WO2001072834A1 - Genes marqueurs selectionnables - Google Patents

Genes marqueurs selectionnables Download PDF

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Publication number
WO2001072834A1
WO2001072834A1 PCT/EP2001/003543 EP0103543W WO0172834A1 WO 2001072834 A1 WO2001072834 A1 WO 2001072834A1 EP 0103543 W EP0103543 W EP 0103543W WO 0172834 A1 WO0172834 A1 WO 0172834A1
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cells
mmusk
musk
nucleic acid
antibody
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PCT/EP2001/003543
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English (en)
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Susanne Dagmar Pippig
Gabor Veres
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Novartis Ag
Novartis-Erfindungen
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Priority to JP2001571765A priority Critical patent/JP2003533183A/ja
Priority to EP01940269A priority patent/EP1272522A1/fr
Priority to AU7390601A priority patent/AU7390601A/xx
Priority to NZ521634A priority patent/NZ521634A/en
Priority to AU2001273906A priority patent/AU2001273906B2/en
Priority to CA002403852A priority patent/CA2403852A1/fr
Priority to IL15199601A priority patent/IL151996A0/xx
Publication of WO2001072834A1 publication Critical patent/WO2001072834A1/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • This invention relates to a method of identifying genetically modified mammalian cells, particularly human cells using a muscle specific tyrosine kinase receptor molecule (MuSK-R) or a mutated MuSK (mMuSK-R) thereof as a selectable cell marker.
  • a muscle specific tyrosine kinase receptor molecule MuSK-R
  • mMuSK-R mutated MuSK
  • selectable markers are well known for the identification of prokaryotic and eukaryotic cells, and the use of these markers is essential because frequently when a DNA sequence of interest is introduced into a cell it will not necessarily lead to a phenotype that is readily determined.
  • the number of selectable markers used in identifying eukaryotic cells and especially mammalian cells has been limited.
  • selectable markers that conferred drug resistance have been employed (i.e. G-418 and hygromycin).
  • selectable markers that are combined with fluorescence activated cell sorting (FACS) have been used, for example, green fluorescent protein (GFP).
  • FACS fluorescence activated cell sorting
  • FACS fluorescence activated cell sorting
  • antibodies that recognize a cell surface molecule may be coupled to a fluorophore to help identify the cells of interest.
  • NGFR Nerve Growth Factor Receptor
  • Several cell surface molecules have been used as selectable cell markers including murine CD8, CD24, and human Low- Affinity Nerve Growth Factor Receptor (NGFR).
  • NGFR Nerve Growth Factor Receptor
  • Cell surface selectable markers offer an advantage over drug resistance cell markers in that identification and selection of the genetically modified cells may be performed in a shorter time frame.
  • a selectable marker is a human protein it may prevent an immune reaction in a human treated with cells expressing the selectable marker. Therefore, it is an object of the present invention to provide a method of identifying genetically modified mammalian cells with a cell surface receptor molecule wherein the cell surface receptor would function as a selectable marker, would have a restricted expression pattern, would not be active in the target cell, and could be identified and selected with anti-marker antibodies. This object has been accomplished by a method of identifying genetically modified cells expressing a MuSK-R or a mMuSK- R.
  • the invention provides for a method of identifying genetically modified mammalian cells comprising introducing a nucleic acid sequence encoding a mutated muscle specific tyrosine kinase receptor (mMuSK-R) operatively linked to a promoter into a mammalian cell to form a genetically modified cell; allowing expression of the mMuSK- R in the genetically modified cell; and identifying the cells expressing the mutant MuSK-R.
  • the mMuSK-R is a MuSK-R sequence having at least 150 amino acids deleted from the intracellular domain.
  • the mMuSK-R is a MuSK-R sequence having the kinase catalytic site deleted.
  • a leader sequence is added to the mMuSK-R.
  • a preferred mMuSK-R is derived from the hMuSK-R sequence illustrated in SEQ ID NO. 1 and SEQ ID NO. 2.
  • the mMuSK-R is mMuSK-RI or mMuSK-RII.
  • the identifying step is accomplished by contacting the genetically modified cells with an antibody.
  • the nucleic acid sequence encoding the mMuSK-R is introduced into the mammalian cells by a vector, preferably a retroviral vector.
  • Hematopoietic cells are the preferred target cells, particularly hematopoietic stem cells and T-cells.
  • the invention provides a vector comprising a nucleic acid sequence encoding a mMuSK-R operatively linked to a promoter wherein the mMuSK-R is derived from the sequence ser forth in SEQ ID NO. 1 or a sequence substantially similar to said sequence.
