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WO2018085967A1 - Mammifères humanisés génétiquement exprimant l'albumine sérique humaine et utilisations associées - Google Patents

Mammifères humanisés génétiquement exprimant l'albumine sérique humaine et utilisations associées Download PDF

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WO2018085967A1
WO2018085967A1 PCT/CN2016/104956 CN2016104956W WO2018085967A1 WO 2018085967 A1 WO2018085967 A1 WO 2018085967A1 CN 2016104956 W CN2016104956 W CN 2016104956W WO 2018085967 A1 WO2018085967 A1 WO 2018085967A1
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human
polynucleotide sequence
albumin
mammal
transgenic mammal
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Liangxue LAI
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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Priority to PCT/CN2016/104956 priority Critical patent/WO2018085967A1/fr
Priority to CN201710014128.7A priority patent/CN106866813A/zh
Publication of WO2018085967A1 publication Critical patent/WO2018085967A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • 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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • 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
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present disclosure relates to genetically humanized mammals, and more particularly to genetically humanized mammals expressing human serum albumin and uses thereof.
  • HSA Human serum albumin
  • rHSA recombinant HSA
  • Embodiments here relate to a non-human transgenic mammal whose genome includes a polynucleotide sequence encoding human albumin.
  • the polynucleotide sequence is operably linked to a promoter polynucleotide sequence.
  • the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin includes a modification of human albumin gene.
  • Some embodiments of the present disclosure further relate to a method of producing human albumin from a non-human transgenic mammal.
  • the method may include preparing the non-human mammal whose genome includes a polynucleotide sequence encoding human albumin.
  • the polynucleotide sequence is operably linked to a promoter polynucleotide sequence.
  • the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin includes a modification of human albumin gene.
  • the method may further include obtaining a blood sample from the non-human mammal, and isolating the human albumin from the blood sample.
  • Some embodiments of the present disclosure further relate to a method of preparing a non-human transgenic mammal.
  • the method may include providing a polynucleotide sequence encoding human albumin, and introducing the polynucleotide sequence encoding human albumin to the genome of the non-human mammal thereby preparing the non-human mammal.
  • the polynucleotide sequence encoding the human albumin may include a modification of human albumin gene, and the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the method may further include introducing the polynucleotide sequence encoding human albumin to a somatic cell isolated form the non-human mammal whose genome includes the polynucleotide sequence encoding human albumin, fusing the nucleus of the introduced somatic cell into an enucleated oocyte of the non-human mammal thereby producing an embryo of the non-human mammal, and implanting the embryo into a foster female non-human mammal thereby producing the non-human mammal.
  • Some embodiments of the present disclosure further relate to a method of transplanting a non-human donor tissue into a recipient mammal.
  • the method may include grafting the donor tissue into the recipient mammal.
  • the non-human donor tissue is obtained from a non-human transgenic mammal that does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin may include a modification of human albumin gene.
  • the non-human donor tissue is a liver.
  • the modification may include codon optimization of the human albumin gene and/or the polynucleotide sequence encoding the human albumin may include one or more introns of a mammary albumin gene.
  • the modification may include replacement of 80%or less of codons of human with codons preferred by non-human mammary cells.
  • the polynucleotide sequence encoding the human albumin may include at least part of intron 1 and intron 2 of a human albumin gene or the non-human mammary albumin gene.
  • blood of the non-human transgenic mammal may include about 8 to 30 g/L of the human albumin as measured using BCG (Bromocresol Green) Albumin Assay.
  • BCG Breast Cancer Green
  • blood of the transgenic pig may include not less than 16 g/L of the human albumin as measured using BCG (Bromocresol Green) Albumin Assay, and wherein the non-human transgenic mammal does not express all of the polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • BCG Breast Cancer Green
  • blood of the transgenic pig may include not less than 8 g/L, and wherein the non-human transgenic mammal does not express the part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the non-human transgenic mammal does not express all of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the promoter polynucleotide sequence is an albumin promoter of the non-human mammary.
  • the non-human transgenic mammal is a porcine animal, a cow, a sheep, a goat, a dog or a rabbit.
  • the non-human transgenic mammal is a porcine animal.
  • the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 3 or 4 and/or SEQ ID NO: 5 or 6.
  • the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 7.
  • the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 7 and a polynucleotide sequence of SEQ ID NO: 5 or 6 and SEQ ID NO: 3 or 4.
  • the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 9, 10, 11, or 12 encoding the human albumin in the non-human transgenic mammal.
  • Some embodiments of the present disclosure further relate to a targeting vector for preparing a transgenic porcine animal whose genome includes a polynucleotide sequence encoding human albumin.
  • the vector may include the polynucleotide sequence including at least one of a polynucleotide sequence of SEQ ID NO: 7, a polynucleotide sequence of SEQ ID NO: 3 or 4, or a polynucleotide sequence of SEQ ID NO: 5 or 6.
  • the transgenic porcine animal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the transgenic porcine animal.
  • the polynucleotide sequence is inserted between a 5’ arm of sequence of SEQ ID NO: 14 and a 3’ arm of sequence of SEQ ID NO: 15.
  • the polynucleotide sequence may include the polynucleotide sequence of SEQ ID NO: 7, the polynucleotide sequence of SEQ ID NO: 3 or 4, and the polynucleotide sequence of SEQ ID NO: 5 or 6.
  • the polynucleotide sequence may include the polynucleotide sequence of SEQ ID NO: 13.
