclease to a target site. The site is then cut by the nuclease. TALENs and ZFNs have the nuclease fused to the DNA- binding member. Cas9/CRISPR are cognates that find each other on the target DNA. The DNA- binding member has a cognate sequence in the chromosomal DNA. The DNA-binding member is typically designed in light of the intended cognate sequence so as to obtain a nucleolytic action at nor near an intended site. Certain embodiments are applicable to all such systems without limitation; including, embodiments that minimize nuclease re-cleavage, embodiments for making SNPs with precision at an intended residue, and placement of the allele that is being introgressed at the DNA-binding site. TALENS
The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left- TALEN and a right-TALEN, which references the handedness of DNA or a TALEN-pair.
The cipher for TALs has been reported (PCT Publication WO 2011/072246) wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence. The residues may be assembled to target a DNA sequence. In brief, a target site for binding of a TALEN is determined and a fusion molecule comprising a nuclease and a series of RVDs that recognize the target site is created. Upon binding, the nuclease cleaves the DNA so that cellular repair machinery can operate to make a genetic modification at the cut ends. The term TALEN means a protein comprising a Transcription Activator-like (TAL) effector binding domain and a nuclease domain and includes monomeric TALENs that are functional per se as well as others that require dimerization with another monomeric TALEN. The dimerization can result in a homodimeric TALEN when both monomeric TALEN are identical or can result in a heterodimeric TALEN when monomeric TALEN are different. TALENs have been shown to induce gene modification in immortalized human cells by means of the two major eukaryotic DNA repair pathways, non-homologous end joining (NHEJ) and homology directed repair. TALENs are often used in pairs but monomeric TALENs are known. Cells for treatment by TALENs (and other genetic tools) include a cultured cell, an immortalized cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, a blastocyst, or a stem cell. In some embodiments, a TAL effector can be used to target other protein domains (e.g., non- nuclease protein domains) to specific nucleotide sequences. For example, a TAL effector can be linked to a protein domain from, without limitation, a DNA 20 interacting enzyme (e.g., a methylase, a topoisomerase, an integrase, a transposase, or a ligase), a transcription activators or repressor, or a protein that interacts with or modifies other proteins such as histones.
Applications of such TAL effector fusions include, for example, creating or modifying epigenetic regulatory elements, making site- specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.
The term nuclease includes exonucleases and endonucleases. The term endonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Non-limiting examples of endonucleases include type II restriction endonucleases such as Fokl, Hhal, Hindlll, Notl, Bbv X, EcoKl, Bglll, and Alwl. Endonucleases comprise also rare- cutting endonucleases when having typically a polynucleotide recognition site of about 12-45 basepairs (bp) in length, more preferably of 14-45 bp. Rare-cutting endonucleases induce DNA double- strand breaks (DSBs) at a defined locus. Rare-cutting endonucleases can for example be a targeted endonuclease, a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl or a chemical endonuclease. In chemical endonucleases, a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical
endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences. Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention.
Examples of such endonuclease include I-See I, I-Chu L I-Cre I, I-Csm I, Pi-See L PI-Tti L PI- Mtu I, I-Ceu I, I-See I 1- See III, HO, Pi-Civ I, Pl-Ctr I PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I PI-Mav I PI-Meh I, PI-Mfu I PI-Mfl I, PI-Mga I PI-Mgo I, PI-Min I PI-Mka I PI-Mle I, PI- Mma I, PI- 30 Msh I PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I PI-Rma I, PI- Spb I, PI-Ssp I PI-Fae I PI-Mja I, PI-Pho I Pi-Tag I PI-Thy I, PI-Tko I, PI-Tsp I, I-Msol.
A genetic modification made by TALENS or other tools may be, for example, chosen from the list consisting of an insertion, a deletion, insertion of an exogenous nucleic acid fragment, and a substitution. In general, a target DNA site is identified and a TALEN-pair is created that can specifically bind to the site. The TALEN is delivered to the cell or embryo, e.g., as a protein, mRNA or by a vector that encodes the TALEN. The TALEN cleaves the DNA to make a double-strand break that is then repaired, often resulting in the creation of an indel, or incorporating sequences or polymorphisms contained in an accompanying exogenous nucleic acid that is either inserted into the chromosome or serves as a template for repair of the break with a modified sequence. This template-driven repair is a useful process for changing a chromosome, and provides for effective changes to cellular chromosomes.
Some embodiments involve a composition or a method of making a genetically modified livestock and/or artiodactyl comprising introducing a TALEN-pair into livestock and/or an artiodactyl cell or embryo that makes a genetic modification to DNA of the cell or embryo at a site that is specifically bound by the TALEN-pair, and producing the livestock
animal/artiodactyl from the cell. Direct injection may be used for the cell or embryo, e.g., into a zygote, blastocyst, or embryo. Alternatively, the TALEN and/or other factors may be introduced into a cell using any of many known techniques for introduction of proteins, RNA, mRNA, DNA, or vectors. Genetically modified animals may be made from the embryos or cells according to known processes, e.g., implantation of the embryo into a gestational host, or various cloning methods. The phrase "a genetic modification to DNA of the cell at a site that is specifically bound by the TALEN", or the like, means that the genetic modification is made at the site cut by the nuclease on the TALEN when the TALEN is specifically bound to its target site. The nuclease does not cut exactly where the TALEN-pair binds, but rather at a defined site between the two binding sites.
Some embodiments involve a composition or a treatment of a cell that is used for cloning the animal. The cell may be a livestock and/or artiodactyl cell, a cultured cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, or a stem cell. For example, an embodiment is a composition or a method of creating a genetic modification comprising exposing a plurality of primary cells in a culture to TALEN proteins or a nucleic acid encoding a TALEN or TALENs. The TALENs may be introduced as proteins or as nucleic acid fragments, e.g., encoded by mRNA or a DNA sequence in a vector.
Zinc Finger Nucleases
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to alter the genomes of higher organisms. ZFNs may be used in method of inactivating genes.
A zinc finger DNA-binding domain has about 30 amino acids and folds into a stable structure. Each finger primarily binds to a triplet within the DNA substrate. Amino acid residues at key positions contribute to most of the sequence-specific interactions with the DNA site. These amino acids can be changed while maintaining the remaining amino acids to preserve the necessary structure. Binding to longer DNA sequences is achieved by linking several domains in tandem. Other functionalities like non-specific Fokl cleavage domain (N), transcription activator domains (A), transcription repressor domains (R) and methylases (M) can be fused to a ZFPs to form ZFNs respectively, zinc finger transcription activators (ZFA), zinc finger transcription repressors (ZFR, and zinc finger methylases (ZFM). Materials and methods for using zinc fingers and zinc finger nucleases for making genetically modified animals are disclosed in, e.g., U.S. 8,106,255; U.S. 2012/0192298; U.S. 2011/0023159; and U.S.
2011/0281306.
Vectors and Nucleic acids
A variety of nucleic acids may be introduced into cells, for knockout purposes, for inactivation of a gene, to obtain expression of a gene, or for other purposes. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double- stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
The target nucleic acid sequence can be operably linked to a regulatory region such as a promoter. Regulatory regions can be porcine regulatory regions or can be from other species.
In general, type of promoter can be operably linked to a target nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters responsive or unresponsive to a particular stimulus. In some embodiments, a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used (i.e., a constitutive promoter). For example, a beta-actin promoter such as the chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase (PGK) promoter can be used, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter. In some embodiments, a fusion of the chicken beta actin gene promoter and the CMV enhancer is used as a promoter. See, for example, Xu et al., Hum. Gene Ther. 12:563, 2001; and Kiwaki et al., Hum. Gene Ther. 7:821, 1996.
Additional regulatory regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, can sometimes be obtained without such additional elements.
A nucleic acid construct may be used that encodes signal peptides or selectable expressed markers. Signal peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B- phosphtransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.
In some embodiments, a sequence encoding a selectable marker can be flanked by recognition sequences for a recombinase such as, e.g., Cre or Flp. For example, the selectable marker can be flanked by loxP recognition sites (34-bp recognition sites recognized by the Cre recombinase) or FRT recognition sites such that the selectable marker can be excised from the construct. See, Orban et al., Proc. Natl. Acad. Sci., 89:6861, 1992, for a review of Cre/lox technology, and Brand and Dymecki, Dev. Cell, 6:7, 2004. A transposon containing a Cre- or Flp- activatable transgene interrupted by a selectable marker gene also can be used to obtain transgenic animals with conditional expression of a transgene. For example, a promoter driving expression of the marker/transgene can be either ubiquitous or tissue-specific, which would result in the ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs). Tissue specific activation of the transgene can be accomplished, for example, by crossing a pig that ubiquitously expresses a marker-interrupted transgene to a pig expressing Cre or Flp in a tissue-specific manner, or by crossing a pig that expresses a marker-interrupted transgene in a tissue-specific manner to a pig that ubiquitously expresses Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.
In some embodiments, the exogenous nucleic acid encodes a polypeptide. A nucleic acid sequence encoding a polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAG™ tag (Kodak, New Haven, CT).
Nucleic acid constructs can be introduced into embryonic, fetal, or adult
artiodactyl/livestock cells of any type, including, for example, germ cells such as an oocyte or an egg, a progenitor cell, an adult or embryonic stem cell, a primordial germ cell, a kidney cell such as a PK-15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast, using a variety of techniques. Non-limiting examples of techniques include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells.
In transposon systems, the transcriptional unit of a nucleic acid construct, i.e., the regulatory region operably linked to an exogenous nucleic acid sequence, is flanked by an inverted repeat of a transposon. Several transposon systems, including, for example, Sleeping Beauty (see, U.S. 6,613,752 and U.S. 2005/0003542); Frog Prince (Miskey et al., Nucleic Acids Res. 31:6873, 2003); Tol2 (Kawakami, Genome Biology 8(Suppl. l):S7, 2007); Minos (Pavlopoulos et al., Genome Biology, 8(Suppl. l):S2, 2007); Hsmarl (Miskey et al., Mol Cell Biol., 27:4589, 2007); and Passport have been developed to introduce nucleic acids into cells, including mice, human, and pig cells. The Sleeping Beauty transposon is particularly useful. A transposase can be delivered as a protein, encoded on the same nucleic acid construct as the exogenous nucleic acid, can be introduced on a separate nucleic acid construct, or provided as an mRNA (e.g., an in vitro- transcribed and capped mRNA).
Nucleic acids can be incorporated into vectors. A vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA. A vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non-viral vectors (e.g., transposons) used for gene delivery in animals have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence. Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.
Many different types of vectors are known. For example, plasmids and viral vectors, e.g., retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, ^-elements, Tol-2, Frog Prince, piggyBac).
As used herein, the term nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids, e.g., synthetic bases or alternative backbones. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand). The term transgenic is used broadly herein and refers to a genetically modified organism or genetically engineered organism whose genetic material has been altered using genetic engineering techniques. A knockout artiodactyl is thus transgenic regardless of whether or not exogenous genes or nucleic acids are expressed in the animal or its progeny. Genetically modified animals
Animals may be modified using TALENs or other genetic engineering tools, including recombinase fusion proteins, or various vectors that are known. A genetic modification made by such tools may comprise disruption of a gene. Materials and methods of genetically modifying animals are further detailed in U.S. 8,518,701; U.S. 2010/0251395; and U.S. 2012/0222143 which are hereby incorporated herein by reference for all purposes; in case of conflict, the instant specification is controlling. The term trans-acting refers to processes acting on a target gene from a different molecule (i.e., intermolecular). A trans-acting element is usually a DNA sequence that contains a gene. This gene codes for a protein (or microRNA or other diffusible molecule) that is used in the regulation the target gene. The trans-acting gene may be on the same chromosome as the target gene, but the activity is via the intermediary protein or RNA that it encodes. Embodiments of trans-acting gene are, e.g., genes that encode targeting
endonucleases. Inactivation of a gene using a dominant negative generally involves a transacting element. The term cis-regulatory or cis-acting means an action without coding for protein or RNA; in the context of gene inactivation, this generally means inactivation of the coding portion of a gene, or a promoter and/or operator that is necessary for expression of the functional gene.
Various techniques known in the art can be used to inactivate genes to make knock-out animals and/or to introduce nucleic acid constructs into animals to produce founder animals and to make animal lines, in which the knockout or nucleic acid construct is integrated into the genome. Such techniques include, without limitation, pronuclear microinjection (U.S.
4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148-6152, 1985), gene targeting into embryonic stem cells (Thompson et al., Cell, 56:313-321, 1989), electroporation of embryos (Lo, Mol. Cell. Biol., 3: 1803-1814, 1983), sperm- mediated gene transfer (Lavitrano et al., Proc. Natl. Acad. Sci. USA, 99: 14230- 14235, 2002; Lavitrano et al., Reprod. Fert. Develop., 18: 19-23, 2006), and in vitro transformation of somatic cells, such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation (Wilmut et al., Nature, 385:810-813, 1997; and Wakayama et al., Nature, 394:369-374, 1998). Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. An animal that is genomically modified is an animal wherein all of its cells have the genetic modification, including its germ line cells. When methods are used that produce an animal that is mosaic in its genetic modification, the animals may be inbred and progeny that are genomically modified may be selected. Cloning, for instance, may be used to make a mosaic animal if its cells are modified at the blastocyst state, or genomic modification can take place when a single-cell is modified. Animals that are modified so they do not sexually mature can be homozygous or heterozygous for the modification, depending on the specific approach that is used. If a particular gene is inactivated by a knock out modification, homozygousity would normally not be sufficient. If a particular gene is inactivated by an RNA interference or dominant negative strategy, then heterozygosity is often adequate.