  • the mMuSK-R is the molecule designated mMuSK-RI or mMuSK-RII.
  • the invention includes a method of identifying genetically modified human hematopoietic cells comprising introducing a nucleic acid sequence encoding a muscle specific tyrosine kinase receptor (MuSK-R) into a human hematopoietic cell; allowing expression of the MuSK-R in said cells; and identifying the genetically modified hematopoietic cells from the non-modified hematopoietic cells.
  • a method of identifying genetically modified human hematopoietic cells comprising introducing a nucleic acid sequence encoding a muscle specific tyrosine kinase receptor (MuSK-R) into a human hematopoietic cell; allowing expression of the MuSK-R in said cells; and identifying the genetically modified hematopoietic cells from the non-modified hematopoietic cells.
  • MoSK-R muscle specific tyrosine kinase receptor
  • the invention provides a method of identifying genetically modified human hematopoietic cells comprising incorporating a nucleic acid sequence encoding a mMuSK-R into a population of human hematopoietic cells; introducing a heterologous DNA sequence which encodes a protein of interest into the population of human hematopoietic cells; allowing expression of the mMuSK-R in said cells; and identifying the genetically modified cells expressing the mMuSK-R.
  • the heterologous DNA sequence encoding the protein of interest and the nucleic acid sequence encoding the mMuSK-R are introduced into the cells on the same vector, preferably a retroviral vector.
  • a further aspect of the invention pertains to a method for the immunoselection of transduced mammalian cells comprising transducing cells with a nucleic acid sequence encoding a mMuSK-R; incubating the cells with an antibody which recognizes and binds specifically to the mMuSK-R; and identifying the bound transduced cells.
  • Another aspect of the invention includes a method of identifying mammalian cells expressing a protein of interest, comprising introducing into a population of mammalian cells a nucleic acid sequence encoding a mMuSK-R, wherein said mMuSK-R can not effect signal transduction; introducing a heterologous DNA sequence encoding a protein of interest into said population; culturing the mammalian cells under conditions which favor growth and expansion of said cells; and identifying cells which express the mMuSK-R thereby obtaining cells which express the protein of interest.
  • Another aspect of the invention pertains to a method of identifying mammalian cells comprising introducing a nucleic acid sequence encoding a mutated muscle specific tyrosine kinase receptor (mMuSK-R) operatively linked to a promoter into a mammalian cell to form a genetically modified cell; allowing expression of the mMuSK-R; exposing the cells to a monoclonal antibody wherein said antibody recognizes and binds to the cells expressing the mMuSK-R and does not bind to the cells lacking expression of mMuSK-R; and separating the cells that bind to the monoclonal antibody from cells that do not bind to the antibody.
  • mMuSK-R mutated muscle specific tyrosine kinase receptor
  • Figure 1 is a schematic representation of a wild type MuSK-R and a mMuSK-R wherein the cytoplasmic domain has been truncated. A leader sequence has been added to the 5' end of the sequence as a tag.
  • Figure 2 illustrates a MuSK-R designated hMuSK-R and corresponds to the nucleic acid sequence as set forth in SEQ ID NO: 1 and the amino acid sequence as set forth in SEQ ID NO: 2.
  • the signal peptide includes amino acid residues 1 - 19.
  • the extracellular domain is represented by amino acid residues 20 - 493.
  • the transmembrane domain includes amino acid residues 494 - 515, and the cytoplasmic domain includes amino acid residues 516 - 869.
  • Figure 3 is a schematic representation of the pSeqTag2bhMuSK-R.
  • Figure 4 illustrates the expression of MuSK-R on CEMSS cells and CEMSS MuSK-R cells using the monoclonal antibodies HI (B.), H2 (C.) and H4 (D.).
  • Figure 5 illustrates expression of hMuSK-R (SEQ ID NO: 1) in nontransduced CEMSS cells (A.) and the expression of hMuSK-R (B.) and mMuSK-RII (D.) on CEMSS cells transduced with PPA-6 supernatants that express hMuSK-R or mMuSK-RII respectively. Both popuations were enriched after immuno-magnetic bead selection using monoclonal antibody H2 as illustrated for hMuSK-R (C.) and mMuSK-RII (E.).
  • a stem cell includes a plurality of stem cells.