  • Some embodiments of the present disclosure further relate to an isolated host cell including the targeting vector.
  • FIGS. 1A, 1B, 1C, 1D, and 1E illustrate TALEN-mediated knock-in of human albumin in the porcine albumin locus.
  • FIG. 1A is a schematic overview depicting the gene targeting strategy in the porcine albumin locus.
  • Proper recombination event resulted in the insertion of the human albumin gene and the PGK promoter-puromycin resistance cassette flanked by loxP sites (white triangles) in the exon 1 of the porcine albumin locus. This event disrupted the expression of the endogenous porcine albumin gene.
  • Human albumin was driven by the endogenous porcine albumin promoter and directed human albumin expression to the liver.
  • TALEN target site and PCR primers (5F1, 5R1, 3F2, 3R2, F, and R) are indicated.
  • FIG. 1B shows schematic of TALENs targeting the exon 1 and intron 1 of the porcine albumin locus.
  • TALEN repeats are colored differently to represent the four repeat variable di-residue (RVD) .
  • FIG. 1C shows detection of TALENs cleavage activity using EGFP SSA assay, and EGFP expression was observed under a fluorescence microscope.
  • FIG. 1D shows detection of TALENs cleavage activity using EGFP SSA assay, and the ratio of the EGFP-positive cells was measured by flow cytometry (D) .
  • FIG. 1E shows 5′-junction (4748 bp) (5F1+5R1) and 3′-junction (3623 bp) (3F2+3R2) PCR analysis to identify individual colonies with stable knock-in at the porcine albumin locus. PCR used primers F and R to distinguish between monoallelic and biallelic targeting.
  • FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show production of albumin humanized pigs via somatic cell nuclear transfer (SCNT) .
  • SCNT somatic cell nuclear transfer
  • FIG. 2A shows newborn cloned piglets derived from SCNT using albumin humanized colonies as nuclei donors.
  • FIG. 2B shows PCR analysis confirmed the correct homologous recombination at the porcine albumin locus in of the 8/9 cloned piglets. Eight piglets were all monoallelic modifications as detected by PCR (F+R) , which is consistent with those of cells chosen as nuclear donors.
  • FIG. 2C shows confirmation of HSA expression in the blood of the four cloned piglets (three positive and one wild-type cloned piglets) by Western blot analysis. 4#, 5#, 8#, 9#: serum of cloned piglets; PC: Positive control; M: protein marker.
  • FIG. 2D shows five F1 albumin humanized piglets, generated through crossbreeding heterozygous male founders and wild-type female pigs.
  • FIG. 2E shows. PCR screening of individuals of F1 generations.
  • FIG. 2F shows expression of HSA in F1 generations as characterized using Western blot analysis.
  • 1–17 serum of F1 piglets
  • PC serum of cloned heterozygote as a positive control
  • NC serum of wild-type piglets as a negative control
  • M protein marker.
  • FIG. 2G shows H&E staining of the liver of sacrificed wild-type and albumin humanized piglets.
  • FIGS. 3A, 3B, 3C, 3D, 3E, and 3F illustrates characterization of human albumin expressed in the serum of albumin humanized pigs.
  • FIG. 3A shows qualitative analysis of HSA expression levels in albumin humanized founders (A) . Approximately 0.2, 0.4, 0.8, 1, 2, and 4 ⁇ g of human albumin standard were used as control.
  • FIG. 3B shows qualitative analysis of HSA expression levels in F1 pigs. Approximately 0.2, 0.4, 0.8, 1, 2, and 4 ⁇ g of human albumin standard were used as control.
  • FIG. 3C shows SDS-PAGE analysis of albumin extracted from the serum of albumin humanized pigs via ammonium sulfate precipitation.
  • Lane 1–3 extracted samples from three pigs
  • lane 4 protein ladder
  • lane 5 human albumin standard.
  • FIG. 3D shows MS analysis of albumin purified by SDS-PAGE.
  • FIG. 3E illustrates a portion of peptide mapping of purified albumin. Peptide sequences highlighted in green indicate the amino acids in purified albumin matched with database human and porcine albumin sequences.
  • FIG. 3F illustrates another portion of peptide mapping of purified albumin. Peptide sequences highlighted in green indicate the amino acids in purified albumin matched with database human and porcine albumin sequences.
  • FIG. 4 is a schematic diagram illustrating exon 1, intron 1, exon 2, and intron 2 of human albumin gene.
  • HSA genetically engineered animals expressing HSA provides a suitable solution for large-scale production of HSA.
  • transgenic animals expressing HSA using current techniques have problems. For example, while ectopic overexpression of HSA in mouse, pig and bovine milk has been reported, a variety of other proteins with similar molecular weights to HSA in milk complicate the procurement of purified HSA. A study has been reported the ectopic overexpression of HSA in pig blood. However, the coexisting endogenous porcine serum albumin in pig blood makes HSA impossible to distinguish from porcine serum albumin. Another study has been reported that one-cell embryo injection and DNA donor was employed to insert human albumin cDNA in porcine ALB locus.
  • Embodiments of the present disclosure provide compositions, methods, and systems for generation of humanized animals that exclusively express human albumin but not animals’ albumin in blood.
  • some embodiments herein including knocking-in the modified HSA gene into the pig albumin locus and together with disruption of endogenous porcine serum albumin expression.
  • the modified HSA gene includes at least one of codon optimization of the human albumin gene, intron 1 of albumin gene, or intron 2 of albumin gene.