Typically, in pronuclear microinjection, a nucleic acid construct is introduced into a fertilized egg; 1 or 2 cell fertilized eggs are used as the pronuclei containing the genetic material from the sperm head and the egg are visible within the protoplasm. Pronuclear staged fertilized eggs can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of donor animals). In vitro fertilized eggs can be produced as follows. For example, swine ovaries can be collected at an abattoir, and maintained at 22-28°C during transport. Ovaries can be washed and isolated for follicular aspiration, and follicles ranging from 4-8 mm can be aspirated into 50 mL conical centrifuge tubes using 18 gauge needles and under vacuum. Follicular fluid and aspirated oocytes can be rinsed through pre-filters with commercial TL-HEPES (Minitube, Verona, WI). Oocytes surrounded by a compact cumulus mass can be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, WI) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 μΜ 2- mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for approximately 22 hours in humidified air at 38.7°C and 5% C02. Subsequently, the oocytes can be moved to fresh TCM-199 maturation medium, which does not contain cAMP, PMSG or hCG and incubated for an additional 22 hours. Matured oocytes can be stripped of their cumulus cells by vortexing in 0.1%
hyaluronidase for 1 minute.
For swine, mature oocytes can be fertilized in 500 μΐ Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, WI) in Minitube 5-well fertilization dishes. In preparation for in vitro fertilization (IVF), freshly -collected or frozen boar semen can be washed and resuspended in PORCPRO IVF Medium to 4 x 10^ sperm. Sperm concentrations can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, WI). Final in vitro insemination can be performed in a ΙΟμΙ volume at a final concentration of
approximately 40 motile sperm/oocyte, depending on boar. Incubate all fertilizing oocytes at 38.7°C in 5.0% C02 atmosphere for 6 hours. Six hours post-insemination, presumptive zygotes can be washed twice in NCSU-23 and moved to 0.5 mL of the same medium. This system can produce 20-30% blastocysts routinely across most boars with a 10-30% polyspermic insemination rate.
Linearized nucleic acid constructs can be injected into one of the pronuclei. Then the injected eggs can be transferred to a recipient female (e.g., into the oviducts of a recipient female) and allowed to develop in the recipient female to produce the transgenic animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 X g for 5 minutes to sediment lipids allowing visualization of the pronucleus. The embryos can be injected with using an Eppendorf FEMTOJET injector and can be cultured until blastocyst formation. Rates of embryo cleavage and blastocyst formation and quality can be recorded.
Embryos can be surgically transferred into uteri of asynchronous recipients. Typically,
100-200 (e.g., 150-200) embryos can be deposited into the ampulla-isthmus junction of the
(g)
oviduct using a 5.5-inch TOMCAT catheter. After surgery, real-time ultrasound examination of pregnancy can be performed.
In somatic cell nuclear transfer, a transgenic artiodactyl cell (e.g., a transgenic pig cell or bovine cell) such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell that includes a nucleic acid construct described above, can be introduced into an enucleated oocyte to establish a combined cell. Oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area. Typically, an injection pipette with a sharp beveled tip is used to inject the transgenic cell into an enucleated oocyte arrested at meiosis 2. In some conventions, oocytes arrested at meiosis-2 are termed eggs. After producing a porcine or bovine embryo (e.g., by fusing and activating the oocyte), the embryo is transferred to the oviducts of a recipient female, about 20 to 24 hours after activation. See, for example, Cibelli et al., Science 280: 1256-1258, 1998, and U.S. 6,548,741. For pigs, recipient females can be checked for pregnancy approximately 20-21 days after transfer of the embryos.
Standard breeding techniques can be used to create animals that are homozygous for the exogenous nucleic acid from the initial heterozygous founder animals. Homozygosity may not be required, however. Transgenic pigs described herein can be bred with other pigs of interest.
In some embodiments, a nucleic acid of interest and a selectable marker can be provided on separate transposons and provided to either embryos or cells in unequal amount, where the amount of transposon containing the selectable marker far exceeds (5-10 fold excess) the transposon containing the nucleic acid of interest. Transgenic cells or animals expressing the nucleic acid of interest can be isolated based on presence and expression of the selectable marker. Because the transposons can integrate into the genome in a precise and unlinked way (independent transposition events), the nucleic acid of interest and the selectable marker are not genetically linked and can easily be separated by genetic segregation through standard breeding. Thus, transgenic animals can be produced that are not constrained to retain selectable markers in subsequent generations, an issue of some concern from a public safety perspective.
Once transgenic animal have been generated, expression of an exogenous nucleic acid can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether or not integration of the construct has taken place. For a description of Southern analysis, see sections 9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; NY., 1989.
Polymerase chain reaction (PCR) techniques also can be used in the initial screening. PCR refers to a procedure or technique in which target nucleic acids are amplified. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. PCR is described in, for example PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. See, for example, Lewis, Genetic Engineering News 12: 1, 1992; Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874, 1990; and Weiss, Science 254: 1292, 1991. At the blastocyst stage, embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci USA, 99:4495, 2002).
Expression of a nucleic acid sequence encoding a polypeptide in the tissues of transgenic pigs can be assessed using techniques that include, for example, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis,
Western analysis, immunoassays such as enzyme-linked immunosorbent assays, and reverse- transcriptase PCR (RT-PCR).
Interfering RNAs
A variety of interfering RNA (RNAi) are known. Double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous gene transcripts. RNA-induced silencing complex (RISC) metabolizes dsRNA to small 21-23 -nucleotide small interfering RNAs (siRNAs). RISC contains a double stranded RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut 2 or Ago2). RISC utilizes antisense strand as a guide to find a cleavable target. Both siRNAs and microRNAs (miRNAs) are known. A method of disrupting a gene in a genetically modified animal comprises inducing RNA interference against a target gene and/or nucleic acid such that expression of the target gene and/or nucleic acid is reduced.
For example the exogenous nucleic acid sequence can induce RNA interference against a nucleic acid encoding a polypeptide. For example, double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to a target DNA can be used to reduce expression of that DNA. Constructs for siRNA can be produced as described, for example, in Fire et al., Nature 391:806, 1998; Romano and Masino, Mol. Microbiol. 6:3343, 1992; Cogoni et al., EMBO J. 15:3153, 1996; Cogoni and Masino, Nature, 399: 166, 1999; Misquitta and Paterson Proc. Natl. Acad. Sci. USA, 96:1451, 1999; and Kennerdell and Carthew, Cell, 95: 1017, 1998. Constructs for shRNA can be produced as described by Mclntyre and Fanning (2006) BMC Biotechnology 6: 1. In general, shRNAs are transcribed as a single-stranded RNA molecule containing complementary regions, which can anneal and form short hairpins.
The probability of finding a single, individual functional siRNA or miRNA directed to a specific gene is high. The predictability of a specific sequence of siRNA, for instance, is about 50% but a number of interfering RNAs may be made with good confidence that at least one of them can be effective.
Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that express an RNAi directed against a gene, e.g., a gene selective for a developmental stage. The RNAi may be, for instance, selected from the group consisting of siRNA, shRNA, dsRNA, RISC and miRNA.
Inducible systems
An inducible system may be used to control expression of a gene. Various inducible systems are known that allow spatiotemporal control of expression of a gene. Several have been proven to be functional in vivo in transgenic animals. The term inducible system includes traditional promoters and inducible gene expression elements. An example of an inducible system is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of the nucleic acid. In this system, a mutated Tet repressor (TetR) is fused to the activation domain of herpes simplex virus VP 16 trans -activator protein to create a tetracycline-controlled transcriptional activator (tTA), which is regulated by tet or doxycycline (dox). In the absence of antibiotic, transcription is minimal, while in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone or rapamycin systems. Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor and the product of the ultraspiracle gene (USP). Expression is induced by treatment with ecdysone or an analog of ecdysone such as muristerone A. The agent that is administered to the animal to trigger the inducible system is referred to as an induction agent. The tetracycline-inducible system and the Cre/loxP recombinase system (either constitutive or inducible) are among the more commonly used inducible systems. The tetracycline-inducible system involves a tetracycline-controlled transactivator (tTA)/ reverse tTA (rtTA). A method to use these systems in vivo involves generating two lines of genetically modified animals. One animal line expresses the activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter. Another set of transgenic animals express the acceptor, in which the expression of the gene of interest (or the gene to be modified) is under the control of the target sequence for the tTA/rtTA transactivators (or is flanked by loxP sequences).
Mating the two strains of mice provides control of gene expression.
The tetracycline-dependent regulatory systems (tet systems) rely on two components, i.e., a tetracycline-controlled transactivator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner. In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is termed tet-OFF, because tetracycline or doxycycline allows transcriptional down- regulation. Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo. rtTA is a variant of tTA that is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation. This tet system is therefore termed tet-ON. The tet systems have been used in vivo for the inducible expression of several transgenes, encoding, e.g., reporter genes, oncogenes, or proteins involved in a signaling cascade.
The Cre/lox system uses the Cre recombinase, which catalyzes site-specific
recombination by crossover between two distant Cre recognition sequences, i.e., loxP sites. A DNA sequence introduced between the two loxP sequences (termed floxed DNA) is excised by Cre-mediated recombination. Control of Cre expression in a transgenic animal, using either spatial control (with a tissue- or cell-specific promoter) or temporal control (with an inducible system), results in control of DNA excision between the two loxP sites. One application is for conditional gene inactivation (conditional knockout). Another approach is for protein over- expression, wherein a floxed stop codon is inserted between the promoter sequence and the DNA of interest. Genetically modified animals do not express the transgene until Cre is expressed, leading to excision of the floxed stop codon. This system has been applied to tissue- specific oncogenesis and controlled antigene receptor expression in B lymphocytes. Inducible Cre recombinases have also been developed. The inducible Cre recombinase is activated only by administration of an exogenous ligand. The inducible Cre recombinases are fusion proteins containing the original Cre recombinase and a specific ligand-binding domain. The functional activity of the Cre recombinase is dependent on an external ligand that is able to bind to this specific domain in the fusion protein.
Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that comprise a gene under control of an inducible system. The genetic modification of an animal may be genomic or mosaic. The inducible system may be, for instance, selected from the group consisting of Tet-On, Tet-Off, Cre-lox, and Hiflalpha. An embodiment is a gene set forth herein.
Dominant Negatives
Genes may thus be disrupted not only by removal or RNAi suppression but also by creation/expression of a dominant negative variant of a protein which has inhibitory effects on the normal function of that gene product. The expression of a dominant negative (DN) gene can result in an altered phenotype, exerted by a) a titration effect; the DN PASSIVELY competes with an endogenous gene product for either a cooperative factor or the normal target of the endogenous gene without elaborating the same activity, b) a poison pill (or monkey wrench) effect wherein the dominant negative gene product ACTIVELY interferes with a process important for normal gene function, c) a feedback effect, wherein the DN ACTIVELY stimulates a negative regulator of the gene function. Founder animals, animal lines, traits, and reproduction
Founder animals (F0 generation) may be produced by cloning and other methods described herein. The founders can be homozygous for a genetic modification, as in the case where a zygote or a primary cell undergoes a homozygous modification. Similarly, founders can also be made that are heterozygous. The founders may be genomically modified, meaning that the cells in their genome have undergone modification. Founders can be mosaic for a modification, as may happen when vectors are introduced into one of a plurality of cells in an embryo, typically at a blastocyst stage. Progeny of mosaic animals may be tested to identify progeny that are genomically modified. An animal line is established when a pool of animals has been created that can be reproduced sexually or by assisted reproductive techniques, with heterogeneous or homozygous progeny consistently expressing the modification.
In livestock, many alleles are known to be linked to various traits such as production traits, type traits, workability traits, and other functional traits. Artisans are accustomed to monitoring and quantifying these traits, e.g., Visscher et al., Livestock Production Science, 40: 123-137, 1994, U.S. 7,709,206, U.S. 2001/0016315, U.S. 2011/0023140, and U.S.
2005/0153317. An animal line may include a trait chosen from a trait in the group consisting of a production trait, a type trait, a workability trait, a fertility trait, a mothering trait, and a disease resistance trait. Further traits include expression of a recombinant gene product.
Recombinases
Embodiments of the invention include administration of a targeted nuclease system with a recombinase (e.g., a RecA protein, a Rad51) or other DNA-binding protein associated with DNA recombination. A recombinase forms a filament with a nucleic acid fragment and, in effect, searches cellular DNA to find a DNA sequence substantially homologous to the sequence. For instance a recombinase may be combined with a nucleic acid sequence that serves as a template for HDR. The recombinase is then combined with the HDR template to form a filament and placed into the cell. The recombinase and/or HDR template that combines with the recombinase may be placed in the cell or embryo as a protein, an mRNA, or with a vector that encodes the recombinase. The disclosure of U.S. 2011/0059160 (U.S. Patent Application No. 12/869,232) is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling. The term recombinase refers to a genetic recombination enzyme that enzymatically catalyzes, in a cell, the joining of relatively short pieces of DNA between two relatively longer DNA strands. Recombinases include Cre recombinase, Hin recombinase, RecA, RAD51, Cre, and FLP. Cre recombinase is a Type I topoisomerase from PI
bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria
Salmonella. Hin belongs to the serine recombinase family of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination. RAD51 is a human gene. The protein encoded by this gene is a member of the RAD51 protein family which assists in repair of DNA double strand breaks. RAD51 family members are homologous to the bacterial RecA and yeast Rad51. Cre recombinase is an enzyme that is used in experiments to delete specific sequences that are flanked by loxP sites. FLP refers to Flippase recombination enzyme (FLP or Flp) derived from the 2μ plasmid of the baker's yeast Saccharomyces cerevisiae.