  • the selectable marker of the instant invention is a muscle specific tyrosine kinase receptor molecule (MuSK-R) or a mutation thereof (mMuSK-R). MuSK-R is believed to initiate the formation of neuromuscular junctions in response to agrin (Glass, et al. Cell 85:513 (1996)).
  • the domain structure of a MuSK-R is schematically illustrated in Figure 1.
  • MuSK-R is comprised of a signal sequence or leader sequence that targets the protein to the secretory pathway.
  • the extracellular domain follows the signal sequence. This domain is made up of several hundred amino acids, and while the exact number of amino acid residues vary, typically the extracellular domain includes around 500 amino acids.
  • the extracellular domain is the part of the receptor that normally projects from the cell into the extracellular environment and includes a ligand binding region.
  • the extracellular domain is one of the most distinctive features of the kinase receptors.
  • the extracellular domain contains immunoglobulin-like (Ig-like) regions. Typically four Ig-like regions are found. However there are reports of MuSK-Rs with three Ig-like regions.
  • the extracellular domain may include 6 contiguous cysteine residues known as a C6-box. While the location of the C6-box may vary depending on the particular MuSK-R, in certain MuSK-Rs it is found approximately at amino acid residues 373 - 382.
  • the transmembrane domain is generally localized in the cell membrane and consists of a stretch of hydrophobic residues followed by several basic residues.
  • the intracellular domain (used interchangeably with the cytoplasmic domain) includes the catalytic part of themolecule and is positioned within the cell.
  • MuSK-Rs are also known in the art as denervated muscle kinase receptors and have been referred to as DmKs (see U.S. Pat. No. 5,656,473 and particularly SEQ ID NOS: 16 and 17 therein). MuSK-R sequences have been isolated and identified from humans, rats, mice, and xenopus.
  • MuSK-Rs available from public depositories such as GeneBank and ATCC include accession numbers: NM005592; AF006464; A448972; AI800924; AI700028; AI341265; AI341122; AI302067; U34985; AA448972; and ATCC 75498. As mentioned above MuSK-R is specific to the skeletal muscle lineage.
  • MuSK-R as used in the present specification and claims is broadly defined to include the known MuSK-Rs (including DmK receptors), isoforms or variants of known MuSK-Rs having similar structure, tyrosine kinase receptors that are functionally similar to known MuSK-Rs and novel MuSK-Rs not previously described that are identified using screening techniques well known to those in the art. Such techniques may include the use of degenerate oligodeoxyribonucleotide primers. Accordingly, the term MuSK-R when referring to a nucleic acid molecule includes
  • MuSK-R includes not only naturally occurring MuSK-Rs but also may include genetically engineered MuSK-Rs.
  • MuSK-R or “mMuSK-R” refer to nucleic acid sequences or protein as appropriate from context.
  • Polynucleotides or nucleic acids of the invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA or synthetic DNA.
  • MuSK-R when referring to a polypeptide encompasses known MuSK receptors, isoforms or variants of MuSK-Rs, and functionally equivalent receptors.
  • a functionally equivalent receptor is a MuSK-R that can compete with a known MuSK-R for binding. More specifically, a functionally equivalent MuSK-R has at least 40%, preferably at least 60%, and more preferably at least 80% identical amino acids to the sequence set forth in SEQ ID NO:2 and can compete with the MuSK-R illustrated in SEQ ID NO: 2 for ligand or substrate binding.
  • MuSK-R or mMuSK-Rs are used as selective markers to identify genetically modified cells.
  • the marker is introduced on a nucleic acid construct into a target cell that normally does not express a MuSK-R.
  • introduction is broadly used herein to include inserted, incorporated and the like.
  • the MuSK-R or mMuSK-Rs are used as selectable markers the molecule no longer possesses signaling activity.
  • Signaling activity has be generally defined as triggering a response pathway in the cytosol to the nucleus which ultimately leads to activation of transcription.
  • the lack of signaling activity may be due to a) use of a MuSK-R in tissue or cells other than muscle (Glass et al., Cell 85:513-523 (1996)) or b) use of a mMuSK-R
  • MuSK-R While modifications of MuSK-R may be known, the method of identifying genetically modified cells comprising using a MuSK-R or mMuSK-R as a selectable marker is not known.
  • MuSK-R may be used as a selective marker in tissue other than muscle.
  • the selectable marker of the invention is a mMuSK-R.
  • the modifications to a MuSK-R encompassing mMuSK-Rs include truncations and/or deletions of MuSK-Rs.