  • the expression level of human albumin in homozygous piglets reaches 8-30 g/L. Accordingly, a population of 130,000–170,000 homozygous pigs produced in accordance with some embodiments herein may potentially satisfy the annual demand of 500 metric tons worldwide. This overcomes the issue on purification and large-scale production.
  • an element means one element or more than one element.
  • binding means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • complementarity refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “A-G-T, ” is complementary to the sequence “T-C-A. ”
  • Complementarity may be “partial, ” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” or a physiologically significant amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc. ) an amount or level described herein.
  • targeted gene may also be accomplished by targeting the mRNA of that gene, such as by using various antisense technologies (e.g., antisense oligonucleotides and siRNA) known in the art. Accordingly, targeted genes may be considered “non-functional” when the polypeptide or enzyme encoded by that gene is not expressed by the modified cell, or is expressed in negligible amounts, such that the modified cell produces or accumulates less of the polypeptide or enzyme product (e.g., albumin) than an unmodified or differently modified cell.
  • the polypeptide or enzyme product e.g., albumin
  • exogenous refers to a polynucleotide sequence that does not naturally-occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques.
  • exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein.
  • endogenous or “native” refers to naturally-occurring polynucleotide sequences that may be found in a given wild-type cell or organism.
  • polynucleotide sequences that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide with respect to the second organism.
  • polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.
  • the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
  • gene is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5’ and 3’ untranslated sequences) .
  • Homology refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. It is contemplated that the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • homologous refers to the relationship between proteins that possess a “common evolutionary origin, ” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc. ) (Reeck et al., Cell 50: 667, 1987) .
  • proteins and their encoding genes
  • sequence homology as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.
  • host cell includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector (s) or isolated polynucleotide of the present disclosure.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the present disclosure.
  • a host cell which comprises a recombinant vector of the present disclosure is a recombinant host cell.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide” refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an “isolated peptide” or an “isolated polypeptide” and the like, as used herein refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.
  • labeled with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody.
  • locus is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome.
  • locus usually refers to the specific physical location of a target sequence on a chromosome.
  • a sample such as, for example, a polynucleotide or polypeptide is isolated from, or derived from, a particular source, such as a desired organism or a specific tissue within a desired organism.
  • Obtained from can also refer to the situation in which a polynucleotide or polypeptide sequence is isolated from, or derived from, a particular organism or tissue within an organism.
  • a polynucleotide sequence encoding a reference polypeptide described herein may be isolated from a variety of prokaryotic or eukaryotic organisms, or from particular tissues or cells within certain eukaryotic organism.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA and RNA.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51%to at least 99%and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein.
  • polynucleotide variant and variant also include naturally-occurring allelic variants and orthologs that encode these enzymes.
  • a targeted gene may be rendered “non-functional” by changes or mutations at the nucleotide level that alter the amino acid sequence of the encoded polypeptide, such that a modified polypeptide is expressed, but which has reduced function or activity with respect to its activity (e.g., introducing transportation of albumin) , whether by modifying that polypeptide’s active site, its cellular localization, its stability, or other functional features apparent to a person skilled in the art.
  • modifications to the coding sequence of a polypeptide involved in albumin expression may be accomplished according to known techniques in the art, such as site directed mutagenesis at the genomic level and/or natural selection (i.e., directed evolution) of a given cell.
  • Polypeptide, ” “polypeptide fragment, ” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • polypeptide variant refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue.
  • a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative.
  • the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide.
  • Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.
  • reference sequence refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Sequence Listing.
  • sample is used herein in its broadest sense.
  • a sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like.
  • samples include saliva, serum, biopsy specimens, blood, urine, and plasma.
  • sequence identity or, for example, comprising a “sequence 50%identical to, ” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp,
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing) , typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence” , “comparison window” , “sequence identity” , “percentage of sequence identity” and “substantial identity” .
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20%or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • BESTFIT Pearson FASTA
  • FASTA Pearson's Alignment of sequences
  • TFASTA Pearson's Alignin
  • Statistical significance By “statistically significant, ” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • substantially or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99%or greater of some given quantity.
  • TALEN is intended a protein comprising a Transcription Activator-like (TAL) effector binding domain and an endonuclease domain, the fusion of both domains resulting in a “monomeric TALEN” .
  • Some monomeric TALEN can be functional per se and others 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.
  • Two monomeric TALEN are different when, for example, their RVDs numbers are different, and/or when the content (i.e amino acid sequence) of at least one RVD is different.
  • TAL effector-DNA modifying enzyme is intended a protein comprising a Transcription Activator-Like effector binding domain and a DNA-modifying enzyme domain.
  • Transformation refers to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome; also, the transfer of an exogenous gene from one organism into the genome of another organism.
  • the term “genome” as used herein, can refer to sequences, either DNA, RNA or cDNA derived from a patient, a tissue, an organ, a single cell, a tumor, a specimen of an organic fluid taken from a patient, freely circulating nucleic acid, a fungus, a prokaryotic organism and a virus.
  • the terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an “expression product” such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed. ”
  • An expression product is, in various aspects, characterized as intracellular, extracellular or secreted.
  • intracellular means inside a cell.
  • extracellular means outside a cell, such as a transmembrane protein.
  • a substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • transfection refers to the introduction of a foreign nucleic acid into a cell.