Herein, "RecA" or "RecA protein" refers to a family of RecA-like recombination proteins having essentially all or most of the same functions, particularly: (i) the ability to position properly oligonucleotides or polynucleotides on their homologous targets for subsequent extension by DNA polymerases; (ii) the ability topologically to prepare duplex nucleic acid for DNA synthesis; and (iii) the ability of RecA/oligonucleotide or
RecA/polynucleotide complexes efficiently to find and bind to complementary sequences. The best characterized RecA protein is from E. coli; in addition to the original allelic form of the protein a number of mutant RecA-like proteins have been identified, for example, RecA803.
Further, many organisms have RecA-like strand-transfer proteins including, for example, yeast, Drosophila, mammals including humans, and plants. These proteins include, for example, Reel, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2 and DMC1. An embodiment of the recombination protein is the RecA protein of E. coli. Alternatively, the RecA protein can be the mutant RecA-803 protein of E. coli, a RecA protein from another bacterial source or a homologous recombination protein from another organism.
Compositions and kits
The present invention also provides compositions and kits containing, for example, nucleic acid molecules encoding site-specific endonucleases, CRISPR, Cas9, ZNFs, TALENs, RecA-gal4 fusions, polypeptides of the same, compositions containing such nucleic acid molecules or polypeptides, or engineered cell lines. An HDR may also be provided that is effective for introgression of an indicated allele. Such items can be used, for example, as research tools, or therapeutically. EXAMPLES
Methods are as follows unless otherwise noted.
Tissue culture and transfection.
Pig were maintained at 37 at 5% C02 in DMEM supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin and streptomycin, and 2mM L-Glutamine. For transfection, all
TALENs and HDR templates were delivered through transfection using the NEON Transfection system (Life Technologies). Briefly, low passage Ossabaw, Landrace reaching 100% confluence were split 1 :2 and harvested the next day at 70-80% confluence. Each transfection was comprised of 500,000-600,000 cells resuspended in buffer "R" mixed with TALEN mRNA and oligos and electroporated using the ΙΟΟμΙ tips that provide a 100 μΐ working volume by the following parameters: input Voltage; 1800V; Pulse Width; 20ms; and Pulse Number; 1.
Typically, 1 -2 μ g of TALEN mRNA and 1 -4 μΜ of HDR templates (single stranded oligonucleotides) specific for the gene of interest were included in each transfection. Deviation from those amounts is indicated in he figures and legends. After transfection, cells were plated in a well of a 6-well dish for three days and cultured at either 30°C. After three days, cell populations were plated for colony analysis and/or expanded and at 37°C until at least day 10 to assess stability of edits.
Surveyor mutation detection andRFLP analysis
PCR flanking the intended sites was conducted using PLATINUM Taq DNA polymerase HiFi (Life Technologies) with 1 μΐ of the cell lysate according to the manufacturer's recommendations. The frequency of mutation in a population was analysed with the SURVEYOR Mutation Detection Kit (Transgenomic) according to the manufacturer's recommendations using 10 μΐ of the PCR product as described above. RFLP analysis was performed on 10 μΐ of the above PCR reaction using the indicated restriction enzyme. Surveyor 5 and RFLP reactions were resolved on a 10% TBE poly aery lamide gels and visualized by ethidium bromide staining. Densitometry measurements of the bands were performed using IMAGEJ; and mutation rate of Surveyor reactions was calculated as described in Guschin et al., 2010(1). Percent homology directed repair (HDR) was calculated by dividing the sum intensity of RFLP fragments by the sum intensity of the parental band + RFLP fragments. RFLP analysis0 of colonies was treated similarly except that the PCR products were amplified by IX MYTAQ RED MIX (Bioline) and resolved on 2.5% agarose gels.
Dilution cloning:
Three days post transfection, 50 to 250 cells were seeded onto 10 cm dishes and cultured5 until individual colonies reached circa 5mm in diameter. At this point, 6 ml of TRYPLE (Life Technologies) 1:5 (vol/vol) diluted in PBS was added and colonies were aspirated, transferred into wells of a 24-well dish well and cultured under the same conditions. Colonies reaching confluence were collected and divided for cryopreservation and genotyping. 0 Sample preparation:
Transfected cells populations at day 3 and 10 were collected from a well of a 6-well dish and 10-30% were resuspended in 50 μΐ of IX PCR compatible lysis buffer: 10 mM Tris-Cl pH 8.0, 2 mM EDTA, 0.45% TRYTON X-100(vol/vol), 0.45% TWEEN-20(vol/vol) freshly supplemented with 200 μg/ml Proteinase K. The lysates were processed in a thermal cycler5 using the following program: 55°C for 60 minutes, 95°C for 15 minutes. Colony samples from dilution cloning were treated as above using 20-30 μΐ of lysis buffer.
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Example 1: Multiplex Gene Editing of pig RAG2 and IL2Ry
Six conditions of TALEN mRNA and HDR templates directed to pig RAG2 and IL2Ry were co-transfected into pig fibroblasts. A fixed quantity of RAG2 mRNA and template were used for each transfection whereas the quantity of IL2Rg TALEN mRNA and HDR template is altered for each condition as indicated. The dosage of TALEN mRNA and HDR template has both on and off target effects. An increase in TALEN mRNA for IL2Ry led to an increase in both NHEJ and HDR for IL2Ry while NHEJ levels for RAG2 were unchanged. An increase in IL2Ry HDR template reduced HDR at the RAG2 locus suggesting a nonspecific inhibition of homology directed repair by escalation of the concentration of oligonucleotide. Colonies with bi-allelic HDR at RAG2 and IL2Ry were obtained at four and two percent from two conditions (FIGs. 4C and 4D) which is at and above the expected frequency of two percent. The expected frequency is calculated by multiplication of day 3 HDR levels which treats each HDR allele as an independent event. Referring to FIGs. 4A-4D, Multiplex gene editing of swine RAG2 and IL2Ry . FIG. 4A SURVEYOR and RFLP analysis to determine the efficiency of non- homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection. FIG. 4B RFLP analysis for homology dependent repair on cell populations 11 days post transfection. FIG. 4C Percentage of colonies positive for HDR at IL2Ry, RAG2 or both. Cells were plated from the population indicated by a "C" in FIG. 4A. Distribution of colony genotypes is shown below. FIG. 4D Colony analysis from cells transfected with TALEN mRNA quantities of 2 and 1 μg for IL2Ry and RAG2 and HDR template at 1 μΜ for each. Distribution of colony genotypes is shown below.
Example 2: Multiplex Gene Editing of pig RAG2 and IL2Ry
Four conditions of TALEN mRNA and HDR templates directed to pig APC and p53 were co-transfected into pig fibroblasts. The quantity of APC mRNA was sequentially reduced from left to right (FIG.5B); the remaining of the quantities remained constant as indicated. Percent HDR reduced in a linear manor with reduction of APC mRNA. There was little effect on p53 HDR with altered dosage of APC TALENs. Genotyping of colonies revealed a higher than expected union of clones with HDR allele in both APC and p53 relative to the day 11 values; 18 and 20 percent versus 13.7 and 7.1 percent for FIGs. 5C and 5D, respectively.
Referring to FIGs. 5A-5D Multiplex gene editing of swine APC and p53. FIG. 5A Surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology depended repair HDR on cell populations 3 days post transfection. FIG. 5B RFLP analysis for homology dependent repair on cell populations 11 days post transfection. FIGs. 5C and 5D. Percentage of colonies positive derived from the indicated cell population (indicated in FIG. 5 A, "C" and "D") for HDR at APC, p53 or both. Colonies with 3 or more HDR alleles are listed below.
Example 3: Multiplex with at least three genes
In Example 1, a non-specific reduction in HDR was observed at high concentration of
HDR oligo, thus it was unknown whether 2+ HDR oligos could be effective without nonspecific inhibition of HDR. Two concentrations were tested, 1 uM and 2 uM for each target site. While TALEN activity was not significantly altered between the two conditions, HDR was blunted significantly at 2 uM concentration for each template. Clones derived from the 1 uM condition had a variety of genotypes, some of those with edits in each gene and up to 7 alleles (FIGs. 7A and 7B). If treated as independent events, the expected frequency of the genotype denoted by an "a", with 7 alleles edited, is 0.001 percent. Binomial distribution predicts the likelihood of identifying 2+ colonies of such a genotype in a sample size of 72, as was done here, is less than 0.000026 percent. This high rate of success could not be predicted and is unexpected and surprising. This result was replicated with two addition combinations of
TALENs/HDR template (FIGs. 8A and 8B, and 9A and 9B). As with the results the first trial, colonies were obtained with HDR edits in up to seven alleles and up to four genes (Table A). Several genotypes were recovered at a frequency far greater than anticipated by chance.
Although a concern regarding simultaneous double strand break at several loci is induction of unintended chromosomal rearrangements, 50 of 50 karyotypes tested from trial 3 cells were normal (data not shown).
Referring to FIGs. 6A and 6B: Effect of Oligonucleotide HDR template concentration on 5-gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA directed to swine RAG2, IL2Rg, p53, APC and LDLR were co-transfected into pig fibroblasts along with 2 uM (FIG. 6A) or 1 uM (FIG. 6B) of each cognate HDR template. Percent NHEJ and HDR were measured by Surveyor and RFLP assay. Referring to FIGs. 7A and 7B: Colony genotypes from 5-gene multiplex HDR. Colony genotypes were evaluated by RFLP analysis. In FIG. 7A, each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 7B provides a tally of the number of colonies edited at 0-5 loci. Referring to FIGs. 8A-8B: Colony genotypes of a second 5-gene multiplex trial. FIG. 8A: Each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 8B: A tally of the number of colonies edited at 0-5 loci. Referring to FIGs. 9A and 9B: Colony genotypes a third 5-gene multiplex trial. FIG. 9A: Each line represents the genotype of one colony at each specified locus. Three genotypes could be identified; those with the expected RFLP genotype of heterozygous or homozygous HDR as well as those with an RFLP positive fragment, plus a second allele that has a visible shift in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with an edit at the specified locus is indicated below each column. FIG. 9B A tally of the number of colonies edited at 0-5 loci.
Examples 4A-4D
Example 4A: Develop RAG2/IL2Rg null (RG-KO) pig fibroblasts by multiplex gene editing. Male pig fetal fibroblasts are transfected with TALENs and oligonucleotide templates for disruption of RAG2 and IL2Rg using previously defined methods (Tan, W., et al., Efficient nonmeiotic allele introgression in livestock using custom endonucleases. PNAS,
110(41): 16526-16531, 2013.) RG-KO candidates are identified by, e.g., RFLP, as confirmed by sequencing. At least about 5 validated RG-KO colonies arepooled as a resource for cloning and chimera production. Example 4B: Production of chimeric embryos using RG-KO host blastocysts.
Host RG-KO embryos and female EGFP-labeled donor cells are produced using chromatin transfer technology followed by in vitro culture to the blastocyst stage. RG-KO cells from Example 1 may be used. Day-7 inter cell mass clumps from EGFP blastocysts are injected into day-6 RG-KO embryos prior to embryo transfer to a synchronized sow. Using this approach, Nagashima and colleagues observed chimerism in >50 percent of liveborn piglets
(Nagashima H. et al., Sex differentiation and germ cell production in chimeric pigs produced by inner cell mass injection into blastocysts. Biol Reprod, 70(3): 702-707, 2004). The male phenotype is dominant in injection chimeras for both mice and pigs. Therefore, XY RG-KO hosts injected with female donor cells exclusively transmit male host genetics. Pregnancy checks are conducted at appropriate times, e.g., days 25, 50, and 100. Pregnant sows at about 100 days of gestation are monitored 4 times daily prior to C-section derivation of piglets by about day 114.
Example 4C: Determine if non-chime ric offspring are deficient for T, B and NK cells.
Non-chimeric offspring is tested to determine if they deficient for T, B and NK cells.
The following process is one technique for the same. C-section derivation is conducted on each sow carrying presumptive chimeras and one bred sow carrying wild-type piglets. Umbilical cord blood is isolated from each piglet immediately after C-section derivation. Cord blood leukocytes are evaluated by fluorescence-activated cell sorting (FACS) for T, B and NK cell populations as well as donor derived EGFP expression. In addition, chimerism is evaluated by PCR from cord blood, ear and tail biopsy. This initial analysis is completed within 6 hours of birth, such that non-chimeric piglets can be monitored closely and humanely euthanized with signs of infection. A portion of non-chimeric animals, or those lacking immune cells, is euthanized for necropsy.
Example 4D: Identify chimeric pigs and determine origin of T, B and NK cells.