  • transformation refers to the introduction of a “foreign” (i.e. exogenous, heterologous, extrinsic or extracellular) gene, DNA or RNA sequence to an embryonic stem (ES) cell or pronucleus, so that the cell will express the introduced gene or sequence to produce a desired substance in a transgenic animal.
  • ES embryonic stem
  • a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a coding sequence.
  • the promoter sequence is bound at its 3′ terminus by a transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease S 1) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • the promoter may be operatively associated with other expression control sequences, including enhancer and repressor sequences.
  • promoters used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839 and No. 5,168,062) , the SV40 early promoter region (Benoist and Chambon, Nature 290: 304-3101981) , the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22: 787-797, 1980) , the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.
  • CMV cytomegalovirus
  • the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42, 1982) ; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcoho) dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit neuronal or brain specific expression, such as the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., Science 234: 1372-1378, 1986) , the Thy1.2 “pan-neuronal” promoter, and synapsin I promoter (Howland et al., Brain Neurobiol Aging 16: 685-699, (995) , active in neurons.
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcoho) dehydrogenase) promoter
  • the promoter is an endogenous blood clotting factor promoter.
  • the worker of ordinary skill in the art will understand that any promoter known in the art is useful, and that the cell type in which expression is desired can dictate use of a particular promoter.
  • a coding sequence is “under the control of, ” “operably linked to” or “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and translated, in the case of mRNA, into the protein encoded by the coding sequence.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated.
  • Such a vector may comprise specific sequences that allow recombination into a particular, desired site of the host chromosome.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector is preferably one which is operably functional in a host cell, such as a plasmid.
  • the vector can include a reporter gene, such as a green fluorescent protein (GFP) , which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
  • a wild-type gene or gene product e.g., a polypeptide
  • a wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
  • selectable marker refers to a gene encoding an enzyme or other protein that confers upon the cell or organism in which it is expressed an identifiable phenotypic change such as resistance to a drug, antibiotic or other agent, such that expression or activity of the marker is selected for (for example, but without limitation, a positive marker, such as the neo gene) or against (for example, and without limitation, a negative marker, such as the dipteheria gene) .
  • a heterologous selectable marker refers to a selectable marker gene that has been inserted into the genome of an animal in which it would not normally be found.
  • selectable markers include, but are not limited to, an antibiotic resistance gene such as neomycin (neo) , puromycin (Puro) , diphtheria toxin, phosphotransferase, hygromycin phosphotransferase, xanthineguanine phosphoribosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase.
  • an antibiotic resistance gene such as neomycin (neo) , puromycin (Puro) , diphtheria toxin, phosphotransferase, hygromycin phosphotransferase, xanthineguanine phosphoribosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltrans
  • tolerance refers to the lack of an antigen-recipient's immune response which would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient. Tolerance involves, in various aspects, humoral, cellular, or both humoral and cellular responses. Tolerance, as used herein, refers not only to complete immunologic tolerance to an antigen or compound, i.e., no immune response, but also to partial immunologic tolerance, i.e., a limited immune response which does not completely eliminate, inhibit, or otherwise suppress the response to the compound. For instance, in some aspects, a tolerant subject exhibits a detectable immune response to a compound, but it is significantly less than, or decreased compared to, a non-tolerant subject's immune response when exposed to the same compound.
  • Some embodiments here relate to a non-human transgenic mammal whose genome includes a polynucleotide sequence encoding human albumin.
  • the polynucleotide sequence is operably linked to a promoter polynucleotide sequence.
  • the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin includes a modification of human albumin gene.
  • Some embodiments of the present disclosure further relate to a method of producing human albumin from a non-human transgenic mammal.
  • the method may include preparing the non-human mammal whose genome includes a polynucleotide sequence encoding human albumin.
  • the polynucleotide sequence is operably linked to a promoter polynucleotide sequence.
  • the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin includes a modification of human albumin gene.
  • the method may further include obtaining a blood sample from the non-human mammal, and isolating the human albumin from the blood sample.
  • Purification and production of albumin from blood may be implanted by various methods (e.g., crystallization) .
  • Information related to purification and/or production of albumin from blood is provided in US Patent No. 7,087,719, incorporated by reference in its entirety.
  • Some embodiments of the present disclosure further relate to a method of preparing a non-human transgenic mammal.
  • the method may include providing a polynucleotide sequence encoding human albumin, and introducing the polynucleotide sequence encoding human albumin to the genome of the non-human mammal thereby preparing the non-human mammal.
  • the polynucleotide sequence encoding the human albumin may include a modification of human albumin gene, and the non-human transgenic mammal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the method may further include introducing the polynucleotide sequence encoding human albumin to a somatic cell isolated form the non-human mammal whose genome includes the polynucleotide sequence encoding human albumin, fusing the nucleus of the introduced somatic cell into an enucleated oocyte of the non-human mammal thereby producing an embryo of the non-human mammal, and implanting the embryo into a foster female non-human mammal thereby producing the non-human mammal.
  • Some embodiments of the present disclosure further relate to a method of transplanting a non-human donor tissue into a recipient mammal.
  • the method may include grafting the donor tissue into the recipient mammal.
  • the non-human donor tissue is obtained from a non-human transgenic mammal that does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal, and the polynucleotide sequence encoding the human albumin may include a modification of human albumin gene.
  • the non-human donor tissue is a liver.
  • albumin refers to serum albumin, which is an albumin (atype of globular protein) found in vertebrate blood.