Chimeric pigs are tested to determine origin of T, B and NK cells. The following process is one technique for the same. Chimeric piglets are identified using the methods above. Weekly evaluation of circulating lymphocytes and serum immunoglobulin is compared between chimeric, non-chimeric and wild-type piglets over a 2 month period. Populations of sorted T, B and NK cells are evaluated for EGFP expression and microsatellite analysis to confirm donor origin. The maintenance of samples and semen collections from chimeric pigs are supported by RCI until Phase II funding is available.
Sample Procedures for Examples A-D:
Cord and peripheral blood FACS.
Evaluation of blood lymphocytes and EGFP chimerism is performed as previously described (2) with adaptations for porcine specimens. Cord blood is collected from each piglet immediately after C-section delivery. A portion of the cord blood is processed and
cryopreserved for potential allograft treatments while the remainder is used for FACS analysis of lymphocytes. Peripheral blood samples are collected at 2, 4, 6 and 8 weeks of age by standard methods. RBCs are removed and approximately 1-2E+5 cells are distributed into tubes.
Aliquots are labeled with anti-pig antibodies for identification of T cells (CD4 and CD8), B cells (CD45RA ad CD3), NK cells (CD 16 and CD3) and myeloid cells (CD3). Antigen expression is quantified on the LS RII Flow Cytometer (BD Biosciences). Fluorophores are carefully selected to enable multiplex evaluation of donor derived EGFP cells along with surface antigens. Single cell suspensions from the spleen are analyzed by the same methods.
Examinations
All major organs and tissues are grossly examined for appropriate anatomic development and appropriate samples from all major organs and tissues including pancreas, liver, heart, kidneys, lungs, gastrointestinal, immune system (peripheral and mucosal lymph nodes and spleen), and CNS are collected for DNA isolation. Single cell suspensions are prepared from the spleen for FACS analysis. Tissues are prepared for histological examination to further assess chimerism and for any alterations that may be associated with the chimeric state and for the presence of any underlying illness.
Assessment of chimerism
Quantitative PCR is conducted on cord blood, ear, and tail biopsy using primers specific to the EGFP transgene and compared to a standard curve with known ratios of EGFP to wild type-cells. Specimens are also evaluated for RG-KO alleles via the RFLP assay previously described. Engraftment of EGFP+ cells are evaluated macroscopically on whole animals and organs during necropsy. Tissues from the major organs are sectioned for EGFP
immunohistochemistry and counterstained with DAPI (4', 6-diamidino-2-phenylindole) to determine the ratio of donor to host cells.
Microsatellite analysis.
Animals are screened for informative microsatellites for host and donor genetics from those routinely used in the lab. Samples from tissues and blood (sorted lymphocytes or myeloid lineages, EGFP positive and negative) are evaluated. Relative quantities of donor versus host cells are evaluated by multiplexed amplicon sequencing on the MISEQ instrument (Illumina).
Animals
Non-chimeric pigs are made having an absence of T, B and NK cells in cord and peripheral blood. Chimeric pigs have levels substantially similar to nearly wild-type levels. Moreover, T, B and NK cell positive chimeras have substantially normal immune functions and remain healthy when reared in standard conditions. Example 5: CRISPR/Cas9 design and production.
Gene specific gRNA sequences were cloned into the Church lab gRNA vector (Addgene ID: 41824) according their methods. The Cas9 nuclease was provided either by co-transfection of the hCas9 plasmid (Addgene ID: 41815) or mRNA synthesized from RCIScript-hCas9. This RCIScript-hCas9 was constructed by sub-cloning the Xbal-Agel fragment from the hCas9 plasmid (encompassing the hCas9 cDNA) into the RCIScript plasmid. Synthesis of mRNA was conducted as above except that linearization was performed using Kpnl.
Example 6: Multiplex gene editing with targeted endonucleases and HDR.
FIG. 13 A is a schematic of each gene in the multiplex experiment (depicted as a cDNA- exons denoted by alternating shades) and the site targeted by TALENS is indicated. The sequence coding the DNA binding domain for each gene is indicated below. Swine fibroblasts were co- transfected with 1 ug of each TALEN mRNA and 0.1 nMol of each HDR oligo (FIG. 13B), targeting each gene, designed to insert a premature termination codon as well as a novel Hindlll RFLP site for genotyping. A total of 384 colonies were isolated for genotyping. The GATA4 and Nkx2-5 RFLP assays were performed (FIG. 13C) and MESP1 was evaluated by sequencing (not shown). Two colonies (2/384, 0.52%) were homozygous HDR knockouts for all three genes. The triple knockouts are labeled with asterisks (FIG. 13C). Additional genotypes can be observed in FIG. 13C, example colony 49 with no HDR edits; colony 52 and 63 with heterozygous edits to NKX2-5; colony 59 with heterozygous edits to both NKX2-5 and GATA4 and so on.
Example 7: Multiplex gene-editing using a combination of TALENs and RGENs.
See FIG. 14. Swine fibroblasts were co-transfected with TALENS (1 ug EIF4G 14.1 mRNA) + Cas9/CRISPR components (2 ug Cas9 mRNA + 2 ug p65 Gls guide RNA) and 02 nMol of HDR oligo for each gene. Transfected cells were evaluated by RFLP assay revealing HDR at both sites. Cells from this population are plated for colony isolation and isolates with edits in both genes are identified.
Example 8: Human-porcine chimeric blastocysts.
An important first step in creating human organs/cells in the pig using blastocyst complementation is to determine whether human stem cells can be incorporated into the inner cell mass as opposed to the trophectoderm and blastocoele cavity. To determine if human stem cells can become incorporated into the inner cell mass, an assay system was developed using parthenogenetic blastocysts. The parthenotes are created by the electrical activation of pig oocytes resulting in the formation of a diploid cell from the combination of DNA from the maternal pronucleus and the polar body. The single diploid cell then divides and the 6th day after activation becomes a well-formed blastocyst suitable for injection of human stem cells. Ten hUCBSC were injected into individual porcine parthenogenetic blastocysts at day 6 post electrical activation. The distribution of the hUCBSCs was then examined at day 7 and day 8, and the number of human stem cells at each time point was qualified using antibodies that recognize human nuclear antigen (HNA) to visualize individual hUCBSCs. It was found that the vast majority of the hUCBSC were incorporated into the inner cell mass (FIGs. 15A- 15G, and 15F). Moreover, the hUCBSCs continued to proliferate during the two days post-injection into the blastocysts (FIG. 15G). Example 9: Human-porcine chimeric fetus.
Another important step in creating human organs/cells via blastocyst complementation is the demonstration that porcine blastocysts injected with human stem cells can give rise to porcine fetuses containing human cells. To address this issue, hUCBSCs were injected into parthenogenetic blastocysts and transferred the chimeric blastocysts to hormonally synchronized sows. Fetuses were harvested at a gestational age of 28 days (FIG. 16A). Histological analysis of tissue sections revealed HNA-positive cells within internal organs of the chimeric fetus (FIGB). These results demonstrate the ability of hUCBSCs to contribute to the developing porcine fetus.
Human -porcine chimeric fetus derived from complemented PITX3 knockout blastocysts. Porcine nigral dopamine neurons in pig-pig chimeras are also created and characterized; and human nigral dopamine neurons in human-pig chimeras. NURR1, LMX1A, and PITX3 knockout blastocysts are generated using TALEN technology in fibroblasts and cloning. It is determined whether the knockout blastocysts are capable of generating complementation based nigral dopamine neurons by using labeled porcine blastomeres as a source of stem cells. This approach has previously been used to generate exogenic pig-pig pancreas (Matsunari et al, 2013). Fetal pigs are sacrificed at embryonic day 34-35 when the fetuses reach a crown-rump length of about 17 mm. At this stage of development the VM and other brain structures are comparable to the size of fetal rats at day El 5 and the human fetus at mid first trimester and used for cellular transplantation. Confirmation of pig-pig exogenic dopamine neurons in the fetal VM from either NURR1, LMX1 A, or PITX3 blastocysts are a milestone that allows us to proceed with the generation of human-pig chimeras.
TALEN-knockout of LMX1A, PITX3, and NURR1 in pig fibroblasts. TALENs were developed to cleave in exons 1, 2 and 3 respectively for LMXA1, PITX3, and also NURRl, another gene that plays a major role in dopamine neuron development (see FIG. 17A, black triangle). TALENs were co-transfected with a homology dependent repair template designed to introduce a novel stop codon, Hindlll site, and a frame-shift after the novel stop codon to ensure disruption of the targeted allele. Populations of transfected cells were analyzed for Hindlll dependent cleavage produced by a PCR-restriction fragment polymorphism assay (FIG. 17B). The proportion of chromosomes with the novel Hindlll-knockout allele (indicated by cleavage products, open triangles) is indicated on the gel. Individual clones were derived from the populations, and those verified as bi-allelic knockout by RFLP and sequencing were cryopreserved for complementation experiments. Example 10: Complementation of PITX3 knockout porcine blastocysts with human stem cells rescues ocular phenotype.
To determine if human stem cells are capable of complementing PITX3 deficiency in the pig, hUCBSCs were injected into PITX3 knockout porcine blastocysts and transferred blastocysts to hormonally synchronized gilts. Chimeric fetuses were harvested at 62 days in gestation and examined for the status of the eyelids (FIGs. 18A, 18C, and 18E). A portion of the chimeric fetuses displayed open eyelids similar to wild-type pig fetuses while others exhibited closed eyelids. These results suggest that the PITX3 knockout in porcine blastocysts is a suitable model for interrogating human stem cell contribution to ectodermal lineages.
Example 11: ETV2 knockout pig embryos
Etv2 is a master regulatory gene for vascular and hematopoietic lineages, and is an ideal candidate for gene editing studies. The Etv2 gene locus was mutated to generate vascular and hematopoietic deficient pig embryos for several reasons. First, it has been comprehensively demonstrated that Etv2 is a master regulatory gene for vascular and hematopoietic development in mice (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012, Rasmussen 2012, Chan 2013, Rasmussen 2013, Behrens 2014, Shi 2014). Using genetic lineage tracing strategies, it was demonstrated that Etv2 expressing cells give rise to vascular/endothelial and hematopoietic lineages (Rasmussen 2011, Koyano-Nakagawa 2012, Rasmussen 2012). Second, a global gene deletional strategy was undertaken and demonstrated that Etv2 mutant mouse embryos were nonviable as they lacked vascular and hematopoietic lineages (Ferdous 2009, Koyano-Nakagawa 2012, Rasmussen 2012, Rasmussen 2013). Using transcriptome analysis, it was determined that Tie2 was markedly dysregulated in the absence of Etv2 (Ferdous 2009, Koyano-Nakagawa 2012). Moreover, using transgenic technologies and molecular biological techniques
(transcriptional assays, EMSA, ChIP and mutagenesis), it was verified that Spil, Tie2 and Lmo2 were direct downstream targets of Etv2 (Ferdous 2009, Koyano- Nakagawa 2012, Shi 2014). Third, forced overexpression of Etv2 in the differentiating ES/EB system significantly increased the populations of endothelial and hematopoietic lineages, demonstrating that Etv2 is a single factor that has the capacity to govern molecular cascades that induce both lineages (Koyano- Nakagawa 2012).
Example 12: ETV2 knockout pig embryos lack vascular and hematopoietic lineages.
Previous studies have demonstrated that Etv2 is important for vasculogenesis and hematopoiesis in the mouse as embryos lacking Etv2 are lethal at approximately E9.5 with an absence of vasculature and blood (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012).
Without intending to be bound by any theory, it was hypothesized that ETV2 is the key regulator of the vasculature and blood in mammals, and thus, the ETV2 knockout in the pig phenocopies the mouse. To examine the role of ETV2 in the pig, the entire ETV2 coding sequence was removed using two TALEN pairs flanking the gene in porcine fibroblasts (FIGs. 19 A and 19B). The process was 15% efficient at complete gene removal; 79/528 of the genotyped clones were homozygous for the deletion of the ETV2 gene. ETV2 homozygous knockout fibroblast clones were used for nuclear cloning (Somatic Cell Nuclear Transfer; SCNT) to generate ETV2 null embryos which were transferred to surrogate sows. The cloning efficiency was 29%, which was higher than the average success rate of 20%.
Embryos were harvested and analyzed at E18.0 (FIGs. 20A-20H). At E18.0, wild-type (Wt) embryos were vascularized with a well-developed vascular plexus in the allantois (FIG. 20 A) and had evidence of blood development (FIG. 20C). In contrast, ETV2 KO embryos showed clear developmental defects. Growth was retarded in ETV2 KOs relative to the Wt embryo, though both embryos were at the 24-somite stage (FIG 20B), and lacked both blood and vascular lineages (FIGs. 20 C-20H). ETV2 KO embryos lacked cardinal veins, dorsal aortae, and the endocardium, that are clearly developed in the Wt embryos (FIGs. 20 E-20H). These results reflect a similar phenotype and suggest that the function of ETV2 is conserved between mice and pigs. Further, these data strongly support the hypothesis that multiple mutations can be directed into the porcine genome to support growth of chimeric organs that are humanized in more than one cell type.
Example 13: Complementation of ETV2 knockout porcine blastocysts with human iPSCs.
Studies have been further undertaken to determine whether hiPSCs are capable of complementing ETV2 deficiency in the pig. hiPSCs were injected into ETV2 knockout porcine blastocysts and transferred these blastocysts to hormonally synchronized gilts. Chimeric fetuses were harvested at 18 days of gestational age and immunohistochemically examined for the status of the hiPSCs (FIGs. 21A-21C). Human cells were identified by genomic in situ hybridization using the probe to Alu repetitive sequence, as well as staining against human nuclear antigen (HNA). The presence of human cells were observed that expressed human CD31 and human vWF (vascular/endothelial marker) supporting the notion that the ETV2 knockout in porcine blastocysts is an excellent model for interrogating human stem cell contributions to vascular and hematopoietic lineages.
Example 14: Nkx2-5 and Handll as important regulators of cardiogenesis.
Cardiac development is a complex highly-orchestrated event that includes the specification, proliferation, migration and differentiation of cardiac progenitors that become electrically coupled and ultimately form a functional syncytium. These stages of cardiogenesis are governed by transcriptional networks, which have been shown, using gene disruption technology, to be important for heart formation and viability (Lyons 1995, Srivastava 1997, Tanaka 1999, Bruneau 2001, Yamagishi 2001, Garry 2006, Ferdous 2009, Caprioli 2011) (Table 1). Nkx2-5 is the vertebrate homolog of the Drosophila homeodomain protein, Tinman (Csx). The Tinman mutation results in the absence of heart formation in the fly (Bodmer 1993). Nkx2-5 is one of the earliest transcription factors expressed in the cardiac lineage. Targeted disruption of NL·2-5 results in perturbed heart morphogenesis, severe growth retardation and embryonic lethality at approximately E9.5 (Lyons 1995, Tanaka 1999). Handll (dHand) is a bHLH transcription factor that has also been shown to be important for cardiac morphogenesis. Handll mutant embryos are lethal during early embryogenesis and have severe right ventricular hypoplasia and aortic arch defects (Srivastava 1997). Moreover, mice lacking both Λ¾χ2-5 and Handll demonstrate ventricular agenesis and have only a single atrial chamber (FIGs. 22A-22D) (Y amagishi 2001). These gene disruption studies in the mouse model illustrate the effectiveness of using a gene editing strategy in the pig model.
Example 15: Multiplex knockout of porcine NKX2-5 and HANDII genes.
A combination of TALEN stimulated HDR were used to generate NKX2-5/HANDII mutant porcine fibroblasts. Each gene was targeted either within or immediately prior to their conserved transcription factor/DNA binding domains (FIG. 23 A). This strategy was favored over targeting the gene near the transcription start site to reduce the chance of producing a functional peptide by initiation at a downstream AUG. For NKX2-5, a homology template was provided to generate a novel in-frame stop codon, restriction site for RFLP screening, and an additional five base insertion after the stop codon to prevent a functional read-through protein. Double mutants were identified (FIG. 23 B). The ability to reliably produce double null pig fibroblast cell lines in a single shot is unique and a transformative technology important for complementation.
Example 16: Perturbed cardiogenesis in triple knockout pig embryos.
Preliminary studies have targeted a number of important transcription factors (i.e. MESP1, GATA4, NKX2-5, HANDII, TBX5, etc.) that result in perturbed cardiogenesis and would provide important new models for the study and potential treatment of congenital heart disease in the pig. Here it was demonstrated, as proof-of-concept successful targeting and generation of clones homozygous for the deletion of NKX2-5/HANDII/TBX5 genes. Triple knockout fibroblast clones were used for nuclear cloning (SCNT) to generate NKX2- 5/HANDII/TBX5 null porcine embryos, which were transferred to surrogate sows. Embryos were harvested and analyzed at E18, which is equivalent to El 1 of the mouse. At E18, the triple knockout porcine embryos have vasculature, skeletal muscle and blood but essentially lack a heart (minimal GATA4 immunohisto-chemically positive cardiomyo-cytes) (FIGs. 24A-24C) compared to the wildtype control porcine embryo. These data support the rationale and feasibility of utilizing NKX2-5/HANDII double knockout porcine model to limit the involvement of other lineages (i.e. neuronal lineage in the TBX5 KO) and be more reflective of congenital heart disease models (i.e. hypoplastic right and left heart defects). This approach results in the engineering of humanized biventricular hearts in the porcine model.
Example 17: Myf5, Myod and Mrf4 as important regulators of myogenesis.
The discovery of the Myod family including Myod, Myf5, Mrf4, and Myog, provided the fundamental platform for understanding the regulatory mechanisms of skeletal muscle myogenesis (FIGs. 25A and 25B).
Multiple strategies have been employed to investigate the regulatory network of the Myod family during myogenesis, such as transcriptome analysis, promoter analysis and ChlP- seq . Myod family members are master myogenic regulators as they transactivate a broad spectrum of gene families, including muscle specific genes, transcription factors, cell cycle genes, etc. to promote a myogenic cell fate . Previous gene disruption studies have demonstrated that mice lacking Myf5/Myod/MRF4 lack skeletal muscle and are lethal early following birth presumably due to their inability for respiration (due to the absence of a diaphragm). These gene disruption studies in the mouse illustrate the effectiveness of using gene editing strategies in the pig.
Utilizing TALENs and homology -dependent repair (HDR) to knockout TO MYF5, and MRF4. To examine the role of MYF5/MY OD/MRF4 (aka MYF6) in the pig, disrupted each coding sequence using TALEN stimulated HDR (FIGs. 26A-26C).
MYF5/MYOD/MRF4 knockout pig embryos lack skeletal muscle lineages. Embryos were harvested and analyzed at E18.0 (FIGs. 27A and 27B). The results in the mouse and pig reflect a similar phenotype and support the notion that the function of MYF5/MY OD/MRF4 are conserved between mice and pigs as mutant embryos lack skeletal muscle. Further, these data strongly support the hypothesis that direct multiple mutations into the porcine genome to support growth of chimeric organs that are humanized in more than one cell type.
Example 18: Complementation of MYF5/MYOD/MRF4 knockout phenotype with GFP WT pig blastomeres.
Porcine MYF5/MYOD/MRF4 null blastocysts were generated using SCNT, and injected with GFP -labeled porcine blastomeres (since no validated porcine ES cells are available, blastomeres were utilized for this experiment). The resulting chimeras were implanted in pseudopregnant sows and examined at E20. The feasibility of complementation was demonstrated as liver and yolk sac were GFP positive. Additionally, it was estimated that approximately 10% of porcine MYF5/MYOD/MRF4 null blastocysts were GFP labeled (FIGs. 28A-28C). These data support pig;pig complementation in this porcine mutant host. These data further support creating a triple knockout in the porcine model devoid of skeletal muscle that ultimately creates a niche for the formation of complemented tissues. This is used throughout these studies for creating humanized skeletal muscle in the pig.
Example 19: PDXl knockout results in apancreatic fetal pigs.
Pdxl~l~ mice are apancreatic and die shortly after birth due to the inability of the pancreatic bud to develop into the mature organ (Offield et al, 1996). Rescue of the mouse
Pdxl"^" phenotype by blastocyst complementation has been demonstrated by injecting wild-type mouse or rat iPSCs into Pdxl^' mouse blastocysts, producing mice that had normal functioning pancreases, derived from the donor cells (Kobayashi et al, 2010). Blastocyst complementation of Pdxl deficiency was also recently described in the pig where a functional pancreas was produced in a trans-genic apancreatic pig following the injection of labeled WT blastomere cells into pig blastocysts expressing the dominant Pdxl:hesl transgene (Matsunaria et al., 2013). Cloned Pdxl knockout pigs are not susceptible to the unpredictable nature of position effects or expression levels seen when using transgenes and offer a more consistent platform for the production of pancreas ablated pigs. TALEN technology has been used extensively to biallelically knockout the PDXl gene in pig fibroblasts (FIG. 29A) using a TALEN pair that targets the important homeobox domain of the PDXl gene, and an HDR construct to introduce a STOP codon, frameshift, and novel restriction enzyme site. Homozygous PDXl knockouts were obtained at a rate of 41% (76/184 clones) (FIG. 29B). These PDXl-/- fibroblasts and chromatin transfer cloning techniques have been used to generate PDXl-/- blastocysts and demonstrated pancreas ablation in PDXl-/- pig embryos harvested at E30 (FIGs. 29C and 29D). Nascent β- cells expressing Pdxl and insulin are present in the pig pancreas in wild-type embryos harvested at E32 (FIG. 29E). Example 20: HHEX knockout results in loss of liver in fetal pigs
Generation of HHEX KO clones. Without intending to be bound by any theory, it is hypothesized that HHEX regulates liver development (FIG. 70) In the initial studies, HHEX KO clones were generated to test the efficiency of this gene-editing method. Constructs were developed to cleave exon 2 of HHEX gene (see FIG. 3 OA black triangle) within the N-terminus of the homeo-domain-like region important for DNA binding. Fibroblasts were transfected with vector constructs and a homology dependent repair template designed to introduce a novel stop codon, a Hindlll site, and a frame- shift mutation after the novel stop codon to ensure disruption of the targeted allele. Over 50% the transfected population was positive for the Hindlll KO allele by PCR-restriction fragment polymorphism assay (FIG. 30B) and several individual clones derived from the population were either heterozygous or homozygous for the KO allele (FIG. 30C). In total, 22 clones with sequence validated KO alleles were cryopreserved. The same vector constructs are used to generate both HHEX and Ubc KO blastocysts.
HHEXKO is embryonic lethal in pigs._To determine the effect of HHEXKO in pigs, HHEX-/- fibroblasts were cloned SCNT and transferred to a synchronized recipient. At 30-32 days in gestation the embryos were harvested and assessed for the development of the liver. All embryos were genotyped and confirmed for knockout of HHEX. All specimens exhibited delayed development with a clear absence of the liver (FIG. 3 IB). Samples were taken from each specimen to grow fibroblasts as a source of HHEX knockout cells for future experiments to combine this knockout with editing of other targeted genes such as ETV2 to create human liver with human vasculature.
Example 21 : Summary of preliminary studies on porcine gene knockouts and
incorporation of human stem cells in the fetal pig.
Preliminary studies demonstrated the ability of targeted gene knockouts in the pig to disrupt the development of the eye, heart, lung, liver, skeletal muscle, pancreas, vasculature, hematopoietic cells, and dopamine neurons. It was also demonstrated that human stem cells injected into the porcine morula/blastocysts can result in their integration within the inner cell mass and contribute to developing fetal pigs. Importantly, it was also observed the contribution of human stem cells in fetal pigs within the context of blastocyst complementation. These results provide strong evidence of the feasibility to engineer human organs and cells within swine.
Example 22: MR imaging of fetal porcine organs at 16.4T.
The imaging of organs generated in the pig via blastocyst complementation are facilitated using high field MRI. The 16.4T magnet at the UMN Center for Magnetic Resonance Research is currently the most powerful magnet in the world for imaging. FIG. 33 shows a fetal pig 30 days in gestation (20 mm crown-rump length) where all of the internal organs are quite visible in great detail. The pulse sequence used in this figure was optimized for visualizing the liver. Other pulse sequences are developed to optimize contrast for other organs for quantitation of parameters such as organ volume in addition to 3D morphology to provide important information regarding the anatomical features of complemented organs. This provides a rapid quantitative approach for determine the success of complementation following the knockout of target genes to generate specific organs.
Example 23: Engineer Dopamine Neurons to Restore Motor Function
Parkinson's disease is a movement disorder that is caused by the progressive loss of dopamine neurons in the substantial nigra area of the brain. Current treatments include medications such as L-DOPA, a precursor for the synthesis of dopamine. L-DOPA therapy is effective for a period of time, but ultimately medication-induced dykinesias occur making this therapy undesirable. Clinical trials of human fetal dopamine cell transplantation for treating Parkinson disease demonstrate the amelioration of motor deficits (Lindvall et al., 1989; Freed et al., 2001; Olanow et al., 2003). Moreover, long-term follow up studies of transplant recipients up to 18 years after transplantation report the discontinuation of medication for treating their parkinsonian symptoms (Kefalopoulou et al., 2014). A major hurdle in the adoption of this form of cell therapy for the generalized treatment of Parkinson disease is the limited access to human fetal brain tissue as a source of donor dopaminergic neurons. Current iPSC approaches are unable to generate completely authentic nigral dopamine neurons. Genes important to the development of dopamine neurons in the substantia nigral have been identified that can be targeted for knockout for the generation of authentic neurons via blastocyst complementation.
Blastocysts are generated from each type of gene knockout mouse. GFP -labeled mouse iPSC cells are injected into blastocysts and transferred to surrogate dams. Offspring are analyzed for histological evidence of GFP -positive cells in the brain. For the pig studies porcine blastocysts are generated from each type of gene knockout. GFP -labeled pig blastomere cells are injected into blastocysts and transferred to surrogate sows/gilts. Fetuses are analyzed at day 60 in gestation for histological evidence of GFP -positive cells in the brain. Human stem cells are injected into blastocysts to assess human-porcine complementation.