  • Human albumin is a globular unglycosylated serum protein (Mr 69.38 Kilodaltons) synthesized by the liver. Its three-domain structure is thought to have arisen via triplication of a single primordial domain. It has an exceptional binding capacity for diverse substances and has recently been implicated in catabolism of prostaglandins.
  • the human albumin gene spans 16,961 nucleotides from the putative “Cap” site to the first poly (A) addition site, and it is split into 15 exons by 14 introns.
  • a “transgenic animal” is a non-human animal in which one or more, and preferably essentially all, of the cells of the animal contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • a “transgene” is a gene or genetic material that has been transferred from one organism to another. Typically, the term describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.
  • a humanized animal or a genetically humanized animal refers to a non-human animal that expresses a human protein but does not express an endogenous protein of the animal or expresses reduced amount of endogenous protein of the animal as compared to a wild-type animal.
  • an albumin humanized pig refers to a pig that express the human albumin but does not express pig albumin or express reduced amount of pig albumin as compared to a wild-type pig.
  • non-human animal is meant to include any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovine) , deer, mules, horses, monkeys, dogs, cats, rats, and mice.
  • genetically altered pigs and methods of production thereof are provided.
  • the animals of the present disclosure are “genetically modified” or “transgenic, ” which means that they have a transgene, or other foreign DNA, added or incorporated, or an endogenous gene modified, including, targeted, recombined, interrupted, deleted, disrupted, replaced, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect in at least one cell of the animal, and typically into at least one germ line cell of the animal.
  • animals may have the transgene integrated on one allele of the genome of the animals (heterozygous transgenic) .
  • animals may have the transgene on two alleles of the genome of the animals (homozygous transgenic) .
  • a “donor” is meant to include any non-human organism that may serve as a source of donor tissue or cells for xenotransplantation including, but not limited to, mammals, birds, chickens, reptiles, fish, and insects.
  • the donor may be in any stage of development, including, but not limited to fetal, neonatal, young and adult.
  • the modification may include codon optimization of the human albumin gene and/or the polynucleotide sequence encoding the human albumin may include one or more introns of a mammary albumin gene.
  • the modification may include replacement of at least a portion of codons of human with codons preferred by non-human mammary cells.
  • the polynucleotide sequence encoding the human albumin may further include intron 1 and intron 2 of human albumin gene.
  • the nucleotide sequence of the human alpha-albumin gene has been determined from three overlapping lambda phage clones.
  • the sequence spans 22, 256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns.
  • FIG. 4 illustrates the exon 1, the exon 2, the intron 1 and the intron 2 of the human albumin gene.
  • the modification may include replacement of 80%or less of codons of human with codons preferred by non-human mammary cells.
  • the polynucleotide sequence encoding the human albumin may include at least part of intron 1 and intron 2 of a human albumin gene or the non-human mammary albumin gene.
  • blood of the non-human transgenic mammal may include about 8 to 30 g/L of the human albumin as measured using BCG (Bromocresol Green) Albumin Assay.
  • blood of the transgenic pig may include not less than 16 g/L of the human albumin as measured using BCG Albumin Assay, and wherein the non-human transgenic mammal does not express all of the polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • blood of the transgenic pig may include not less than 8 g/L of the human albumin as measured using BCG Albumin Assay, and wherein the non-human transgenic mammal does not express the part of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the non-human transgenic mammal does not express all of a polynucleotide sequence encoding endogenous albumin of the non-human transgenic mammal.
  • the non-human transgenic mammal does not express any endogenous albumin of the non-human transgenic mammal.
  • the promoter polynucleotide sequence is an albumin promoter of the non-human mammary.
  • the non-human transgenic mammal is a porcine animal, a cow, a sheep, a goat, a dog or a rabbit.
  • the terms “porcine” , “porcine animal” , “pig” and “swine” are generic terms referring to the same type of animal without regard to gender, size, or breed.
  • the non-human transgenic mammal is a porcine animal.
  • the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 3 or 4 and/or SEQ ID NO: 5 or 6. In some embodiments, the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 7 and a polynucleotide sequence of SEQ ID NO: 5 or 6 and SEQ ID NO: 3 or 4. In some embodiments, the polynucleotide sequence encoding human albumin may include a polynucleotide sequence of SEQ ID NO: 9, 10, 11, or 12 encoding the human albumin in the non-human transgenic mammal.
  • Some embodiments of the present disclosure further relate to a targeting vector for preparing a transgenic porcine animal whose genome includes a polynucleotide sequence encoding human albumin.
  • the vector may include the polynucleotide sequence including at least one of a polynucleotide sequence of SEQ ID NO: 7, a polynucleotide sequence of SEQ ID NO: 3 or 4, or a polynucleotide sequence of SEQ ID NO: 5 or 6.
  • the transgenic porcine animal does not express all or a part of a polynucleotide sequence encoding endogenous albumin of the transgenic porcine animal.
  • the polynucleotide sequence is inserted between a 5’ arm of sequence of SEQ ID NO: 14 and a 3’ arm of sequence of SEQ ID NO: 15.
  • the polynucleotide sequence may include the polynucleotide sequence of SEQ ID NO: 7, the polynucleotide sequence of SEQ ID NO: 3 or 4, and the polynucleotide sequence of SEQ ID NO: 5 or 6.
  • the polynucleotide sequence may include the polynucleotide sequence of SEQ ID NO: 13.
  • TALENs targeting the exon 1 and intron 1 of the porcine albumin gene were designed and constructed through Golden Gate TALEN assembly as previously described.