Without intending to be bound by any theory, it is hypothesized PITX3 functions in the development of dopamine neuron/eye lens (FIG. 34). The PITX3 gene was successfully removed using TALENs (FIGs. 17 and 34). FIG. 35 shows the procedure used to introduce donor hiPSC or human umbilical cord blood stem cell into PITX3 KO porcine morula. Chimeric fetuses were generated as shown in FIG. 36. Complementation of PITX3 knockout in porcine blastocysts with human stem cells rescued the ocular development defects (FIG. 18 and FIG. 37). Porcine PITX3 knockout also resulted in loss of dopamine neurons in fetal substantia nigra (FIGs. 38 A and 38B). Complementation with human source neurons led to the incorporation of human source neurons in the chimeric fetuses and rescued the loss of dopamine neuron due to the knockout of PITX3 (FIGs. 39A-39D). hiPSC complementation also induced survival of substantia nigra dopamine neurons in chimeric fetuses to a level similar to that of a wild type pig, while the PITX3 knockout caused loss of dopamine neuron (FIGs. 40 and 41).
Without intending to be bound by any theory, it is hypothesized that LMX1A and PITX3 gene are important in dopamine neuron development (FIG. 42). To investigate whether complementation can rescue the phenotypes caused by LMX1A/PITX3 knockout, wild-type blastomeres expressing GFP were introduced into LMX1A~ /PITX3~ ~ blastocysts (FIG. 43). Porcine-porcine complementation in PITX3/LMX1A knockout fetal rescued fetal development (FIGs. 44A-44F). In PITX3 knockout animal, lens was ablated (FIGs. 45A and 45B). Porcine- porcine complementation in PITX3/LMX1A knockout animal reinstated lens development (FIGs. 46A-46C). Cells of donor origin (GFP positive cells) were found in chimeric fetal pig brain (FIGs. 47A-47D). DA neurons (Dopamine Neurons) were found in primordial ventral mesencephalon of the chimeric pigs, suggesting that porcine-porcine complementation results in development of da neurons in primordial ventral mesencephalon (FIGs. 48A-48C).
Table E provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000083_0003
Figure imgf000083_0001
Yellow = Cells are made.
Figure imgf000083_0002
Example 24: Engineer Oligodendroglia Progenitor Cells for Spinal Cord Repair
Neurological disorders such as multiple sclerosis, the leukodystrophies, and traumatic spinal cord injury result in the loss of myelination. These disorders may benefit from cell-based therapies that either inhibit further loss of myelin or restore lost myelin. High purity oligodendrocyte progenitor cells (OPCs) differentiated in vitro from human embryonic stem cells myelinate neurons after spinal cord transplantation (Nistor et al., 2005). This has been highlighted as a potential mechanism of action for the observed functional recovery (Keirstead et al., 2005; Sharp et al., 2010). A potential therapeutic strategy, therefore, is the manufacture of oligodendrocyte progenitor cells (OPCs). Current iPSC-based approaches result in poor yield and incomplete reprogramming of OPCs. Genes involved in the development of OPCs are identified and can be targeted for knockout for large-scale production of authentic human OPCs in the pig brain.
The combined knockout of OLIG1 and OLIG2 in mice is investigated to determine if knockout blastocysts can give rise to murine OPCs following murine-murine complementation, i) OLIG1 and OLIG2 knockout mice are created and interrogated for OPC deficiency, ii) mouse oligodendrocyte progenitor cells (OPCs) are created in young mice via complementation of OLIG1/OLIG2 knockout blastocysts (or NKX2.1, NKX2.2 and/or SOX10 as choosen), iii) functionality of the OPCs are confirmed by transplanting them into a congenitally dysmyelinated mouse and demonstrating myelination.
Without intending to be bound by any theory, it is hypothesized that OLIG1/OLIG2 are important in oligodendrocyte development (FIG. 65). To investigate the functions of
OLIG1/OLIG2, OLIG1/OLIG2 knockout fibroblast were generated (FIGs. 66A-66C). Wild type blastomeres expressing GFP were introduced into OLIG1/OLIG2 knockout blastocysts to generate chimeric pig. GFP expression was observed in whole body fluorescent imaging of the porcine fetus complemented with wild type cells (FIGs. 67A and 67B ). The GFP expressing cells were also incorporated into central nervous system of the chimeric pig (FIGs. 68A and 68B, and 69A and 69B). Specifically, GFP expressing cells were present in both chimeric pig brain (FIGs. 68A and 68B) and spinal cord (FIGs. 69A and 69B).
Table F provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Table F:
Figure imgf000084_0001
Yellow = Cells are made.
Figure imgf000084_0002
Example 25: Engineer Young Blood to Rejuvenate Old Brains
Recent studies demonstrate that the parabiotic coupling of young mice with old mice can rejuvenate brain function in the older animals. However, clinical translation of this approach for treating patients with aged/degenerated brains would require individual patients to receive blood from young donors. The limited availability of young blood would prevent the application of this approach to the vast majority of patients. A source of autologous young blood is generated by editing key genes involved in hematopoiesis. Knockout blastocysts can be used for complementation studies for generating young blood in mice and pigs. Blood from young mice are administered to aged and Alzheimer's mice to rejuvenate brain function. In parallel, generated young mice are coupled with old and Alzheimer's mice by parabiotic surgery as a "proof of concept" demonstrating rejuvenation of brain function though direct sharing of blood circulation. In addition, the efficacy of young human blood generated in pigs are assessed to rescue cognitive and motor function in mouse models of age-related neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Success leads to an unlimited source of autologous young blood for the rejuvenation of aged and/or degenerated brains.
Blastocysts with knockouts of RAG2, IL2rg, C-KIT, and ETV2 alone or in combination can be used to generate murine hematopoiesis via complementation using GFP -labeled murine iPSCs. Immune cells can be identified through a series of antibody panels designed to distinguish between murine and human cells. The T-cell panel use CD3 to isolate the T cell population, CD4 to stain for helper T cells, CD8a for cytotoxic T cells, and CD25 is used as an activation marker for each of these T cell subtypes. In a separate panel, FoxP3 can be used to identify regulatory T cells, and CD25 is used to identify activated regulatory T cells. Microglial cells are analyzed by staining for CD45 and CD1 lb. Also in this panel is CD200, which is a marker of microglial quiescence; and CD163, which is a marker of the alternately activated M2- like microglia. Young mice that exhibit robust complementation are used as the young parabiotic partner for circulatory coupling. Both parabiosis and blood plasma infusion approaches are employed to provide young blood/plasma to old mice and Alzheimer's mice. Young mice engineered to produce human blood are jointed to aged mice or Alzheimer's mice by parabiotic surgery. In parallel, blood plasma is pooled from young mice and intravenously infused to aged or Alzheimer's mice. Three to six months after the surgery /plasma infusion, the mice (the parabiotic pair separated) are subjected to a battery of behavioral tests for the assessment of cognitive function, which include an open field, an elevated plus maze test, a novel object recognition test, and the Morris water maze test. Non-parabiosed and untreated age-matched mice are used as controls. Porcine studies evaluate RAG2, IL2rg, C-KIT, and ETV2 knockouts alone or in combination to generate porcine hematopoietic cells using labeled pig blastomeres. See, FIGs. 71-73. As with the mouse studies, a panel of antibodies are used to determine the cell phenotype in pigs, including (CD45, CD3, δ-chain TCR, CD8, CD4 to identify αβ and γδ T cells: CD3, CD21 and CD79a for B cells; CD16. CD172a, CD2, and CD8 for NK cells and macrophages; and FoxP3 for Tregs). The results from the pig-pig chimeras provide information as to which knockouts are suitable for generating human hematopoiesis in the pig. By the time the studies on the pig-pig chimeras are completed, candidate human stem cells from Technical Aim III are identified for starting studies to engineer human blood in pigs. A panel of antibodies that specifically recognize human blood cells (such as CD45, CD3, CD4, CD8, and CD 19) are used to analyze the phenotype the cells using multicolor flowcytometry and hierarchical analysis where species specific antibodies are not available.
The genotype of host cells (carriers) used are: RAG2"/7IL2rg"/7C-KIT"/"/ETV2"/";RAG2"/"
/IL2rg"/7C-KIT"/";RAG2"/7IL2rg"/7ETV2"/";IL2rg"/7C-KIT"/7ETV2"/";RAG2"/7IL2rg"/";RAG2"/7C- KIT"/";RAG2"/7ETV2"/";IL2rg"/7C-KIT"/7;IL2rg"/7ETV2"/";C-KIT"/7ETV2"/";RAG2"/";IL2rg"/";C- KIT-/-.ETV2-/-.C_KIT-/-
Without intending to be bound by any theory, it is hypothesized RAG2/IL2Rg are important for T cell, B cell, and NK cell development and thymus development (FIGs. 49 and 50). It is also hypothesized that C-KIT gene is important for T cell, B cell, and NK cell development and thymus development. To investigate the effect of complementation in
IL2Rg/RAG2 knockout animals, chimeric animals were generated by introducing wild type blastomeres expressing GFP into IL2Rg/RAG2 knockout blastocysts (FIG. 51). Further, IL2Rg/RAG2/C-KIT animal were generated (FIGs. 77-79). A IL2Rg/RAG2/C-KIT piglet complemented with wild type cell (Recip ID 6035) was harvested and subject to further study. Phenotyping analysis indicated that the complemented animal may have both wild type genome and mutant genome (FIG. 80). Complementation in IL2Rg/RAG2/C-KIT knockout rescued the gene disruptions and resulted in generation of thymus (FIGs. 52A-52B). GFP+ immune cells were observed in cells collected from cord blood, thymus, spleen, peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN) (FIG. 53). Immune cells were generated in the thymus in chimeric animals (FIGs. 54A-54F). In blood and spleen, chimeric pig embryos contained T cells in an amount similar to wild type pig embryos, while the T cells were significantly reduced in IL2Rg/RAG2/C-KIT knockout pig embryos (FIGs. 55 & 56). Similar phenotypes were observed with respect to B cells and NK cells (FIGs. 57-60). In sum, chimeric pig embryos have similar immune cells profiles as wild type pig embryos, while immune cells are significantly reduced in knockout pig embryos. Table G provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000087_0002
Green = the TALENs are made and validated for function.
Yellow = Cells are made.
Figure imgf000087_0001
Example 26: Engineer Hematopoietic Cells to Repair Skin
Carrier (host) genotypes used include: RUNXl^/C-KIT^/FLKl"7"; RUNX 1 _/7FLK 1 _/"; RUNXl^/C-KIT"7"; C-KIT^/FLKl"7; RUNX1"7"; C-KIT"7" ; FLK1"7".
Blastocysts are generated from each type of knockout mouse. GFP -labeled mouse iPSC cells are injected into blastocysts and transferred to surrogate dams. Offspring are analyzed for histological evidence of GFP -positive cells in blood, bone marrow, thymus, lymph nodes and skin.
Histological demonstration of cells in the murine blood, bone marrow, thymus, lymph nodes and skin that are labeled with GFP and express mesodermal and hematopoietic markers CD45, CD3, and PDGFR1 alfa are indications of successful complementation.
FACS plots and photomicrographs showing histological evidence of cells in blood, bone marrow, thymus, lymph nodes and skin of the mouse that are labeled with GFP, CD45, CD3, and PDGFR1 alfa indicate successful complementation.
Blastocysts are generated from each type of knockout pig. GFP-labeled pig blastomere cells are injected into blastocysts and transferred to surrogate sows/gilts. Fetuses are analyzed at day 60 in gestation for histological evidence of GFP -positive cells in blood, bone marrow, thymus, lymph nodes and skin. Histological demonstration of cells in blood, bone marrow, thymus, lymph nodes and skin of the pig fetus that are labeled with GFP and express mesodermal and hematopoietic markers CD45, CD3, and PDGFR1 alfa.
Table H provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000088_0001
Yellow = Cells are made. Example 27: Engineer Blood Vessels to Repair the Vasculature
Vascular disease is extremely common as peripheral artery disease affects more than 10M Americans resulting in more than 150,000 limb amputations each year in the U.S (Hirsch 2013). In addition, more than 300,000 patients have coronary artery bypass grafting (surgical revascularization). These diseases collectively are amplified by the rising incidence of diabetes, obesity and cardiovascular disease. Importantly, these complications result in considerable morbidity and mortality. Current medical therapies for vascular disease include limb amputation, vascular bypass grafting (using the patient's diseased vasculature) or vascular grafts-all these therapeutic interventions have significant limitations. Etv2 was previously discovered as a master regulator of development of the vascular and the hematopoietic lineages, and that Etv2 null embryos were lethal and lacked hematoendothelial lineages (Ferdous 2009, Rasmussen 2011, Koyano-Nakagawa 2012, Rasmussen 2012, Rasmussen 2013, Behrens 2014, Shi 2014). Using gene-editing technologies, it was established that ETV2 mutant porcine embryos also lacked hemato-endothelial lineages. Humanized vasculature in the ETV2 mutant porcine model is generated. This not only provides an unlimited source of humanized vascular grafts, but also serves as a platform to multiplex with other gene editing strategies and engineer various organs with humanized vasculature. The impact on transplantation medicine is tremendous, as a cell surface antigen (galactose-alpha(l,3)galactose) present on porcine endothelial cells are known to be the major cause of hyperacute rejection and intravascular coagulation upon xenotransplantation. ETV2 mutant porcine embryos are generated by SCNT and comprehensively analyzed. ETV2 mutant morulae are injected with GFP -labeled porcine blastomeres. These GFP -labeled blastomeres are also generated using SCNT. Injected morulae are transferred to sows, and embryos are collected at El 8, E24 and neonates are collected at postnatal day 7 (P7).