  • the cleavage activity of each TALEN was rapidly measured using an EGFP single strand annealing (SSA) assay in human HEK293 cells as previously described.
  • SSA EGFP single strand annealing
  • the ratio of the EGFP positive cells reflected the cleavage activity of TALENs.
  • EGFP expression was observed under a fluorescence microscope, and the ratio of EGFP-positive cells was measured through flow cytometry.
  • the targeting donor, 5′ (4.5 Kb) and 3′ (3.4 Kb) homology arms were cloned from genomic DNA of Yorkshire porcine fetal fibroblasts.
  • the HSA coding sequences were obtained from the RT-PCR of human liver cDNA and then inverted between two homology arms into the donor.
  • a loxP-flanked PGK-puromycin cassette was also inserted in the vector pFlexible-DT.
  • the constructed vector was fully sequence verified.
  • the vector was linearized by ApaLI before targeting.
  • PFFs were isolated from 35-day-old Yorkshire porcine fetuses. In brief, the heads, tails, limbs, and viscera were removed. The remaining tissues were minced with sterile scissors and then digested with collagenase-DNase in cell culture medium supplemented with 0.5 mg/mL Collagenase IV (Life Technology) and 100 KU/mL DNaseI (Sigma) for 3–4 h at 37 °C. The isolated PFFs were cultured in 10 cm culture dishes for 24 h and then frozen in fetal bovine serum containing 10 %dimethylsulfoxide. PFFs were thawed and grown in 10 cm culture dishes until 90%confluent before electroporation.
  • Approximately 2 ⁇ 10 6 cells were electroporated using the Neon transfection system (Life technology) at 1350 V with 1 pulse of 30 ms duration in 100 ⁇ L of Buffer B containing 15 ⁇ g of linearized targeting donors and 7 ⁇ g of each TALEN.
  • the transfected cells were divided into sixty 10 cm culture dishes and then recovered for 48 h. After recovery, 1 ⁇ g /mL puromycin (Merck) was added to the cell culture medium. After 8–12 days of selection, puromycin-resistant colonies were picked and cultured in 48-well plates by using cloning cylinders. Cell colonies at 70%–80%confluency were sub-cultured, and a fraction was lysed individually in 10 ⁇ L of NP40 lysis buffer for 60 min at 56 °C and then for 10 min at 95 °C.
  • the lysate was used as a template for PCR screening.
  • PCR screening was performed using Long PCR Enzyme Mix (Thermo Scientific) in accordance with the manufacturer’s instructions.
  • PCR analysis was used to confirm the HDR with the 5′ junction primers (5F1 and 5R1) , the 3′ junction primers (3F2 and 3R2) .
  • HDR-positive colonies were analyzed for the presence of a 680 bp region spanning the TALEN target site using the primers (F and R) .
  • the reconstructed embryos were surgically transferred into the oviducts of surrogates the day after the observed estrus.
  • An ultrasound scanner was used to monitor the pregnancy status of the surrogates weekly after a month of implantation, and the cloned piglets were delivered through natural birth.
  • the genomic DNA extracted from the newborn piglets’ ear was used as a PCR template.
  • the primers used for PCR genotyping were similar to those for cell colony genotyping.
  • the serum samples obtained from the wild-type and heterozygous albumin humanized pigs were diluted in 1 ⁇ SDS sample buffer (62.6 mM Tris-HCl, 10%glycerol, 0.01%bromophenol blue, 2%SDS, pH 6.8) and then boiled for 10 min.
  • the proteins in the porcine serum were fractionated via 10%SDS-PAGE gels and then transferred electrophoretically onto polyvinylidene fluoride membranes (Millipore) .
  • the membranes were blocked with 5%milk in TBST for 2 hours before incubation with the primary antibody for 2 hours (mouse anti-human albumin antibody, Sigma–Aldrich, USA) .
  • the membranes were washed thrice with TBST, incubated for 2 hours at room temperature with HRP-conjugated secondary antibodies, and then washed again.
  • the signal was visualized with ECL plus (Amersham) in accordance with the manufacturer’s instructions.
  • liver tissues obtained from the sacrificed wild-type and heterozygous piglets were fixed in 4%paraformaldehyde for 2 days.
  • the fixed tissues were subsequently dissected, embedded in paraffin wax, and then cross-sectioned at 3 ⁇ M.
  • the sections were deparaffinized with xylene and then rehydrated with a graded series of alcohol (100%, 90%, 80%, 70%, and 50%) , followed by H 2 O.
  • a graded series of alcohol (100%, 90%, 80%, 70%, and 50%) , followed by H 2 O.
  • H&E staining the rehydrated sections were stained with hematoxylin and eosin, differentiated, and then cover-slipped.
  • Porcine blood was obtained from albumin humanized pigs and then rested at 4 °C overnight. The blood was centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was removed and transferred into a new tube. About 1 mL of saturated ammonium sulfate, 800 ⁇ L of H 2 O were added into 200 ⁇ L of supernatant; they were mixed gently by inverting the tube. The mixture was rested for 10 min at room temperature and then centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was decanted and then transferred into a new tube. Solid ammonium sulfate was added to the supernatant slowly until 100%saturation was achieved.
  • the purified albumin from porcine plasma was tested by Sun Yat-sen University. The details were performed using a Thermo Scientific Q Exactive mass spectrometer in accordance with the manufacturer’s instructions.
  • the e-PCR program downloaded from the National Center for Biotechnology Information website ( http: //www. ncbi. nlm. nih. gov/sutils/e-pcr/ ) was used to identify potential off-target sites in the porcine genome.