Contribution of GFP positive cells to the embryos are examined. ETV2 mutant morulae are injected with human stem cells (four separate lines are analyzed. Injected morulae are transferred to sows, and embryos are collected at E18 and E24. Porcine- or human stem cell- complemented ETV2 null pig embryo is generated and examined and the cardiac and vascular functions by MRI and transthoracic echocardiography, respectively. The pig-pig chimeras are analyzed at E18, E24, and P7; and human-pig chimeras are analyzed at E18 and E24.
Host genotypes include: ETV2_/".
Table I provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000089_0001
Example 28: Engineer Blood Hearts to Repair Cardiac Function
Congenital cardiovascular malformation is the most common birth defect and it contributes to advanced or endstage heart failure in the pediatric and adult population.
Congenital heart disease afflicts approximately 1% of all live births and has considerable morbidity and mortality (Garry 2006, Hoffman 1995, Kang 2000, Kramarow 2012, Rasmussen 2011). The only curative therapy for congenital heart disease-induced heart failure is orthotopic heart transplantation. Due to the shortage of organs for transplantation, less than 2% receive such lifesaving therapy. Those that receive a heart transplant require lifelong immunosuppression, which also has deleterious side effects and limits survival. Therefore, there is a need to develop an alternative source of human heart tissues. Humanized biventricular hearts, which serve as an unlimited source of organs for transplantation and provide a paradigm shifting platform for the treatment of congenital heart disease and endstage heart failure, are generated. Previously published studies demonstrate important roles for Nkx2-5 and Hand2 for specification of ventricular myocardium (Y amagishi 2001). Therefore, a NKX2-5/HANDII double knockout porcine embryo can be engineered, using gene editing technologies, which lack ventricular myocardium. Human stem cells, SCNT and blastocyst complementation are used to produce a humanized biventricular heart. In addition to serving as a novel source of human ventricular myocardium for treatment of cardiovascular disease, the humanized pigs also serve as a large animal model to study regeneration of human lineages or response(s) to pharmacological agents and lead to improved therapies for cardiovascular diseases including hypoplastic right and left heart congenital and heart failure diseases.
NKX2-5/HandII mutant morulae are generated using SCNT and comprehensively characterized at E18 and E24. NKX2-5/HandII mutant morulae are injected with GFP -labeled porcine blastomeres. These GFP-labeled blastomeres are also generated using SCNT. Injected morulae are transferred to sows, and embryos are collected at E18, E24, and neonates are collected at postnatal day 7 (P7). Contribution of GFP positive cells to embryos are examined. NKX2-5/HandII mutant morulae are injected with human stem cells (four separate lines are analyzed and they are selected. Injected morulae are transferred to sows, and embryos are collected at E18 and E24. For subtask 1-9.4, porcine- or human stem cell-complemented NKX2- 5/HandII double knockout pig embryos are generated and the cardiac and vascular functions are examined by MRI and transthoracic echocardiography. The pig-pig chimeras are analyzed at E18, E24 and P7; and human-pig chimeras are analyzed at E18 and E24.
Genotypes of host and donor cells used include: NKX2-5-'-/HANDir'-/TBX5-/-; NKX2-5''- /HANDir'-; HANDirl~/TBX5~l~; NKX2-5^ITBX5-'-; HANDir1'; TBX5~'~; NKX2-5^.
Table J provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000090_0001
Green = the TALENs arc made and \ alidatcd for function.
Yellow = Cells are made.
Example 29: Engineer Skeletal Muscle to Restore Limb Function
Myopathic diseases such as muscular dystrophies and aging are common and deadly. While skeletal muscle has a tremendous capacity for regeneration, this potential ultimately fails with disease and aging. No treatments are currently available for terminal muscle diseases and fifty percent of the falls in the elderly lead to their demise. Additionally, U.S. Army soldiers, even light infantry troops, lose substantial training hours due to musculoskeletal injuries. The incidence of these injuries accounts for 56% of all sick-call diagnoses. Prolonged recovery is typically due to the need for long term physical therapies (Smith and Cashman, 2002).
Xenogeneic transplants of porcine extracellular matrices have proven beneficial for muscle regeneration following traumatic injury (Sicari et al., 2014). The long range goal and the clinical significance of this proposal is the production of a humanized skeletal muscle using
MYF5/MYOD/MRF4 (aka Myf6) knockout pigs. These humanized pigs serve as a large animal model to study the regeneration of the human skeletal muscle, and/or response(s) to
pharmacological agents. Skeletal muscle engineered in this way holds potential as a future source for muscle transplantation. Genetically engineered mouse models have been previously generated to define networks that are necessary and sufficient for myogenesis and regeneration.
MYF5/MYOD/MRF4 triple mutant porcine embryos are generated by SCNT and comprehensively analyzed. MYF5/MYOD/MRF4 triple mutant morulae are injected with GFP- labeled porcine blastomeres. These GFP-labeled blastomeres are also generated using SCNT. Injected morulae are transferred to sows, and embryos are collected at E24, E50 and neonates are collected at postnatal day 7 (P7). Contribution of GFP positive cells to the embryos are examined. For subtask 1-10.3, MYF5/MYOD/MRF4 triple mutant morulae can be injected with human stem cells (four separate lines can be analyzed and they are selected as outlined in Technical Area III). Injected morulae are transferred to sows, and embryos are collected at E24 and E50. For subtask 1-10.4, porcine- or human stem cell-complemented MYF5/MYOD/MRF4 null pig embryo is generated and the skeletal muscle functions are examined by MRI, contractility assays and lameness testing. The pig-pig chimeras are analyzed at E24, E50, and P7; and human-pig chimeras are analyzed at E24 and E50.
Genotypes of host and donor cells used include: MYF5"/7MYOD"/7MRF4"/"; MYF5"7" /MYOD"7"; MYF5_/7 MRF4"7"; MYOD"/7MRF4"/"; MYF5"7"; MYOD"7"; MRF4"7"; PAX3^.
Without intending to be bound by any theory, it is hypothesized that PAX3 regulates myogenesis (FIG. 61). To investigate whether complementation rescues phenotypes caused by PAX3 knockout, chimeric mice were generated (FIGs. 62A, 62B, and 74A). In Pax3 mutant mice complemented with GFP expressing wild type iPSC, the GFP positive cells were preferentially distributed to the limb skeletal muscle (FIG. 62B). The complemented mice also developed the muscle tissue/organ structures of hind limb (FIGs. 74B and 74C). Pax3 knockout pigs were also generated (FIGs.75A-75B). Mutant pigs displayed similar phenotype as the mutant mice (FIGs. 75C-75F). PAX3 mutant pigs lost limb skeletal muscles. Table K provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000092_0001
Green = the TALENs are made and validated for function.
Yellow = Cells are made.
Example 30: Engineer Livers to Restore Hepatic Function
Alcohol, obesity, type II diabetes, metabolic syndrome, and viral infections are major causes of chronic liver diseases. The incidence of nonalcoholic fatty liver disease (NAFLD), and hepatocellular carcinoma, is on the verge of becoming a worldwide epidemic. For instance, the prevalence of NAFLD in the USA is estimated to be at 20-30% of the general population, reaching levels as high as 75-100% in obese (BMI > 30) and morbidly obese (BMI > 40) people (Farrell and Larter, 2006). While a number of treatment options are available for these diseases, liver transplantation remains in many cases the most viable option. Although the procedure increased 3.7-fold from 1988 to 2009 (Wertheim et al., 2011), the increasing gap between the demand for and availability of donor livers for orthotopic liver transplantation remains a daunting problem.
Generation of HHEX and Ubc knockout blastocysts - Among the genes that were found to be important for early liver development are the homeobox gene HHEX and the polyubiquitin gene Ubc. (i) knockout either of these genes in mouse zygotes using the TALEN and CRISPR gene editing technologies; (ii) sequence the genome of KO zygotes to determine the absence of full-length HHEX and Ubc genes; and (iii) culture HHEX-KO and Ubc-KO zygotes generated in vitro until they reach the blastocyst stage, and_characterize them extensively.
Creation of human-mouse liver chimeras by blastocyst complementation - hLDPCs originally derived from hepatocytes isolated from healthy human liver biopsies, can be used to create human-mouse chimeras. In preliminary studies hLDPCs have been differentiated into hepatocytes, and found that they can engraft efficiently in mouse livers. Blastocyst
complementation are carried out by injecting hLDPCs into HHEX-KO and Ubc-KO murine blastocysts, and implant them into C57BL/6 female mice. The engraftment of LDPCs are studied after injection into mouse embryos. The livers of pups born to female recipients of blastocysts are analyzed. Of note, HHEX and Ubc mice are each embryonic lethal, and embryos of these mice survive beyond the E14.5 stage only if the hepatocyte and liver epithelial progenitor cell proliferation is complemented by the implanted hLPDCs.
Generation of human-pig liver chimeras - Chimeric livers in pigs, just as in mice are generated, and test the functionality of humanized livers generated in pigs to assess:
• their hepatic function both in vivo and ex vivo;
• human cell chimerism of hepatocytes isolated from the developed livers;
• the contribution of implanted human LDPCs to the formation of liver vasculature; and
• the enzymatic functions of isolated hepatocytes and compare them to primary human hepatocytes.
Genotypes of host and donor cells used include: HHEX'1'! Ubc'1'; HHEX1'; Ubc'1'.
Table L provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000093_0001
Green = the TALENs are made and validated for function.
Yellow = Cells are made. Example 31 : Engineer Pancreas for Transplant and to Treat Diabetes
Treatment of pancreatic diseases and disorders, including diabetes, is currently severely limited by the scarcity of human pancreases available for whole organ or islet transplantation. Blastocyst complementation of apancreatic phenotypes has been demonstrated with exogenic rat iPSCs in PdxV'' mice and syngenic cells in Pdxl .Hesl pigs (Kobayashi et al., 2010, Matsunaria et al, 2013). In both cases the pancreas formed in the complemented animals was derived entirely from progeny of the donor cells. Cloned PdxT " pigs were generated and are used to generate whole human pancreases derived from the progeny of human stem cells in pig-human chimeras, which enable production of an unlimited number of human pancreas organs and islets for transplant and is a technology platform for producing patient-specific pancreases for autologous transplants.
Complementation of the available pancreatic niche in Pdxl" " pigs are first demonstrated by injecting WT, GFP+ve pig blastomeres into Pdxl" " pig blastocysts and analyzing complemented litters for the presence of GFP expressing cells in chimeric embryos at early and mid-gestation and early postnatal timepoints. Complementation by human stem cells of the knockout of the Pdxl gene (and double knockout of the Pdxl and ETV2 genes) in pigs is investigated. Cloned Pdxl"7" or Pdxl"7" Etv2_/" pig blastocysts can be injected with the most optimal complementing human stem cells. Chimeric embryos at mid-gestation, early postnatal neonates and adult complemented pigs are generated to provide human pancreas tissue and islets for testing in vitro and in vivo including testing the ability of the human islets generated in pigs to restore blood glucose homeostasis in rodent and non human primate models of diabetes.
Genotypes of host and donor cells used include: Pdxr/7Etv2"/";Pdxl"/"; Etv2_/".
Without intending to be bound by any theory, it is hypothesized that PDX1 regulates pancreas development (FIG. 63). To investigate the effect of PDX1 gene, PDX1 gene knockout pigs were generated. In PDX1 knockout pigs, pancreas development did not occur (FIGs.29A- 29E).
Table M provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000094_0002
Figure imgf000094_0001
Example 32: Engineer Lungs to Restore Pulmonary Function
Lung disease accounts for 400,000 deaths per year in the US and transplantation is the only "cure" for many end-stage lung diseases, such as chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, primary pulmonary hypertension and cystic fibrosis. However, only 2,000 lung transplants are performed per year. Of particular significance to our military personnel are the recent reports of inhalational lung injuries in 14% of US soldiers returning from Iraq and Afghanistan. The long-term effects of these injuries, which include chronic bronchiolitis and pulmonary vascular remodeling, are certain to increase the incidence of irreversible lung disease requiring transplantation in these veterans. Major obstacles to lung transplantation include the paucity of donor lungs available, chronic rejection since lungs are typically not HLA-matched, the requirement for lifelong immunosuppression and high mortality. A new source of transplantable lung tissue that is not subject to rejection is therefore warranted. Genes involved in lung development are tested to determine which can be targeted for knockout in blastocyst complementation studies that ultimately generate human lungs.
Knockouts are tested of transcription factors active in different stages and cell lineages
(epithelial Nkx2.1, Sox2, Id2, mesenchymal Tbx4) of lung development to determine if the corresponding blastocysts have differential capacity for complementation. Nkx2. r/_ are generated by crossing Nkx2. available from the MMRRC. Other KO fibroblasts are generated using TALENS gene editing. KO mouse blastocysts are injected with wild-type mouse GFP+ ES cells. Successful complementation is repeated using mouse iPS cells. Knockouts are tested of transcription factors active in different stages and cell lineages (epithelial Nkx2.1, Sox2, Id2, mesenchymal Tbx4) of lung development to determine if resulting pig fetuses are lung-deficient. Knockouts that led to successful blastocyst complementation are recreated with KO pig fibroblasts generated using TALENS gene editing. KO pigs are cloned using parthenogenetic porcine blastocysts to evaluate generation of lung-deficient phenotypes. For those KOs with lung development defects confirmed, GFP-expressing porcine blastomeres are injected into KO blastocyst embryos. Donor cell engraftment and complementation of the lung phenotype are determined in the chimeric fetal pigs at 30 and 60 days gestation to check for complementation ability of the KO. To determine whether human pluripotent stem cells can complement lung- defective pig blastocysts, GFP-expressing human iPS cells, naive iPS cells, UCBSCs or MAPCs are injected into KO pig blastocysts. The lungs are evaluated for mosaicism and
complementation at 30 and 60 days gestation. Blastocyst complementation of the most- promising knockouts identified in subtask 1-13.3 are done using the highest engrafting human stem cell lines identified. Lung morphogenesis areevaluated and human cells identified using immunohistochemical techniques and flow cytometry. Successful combinations are further explored at later stages of gestation closer to term for evaluation of lung function (pulmonary function testing), surfactant and mucin production to assess lung maturation.