  • the criteria for identifying off-target sites were up to 12 mismatches, 4-bp gaps in the two effector binding elements EBEs, and ⁇ 100 bp between the two putative off-target sites. A total of 1492 potential off-target sites were identified from those examined.
  • the sites with the spacer region within the range of 38–60 bp for a total of 42 sites were amplified and sequenced.
  • a gene-targeting donor containing recombinant human albumin cDNA, puromycin-resistance gene, and a 4.5 Kb 5′ arm/3.4 Kb 3′ arm for homologous recombination was designed and constructed.
  • the HSA expression is regulated by porcine albumin promoter, and the endogenous porcine albumin expression is disrupted by puromycin-resistance gene (FIG. 1A) .
  • the puromycin-resistance cassette for selection was flanked by two loxP sites, and thus can be removed by Cre recombinase to address the safety concern for future clinical applications.
  • FIG. 1B Two pairs of TALENs targeting the exon I and intron I of porcine albumin were designed and assembled according the Golden Gate assembly method (FIG. 1B) .
  • the activities of the TALENs were validated through the single-strand annealing (SSA) assay.
  • the activity of pALB-TALEN1 (39.4%) was slightly higher than that of pALB-TALEN2 (33.9%) (FIGS. 1C, 1D) .
  • pALB-TALEN1 was chosen to target pALB in pig fibroblasts.
  • Porcine fetal fibroblast (PFF) cells isolated from an E35 Yorkshire pig fetus were used for gene targeting modification.
  • the targeting donor was cotransfected with pALB-TALEN1 into PFFs via electroporation.
  • the transfected cells were then plated at a low-density on 10 cm culture dishes at 37 °C/5%CO 2 . After 48 hours, 1 ⁇ g/mL puromycin was added to select the targeted colonies. Approximately 10 days later, 770 cell colonies were selected and expanded. Two colonies (0.26%, 2/770) contained cells with the correct target as confirmed by 5′-and 3′-arm PCR analysis (FIG. 1E, Table 1) . In both positive colonies (491#, 757#) , knock-in mutation occurred in one allele, and the other allele remained intact. NHEJ-mediated mutation was detected in some colonies without knock-in mutation (FIG. 1E) .
  • Table 1 Summary of pALB-TALEN 1#-mediated albumin humanization at porcine albumin locus via HDR.
  • the two targeted cell lines were used as donors for SCNT.
  • a total of 3012 (1692 from 491#cell colony and 1320 from 757#cell colony) reconstructed embryos were generated and transferred into 14 surrogate mothers (Table 2) .
  • Five surrogates were confirmed pregnant through ultrasound examination at one-month post-transfer. These pregnant surrogates were all developed to term and gave birth to nine male cloned piglets (FIG. 2A, Table 2) .
  • Two piglets died at birth, and three died 2 days after birth.
  • Genomic DNA extracted from all of the nine cloned piglets was used to perform 5′-and 3′-arm PCR analysis, followed by Sanger sequencing.
  • Eight piglets (three live piglets and five dead ones) were confirmed to have one allele with knock-in mutation and the other that remained intact (FIG. 2B) , which is exactly consistent with that of cells used for nuclear donors.
  • Table 2 Summary of somatic cell nuclear transfer results for generating albumin humanized pigs.
  • potential off-target sites were predicted by employing the e-PCR program ( www. ncbi. nlm. nih. gov/sutils/e-pcr ) in scanning the porcine genomic sequence. Using previously reported criteria, 42 potential off-target sites (Table 4) were identified. Genomic DNA extracted from all cloned piglets was used as a PCR template to amplify the potential off-target regions. DNA sequencing results suggested that no mutations occurred in any of the potential off-target sites in all of the eight cloned piglets.
  • the three founder pigs with correct mutation grew up to six maturation age, but one died a year after birth.
  • One transgenic founder pig was mated with two wild-type sows.
  • seven (three male and four female) of the 17 offspring harbored the HSA gene, as confirmed by PCR analysis (FIGS. 2D, 2E) .
  • the serum of the three lived heterozygous founders and seven F1 offspring contained a distinct protein band of 66 KDa, which is exactly the same as that in standard HSA (66 KDa) (FIGS. 2C, 2F) . No band with a similar size was found in the wild-type serum samples.
  • HSA expression level in transgenic pigs was further determined through qualitative Western blot analysis. Approximately 4 ⁇ L and 8 ⁇ L of diluted serum (1: 10 in PBS) from two piglets (one founder, one offspring) , and 0.2, 0.4, 0.8, 1, 2, and 4 ⁇ g of standard HSA (Sigma) were loaded for SDS-PAGE.
  • LC/MS was then performed to further confirm the identity of HSA in the serum of the cloned and F1 piglets.
  • Albumin was extracted by ammonium salt precipitation of pooled blood collected from the albumin humanized and wild-type piglets. About 0.5 mg of standard HSA was used as a positive control. A strong protein band of approximately 66 KDa, which was the same as that of the positive control, was illustrated in the SDS-PAGE of the plasma samples from albumin humanized pigs (FIG. 3C) . The 66 KDa protein band was collected and purified through native-PAGE. LC/MS assays were performed on the purified albumin obtained from albumin humanized pigs to confirm its primary sequence (FIG. 3D) . Two types of peptide sequences were identified in the albumin extracted from the plasma of our transgenic piglets, which corresponded to the expected human and porcine albumin sequence, respectively (FIG. 3E) .