Genotypes of host and donor cells used include: Nkx2.1"/7 Sox2"/7Id2"/7Tbx4"/";Nkx2. V1' /Sox2"/7Id2-/-; Nkx2. l"/7Sox2"/7Tbx4"/"; Nkx2. l"/7Id2"/7 Tbx4_/"; Sox2"/7Id2"/7 Tbx4"A; Nkx2.1"7" /Sox2_/"; Nkx2. l"/7Id2"/"; Nkx2. l"/7Tbx4"/"; Sox2"/7Id2"/" ; Sox2"/7Tbx4"/"; Id2"/7Tbx4"/"; Nkx2.1_/"; SOX2"7"; Id2"A; Tbx4"A.
Without intending to be bound by any theory, it is hypothesized that NKX2.1 regulates lung development (FIG. 64). To investigate the effect of NKX2.1 gene on lung development, NKX2.1 knockout pigs were generated. Pig fibroblasts were knocked out (TALEN gene editing) for the transcription factor Wx2.1, a gene important for lung development. Pig embryos at day 30 were shown to have blunted lung growth at the early pseudoglandular stage (FIGs. 32A-32F). Therefore complementation at the blastocyst stage with human stem cells may be a source of generating human lungs for transplantation or lung disease studies. The NKX2.1 knockout pigs can be used to resolve the issues of shortage of donor lungs for transplantation, and lack of suitable in vivo models of human lung disease.
Table N provides a list of the genotype of edited carriers (host), their genotype of the donor used to complement (rescue) the animal.
Figure imgf000096_0001
Yellow = Cells are made.
FURTHER DISCLOSURE
Patents, patent applications, publications, and articles mentioned herein are hereby incorporated by reference; in the case of conflict, the instant specification is controlling. The embodiments have various features; these features may be mixed and matched as guided by the need to make a functional embodiment. The headings and subheadings are provided for convenience but are not substantive and do not limit the scope of what is described.

Claims

What is claimed is:
1. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
2. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues are disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogenous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue, wherein: the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
3. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
4. A chimeric embryo comprising a non-human embryo having at least one human cell, wherein both alleles of one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted and wherein one or more genes of the human cell responsible for the development of one or more corresponding human organs or tissues complement the function of the one or more disrupted endogneous genes, such that an animal that develops from the chimeric embryo comprises at least one human organ or tissue,
wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ and/or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
5. The chimeric embryo according to claim 1, wherein the endogenous genes comprise
NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons.
6. The chimeric embryo according to claim 1, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes.
7. The chimeric embryo according to claim 1, wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
8. The chimeric embryo according to claim 1, wherein the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
9. The chimeric embryo according to claim 1, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
10. The chimeric embryo according to claim 1, wherein the endogenous genes comprise
MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
11. The chimeric embryo according to claim 1, wherein the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
12. The chimeric embryo according to claim 1, wherein the endogenous genes comprise
Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
13. The chimeric embryo according to claim 1, wherein the endogenous genes comprise HHEX or Ubc and the human organ or tissue comprises human liver cells.
14. The chimeric embryo according to claim 1, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
15. The chimeric embryo according to any one of claims 1-14, wherein the non-human embryo is a non-human vertebrate embryo.
16. The chimeric embryo of claim 15, wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
17. The chimeric embryo according to claim 15, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, crustacean, and fish.
18. The chimeric embryo of claim 16, wherein the vertebrate non-human embryo is a cow, pig, sheep, goat, chicken or rabbit embryo.
19. The chimeric embryo of any one of claims 1-18, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS) , Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNs), molecules encoding site-specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi, CRISPRi or combinations thereof.
20. The chimeric embryo of claim 19, wherein the one or more endogenous genes have been disrupted by Cas9.
21. The chimeric embryo according to any one of claims 1-20, wherein the human cells are derived from at least one donor cell and the at least one donor cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
22. The chimeric embryo according to any of claims 1-21, wherein the disruption comprises a gene edit, a knockout, an insertion of one or more DNA residues, a deletion of one or more bases, or both an insertion and a deletion of one or more DNA residues.
23. The chimeric embryo according to any of claims 1-21, wherein the disruption comprises a substitution of one or more DNA residues.
24. The chimeric embryo of claim 23, wherein the disruption consists of a substitution of one or more DNA residues.
25. An animal that has developed from the chimeric embryo according to any one of claims 1-24.
26. A human tissue or organ harvested from an animal that has developed from the chimeric embryo according to any one of claims 1-25.
27. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
28. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes; the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
29. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
30. A method of producing a chimeric embryo comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein: the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HAND II and TBX5 and the human organ or tissue comprises cardiac muscle cells.
31. The method according to any one of claims 30-32, wherein the endogenous genes comprise NURRl, LMXIA, and/or PITX3 and the human organ or tissue comprise dopamine neurons.
32. The method according to claim 27, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes.
33. The method according to claim 27, wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
34. The method according to claim 27, wherein the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
35. The method according to claim 27, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
36. The method according to claim 27, wherein the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
37. The method according to claim 27, wherein the endogenous genes comprise NKX2-
5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
38. The method according to claim 27, wherein the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
39. The method according to claim 27, wherein the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells.
40. The method according to claim 27, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
41. The method according to any one of claims 27-40, wherein the non-human embryo is a non-human vertebrate embryo.
42. The method of claim 41, wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
43. The method according to claim 41, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
44. The method of any one of claims 27-43, wherein the at least one human donor cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
45. The method of any one of claims 27-44, further comprising implanting the chimeric embryo into a uterus of an animal wherein the chimeric embryo develops into a chimeric animal comprising human cells.
46. The method of claim 45, further comprising harvesting the human cells from the chimeric animal.
47. The method of claim 46, further comprising transplanting the human cells into a human patient in need thereof.
48. The method of claim 47, wherein the at least one human cell is donated by the human patient.
49. The method of any one of claims 27-48, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNS), molecules encoding site-specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi,CRISPRi or combinations thereof.
50. The method of claim 49, wherein the method is Cas9.
51. The method of any one of claims 49-50, further comprising introducing a homology directed repair (HDR) template having a template sequence with homology to one of the endogenous genes, with the template sequence replacing at least a portion of the endogenous gene sequence to disrupt the endogenous gene.
52. The method of claim 51, further comprising introducing a plurality of homology directed repair (HDR) template, each having a template sequence with homology to one of the endogenous genes, with each the template sequences replacing at least a portion of one of the endogenous gene sequences to disrupt the endogenous gene.
53. The method of claim 51 or 52, wherein the disruption comprises a substitution of one or more DNA residues of the endogenous gene.
54. The method of claim 51 or 52, wherein the disruption consists of a substitution of one or more DNA residues of the endogenous gene.
55. A chimeric embryo or chimeric animal created using the method of any one of claims 27-54.
56. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising: a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and
c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
wherein the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, and wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells;
the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
57. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue;
wherein the animal that develops from the chimeric host embryo will comprise the human or humanized organ or tissue, thereby producing a human or humanized organ or tissue in a non-human host animal, wherein:
the endogenous genes comprise NURR1, LMX1A, and/or PITX3 and the human organ or tissue comprise dopamine neurons;
the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes;
the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes comprise RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells;
the endogenous genes comprise PAX3 and the human organ or tissue comprises skeletal muscle cells;
the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells;
the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells; or
the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
58. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising:
a) disrupting both alleles of one or more endogenous genes responsible for the development of an organ or tissue in at least one cell of a non-human embryo;
b) if step a) is performed on a cell of the animal host, cloning the cell to produce an embryo; and
c) producing a chimeric host embryo by introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of a corresponding human organ or tissue; wherein:
the endogenous genes are RAG2 and IL2rg or ETV2 and the human organ or tissue comprises young blood;
the endogenous genes are MYF5, MYOD and MRF4 or PAX3 and the human organ or tissue comprises skeletal muscle cells; or the endogenous genes comprise NKX2-5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
59. A method of producing a human or humanized organ or tissue in a non-human host animal, comprising
a) disrupting both alleles of one or more endogenous genes responsible for the development of one or more organs or tissues in at least one non-human cell or non-human embryo;
b) if step a) is performed on a non-human cell, cloning the cell to produce an embryo; and
c) introducing at least one human cell into the embryo of step a) or step b), wherein the human cell carries one or more genes responsible for the development of the one or more organs or tissues; thereby producing a chimeric embryo, wherein:
the endogenous genes are RAG2 and IL2rg and/or ETV2 and the human organ or tissue comprises young blood;
the endogenous gene is PAX3 and the human organ or tissue comprises skeletal muscle cells; or
the endogenous genes are NKX2-5, HANDII and TBX5 and the human organ or tissue comprises cardiac muscle cells.
60. The method according to claim 56, wherein the endogenous genes comprise NURRl, LMXIA, and/or PITX3 and the human organ or tissue comprise dopamine neurons.
61. The method according to claim 56, wherein the endogenous genes comprise OLIG and/or OLIG2 and the human organ or tissue comprises oligodendrocytes.
62. The method according to claim 56, wherein the endogenous genes comprise RAG2, IL2rg, C-KIT and/or ETV2 and the human organ or tissue comprises young blood.
63. The method according to claim 56, wherein the endogenous genes comprise
RUNX1, C-KIT and/or FLK1 and the human organ or tissue comprises hematopoietic cells.
64. The method according to claim 56, wherein the endogenous genes comprise ETV2 and the human organ or tissue comprises human blood vessel cells.
65. The method according to claim 56, wherein the endogenous genes comprise MYF5, MYOD, MRF4 and/or PAX3 and the human organ or tissue comprises skeletal muscle cells.
66. The method according to claim 56, wherein the endogenous genes comprise NKX2- 5, HANDII and/or TBX5 and the human organ or tissue comprises cardiac muscle cells.
67. The method according to claim 56, wherein the endogenous genes comprise Pdx and/or ETV2 and the human organ or tissue comprises pancreatic cells.
68. The method according to claim 56, wherein the endogenous genes comprise HHEX and/or Ubc and the human organ or tissue comprises human liver cells.
69. The method according to claim 56, wherein the endogenous genes comprise Nkx2.1, Sox2, Id2 and/or Tbx4 and the human organ or tissue comprises lung cells.
70. The method according to any one of claims 56-69, wherein the non-human embryo is a non-human vertebrate embryo.
71. The method of claim 70, wherein the vertebrate non-human embryo is an artiodactyl embryo or a non-human primate embryo.
72. The method according to claim 70, wherein the non-human vertebrate embryo is selected from the group consisting of cattle, horse, swine, sheep, chicken, avian, rabbit, goat, dog, cat, laboratory animals, and fish.
73. The method of any one of claims 56-72, wherein the at least one human cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, a pluripotent stem cell or an induced pluripotent stem cell.
74. The method of claim 56-73, further comprising harvesting the human cells from the chimeric animal.
75. The method of claim 74, further comprising transplanting the human cells into a human patient in need thereof.
76. The method of claim 75, wherein the at least one human cell is donated by the human patient.
77. The method of any one of claims 56-76, further comprising introducing a homology directed repair (HDR) template having a template sequence with homology to one of the endogenous genes, with the template sequence replacing at least a portion of the endogenous gene sequence to disrupt the endogenous gene.
78. The method of claim 77, further comprising introducing a plurality of homology directed repair (HDR) template, each having a template sequence with homology to one of the endogenous genes, with each the template sequences replacing at least a portion of one of the endogenous gene sequences to disrupt the endogenous gene.
79. The method of claim 77 or 78, wherein the disruption comprises a substitution of one or more DNA residues of the endogenous gene.
80. The method of claim 77 or 78, wherein the disruption consists of a substitution of one or more DNA residues of the endogenous gene.
81. The method of any one of claims 56-80, wherein the one or more endogenous genes of the non-human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Transcription Activator-Like Effector Nucleases (TALENS) , Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR associated protein 9 (Cas9), Zinc Finger Nucleases (ZFNS), molecules encoding site-specific endonucleases, synthetic artificial chromosomes, RecA-gal4 fusions, RNAi, CRISPRi or combinations thereof.
82. The method of claim 81, wherein the one or more endogenous genes of the non- human embryo responsible for the development of one or more endogenous organs or tissues have been disrupted by Cas9.
83. A chimeric animal created using the method of any one of claims 56-82.
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KR102684690B1 (en) 2015-03-03 2024-07-15 리젠츠 오브 더 유니버시티 오브 미네소타 ETV2 and its uses
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US11576889B2 (en) 2019-09-23 2023-02-14 The Board Of Regents Of The University Of Texas System Methods of identifying and treating patients with HIF-2 inhibitor resistance

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