  • Table 3 Hematological analysis of pigs among wildtype and albumin humanized pigs (founders and F1 generation) .
  • F1 male pigs with correct mutation grew up to maturation age and was mated with two F1 sows (heterozygote) .
  • PCR analysis confirmed that nine (four male and five female) of the twenty-seven offspring harbored the HSA gene in two alleles (homozygote) .
  • the heterozygous and homozygous piglets were healthy and grew normally.
  • One 50-day-old wildtype piglet (2#) , one 50-day-old heterozygous piglet (4#) and one 50-day-old homozygous piglet (7#) were sacrificed to retrieve the livers and to verify whether the mutant piglets could express HSA.
  • the whole mRNA was extracted from the livers of the three piglets.
  • HSA-mRNA was amplified through RT-PCR and sequencing. Our results showed that, the heterozygote contained both porcine and HSA mRNAs, while the homozygote merely transcribed the HSA mRNA.
  • the serum of the wildtype and heterozygote contained a distinct protein band of 66 KDa, which is exactly the same as that in standard porcine serum albumin (66 KDa) , but no band with a similar size was found in the homozygote serum samples.
  • 66 KDa porcine serum albumin
  • HSA expression level in heterozygous pigs was further determined through human Albumin BCG (BROMOCRESOL GREEN) ALBUMIN ASSAY kit.
  • the expression level of human albumin in one-week-old homozygous piglets were 8-13.6 g/L; fifty-day-old homozygous piglets were 8-20.6 g/L, five-month-old homozygous piglets were 8-30 g/L.
  • SEQ ID NO: 8 coding sequence of human albumin gene
  • SEQ ID NO: 9 ALB-2-1 polynucleotide sequence including intron 1 and intron 2 of as well as exons of human albumin gene
  • SEQ ID NO: 10 ALB-2-2 polynucleotide sequence including intron 1 and intron 2 of pig albumin gene as well as exons of human albumin gene
  • SEQ ID NO: 11 ALB-3-1 polynucleotide sequence including intron 1 and intron 2 of human albumin gene as well as optimized exons of human albumin gene
  • SEQ ID NO: 12 ALB-3-2 polynucleotide sequence including intron 1 and intron 2 of pig albumin gene as well as optimized exons of human albumin gene
  • SEQ ID NO: 14 Porcine Albumin-5-ARM

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Abstract

L'invention concerne des mammifères humanisés génétiquement exprimant l'albumine humaine, ainsi que des utilisations associées. Dans certains modes de réalisation, le génome du mammifère transgénique non humain comprend une séquence polynucléotidique codant pour l'albumine humaine, qui peut comprendre une modification du gène de l'albumine humaine.
PCT/CN2016/104956 2016-11-08 2016-11-08 Mammifères humanisés génétiquement exprimant l'albumine sérique humaine et utilisations associées Ceased WO2018085967A1 (fr)

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PCT/CN2016/104956 WO2018085967A1 (fr) 2016-11-08 2016-11-08 Mammifères humanisés génétiquement exprimant l'albumine sérique humaine et utilisations associées
CN201710014128.7A CN106866813A (zh) 2016-11-08 2017-01-06 人源化基因编辑哺乳动物的制备方法及其用途

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CN116004645A (zh) * 2023-01-13 2023-04-25 新乡医学院 一种利用昆虫细胞生产人血清白蛋白的方法、核苷酸序列及表达载体

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US5633076A (en) * 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
WO2000061725A1 (fr) * 1999-04-14 2000-10-19 Genzyme Transgenics Corp. Purification de proteines heterologues
WO2002083897A1 (fr) * 2001-04-18 2002-10-24 Gene Stream Pty Ltd Animaux transgeniques destines a des etudes pharmacologiques et toxicologiques
EP1557084A2 (fr) * 2002-10-21 2005-07-27 Centro De Ingenieria Genetica Y Biotecnologia Methode de production de proteines de recombinaison dans la glande mammaire de mammiferes non transgeniques
CN105985981A (zh) * 2015-02-05 2016-10-05 安泰吉（北京）生物技术有限公司 人血清白蛋白在哺乳动物细胞中的高效表达

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Publication number Priority date Publication date Assignee Title
US5633076A (en) * 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
WO2000061725A1 (fr) * 1999-04-14 2000-10-19 Genzyme Transgenics Corp. Purification de proteines heterologues
WO2002083897A1 (fr) * 2001-04-18 2002-10-24 Gene Stream Pty Ltd Animaux transgeniques destines a des etudes pharmacologiques et toxicologiques
EP1557084A2 (fr) * 2002-10-21 2005-07-27 Centro De Ingenieria Genetica Y Biotecnologia Methode de production de proteines de recombinaison dans la glande mammaire de mammiferes non transgeniques
CN105985981A (zh) * 2015-02-05 2016-10-05 安泰吉（北京）生物技术有限公司 人血清白蛋白在哺乳动物细胞中的高效表达

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LIANG, ZHENXIN ET AL.: "Progress and Technology in Mammary Gland Bioreactor of Transgenic Animals", CHINA BIOTECHNOLOGY, vol. 35, no. 2, 31 December 2015 (2015-12-31), pages 92 - 98, XP055484391 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116004645A (zh) * 2023-01-13 2023-04-25 新乡医学院 一种利用昆虫细胞生产人血清白蛋白的方法、核苷酸序列及表达载体

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