  • the mutation may occur in the extracellular domain and/or the intracellular domain by means well known in the art. The mutation causes the molecule to be devoid of signaling activity. Preferably the extracellular domain should still be capable of binding an antibody. In general the smallest peptide fragment of the extracellular domain capable of binding an antibody would be approximately 15 amino acid residues, more preferably at least 50 amino acid residues.
  • a preferred MuSK-R according to the invention is the sequence set forth in SEQ ID NOs: 1 and 2, designated herein as hMuSK-R.
  • the extracellular domain is encoded by nucleotides 1 through 1479
  • the transmembrane domain is encoded by nucleotides 1480 through 1545
  • the intracellular domain is encoded by nucleotides 1546 through 2607.
  • Other preferred MuSK-Rs are molecules closely related to the sequences set forth in SEQ ID NOS: 1 and 2. Examples of closely related sequences are the sequences set forth in U.S. Pat No. 5,656,473 particularly SEQ ID NOS: 16 and 17.
  • MuSK-Rs Mutants of MuSK-R (mMuSK-Rs) are known and reference is made to Apel et al., Neuron 18:623 - 635 (1997).
  • preferred modifications to a MuSK- R include modifications to the cytoplasmic domain such as deletions of at least 150, preferably at least 200, more preferably at least 250, more preferably 300, and most preferably at least 350 amino acids of the cytoplasmic domain.
  • the deletions are preferably truncations. Deletions or truncations may include deletion of tyrosine phosphorylation sites in the range of 1 to 19, preferably 2 - 15, more preferably 2 - 10 sites. Additionally the kinase catalytic site may be deleted from a MuSK-R.
  • the kinase catalytic site is found at approximately amino acid residues 672 to 691 of SEQ ID NO.2. As long as the protein is stably expressed, there is no limitation to the number of sequences deleted or truncated in the cytoplasmic domain.
  • mMuSK-Rs useful as selectable markers according to the invention include modifications to the MuSK-R sequence set forth in Figure 2 (SEQ ID NO:2).
  • the MuSK-R is truncated by least 300 amino acid residues in the cytoplasmic domain.
  • One preferred embodiment includes the deletion of amino acid sequence 538 - 869 and is designated mMuSK-RI.
  • Another preferred embodiment includes the deletion of amino acid sequence 577 - 869 and is designated mMuSK-RII.
  • the extracellular domain modification may include deletion of at least about 100 amino acids, preferably at least about 150 amino acids, more preferably at least about 200 amino acids, and still more preferably at least about 250 amino acids.
  • a MuSK-R or mMuSK-R used as a selectable marker according to the invention preferably should contain an antibody-binding site in the extracellular domain.
  • Random methods encompass altering the sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, using chemicals to damage plasmid DNA, and incorporating incorrect nucleotides during in vitro DNA synthesis.
  • site-directed mutagenesis may be a more beneficial tool.
  • Particularly preferred site-directed methods include oligonucleotide-directed mutagenesis and polymerase chain reaction (PCR) amplified oligonucleotide mutagenesis.
  • PCR polymerase chain reaction
  • MuSK-R or mMuSK-R as a selectable marker concerns the ability to select genetically modified cells in vitro, ex vivo and in vivo.
  • the MuSK-R or mMuSK-R may be introduced into a target cell as part of a nucleic acid construct operatively linked to a promoter
  • the selectable marker of the invention is placed in a vector and then introduced into a target cell.
  • operatively linked refers to an arrangement of elements wherein the components are configured so as to perform their usual function.
  • a promoter or other control elements need not be contiguous with the coding sequence.
  • a promoter is well within the skill of one in the art and extends to any prokaryotic, eukaryotic or viral promoter capable of directing gene transcription in a target cell modified with a selectable marker of the invention.
  • the promoter may be a tissue specific promoter, inducible promoter, synthetic promoter or hybrid promoter. More than one promoter may be used.
  • promoters include but are not limited to; the phage lamda (PL) promoter; SV40 early promoter; adenovirus promoters, such as adenovirus major late promoter (Ad MLP); he ⁇ es simplex (HSV) promoter; a cytomegalovirus (CMV) promoter, such as human CMV immediate early promoter; a long terminal repeat (LTR) promoter, such as MoMLV LTR; the U3 region promoter of the Moloney murine sarcoma virus; Granzyme A promoter; regulatory sequences of the metallothionin gene; CD34 promoter; CD8 promoter; thymidine kinase (TK) promoters, B19 parvo virus promoters; PGK promoter; and rous sarcoma virus (RSV) promoter.
  • PL phage lamda
  • SV40 early promoter such as adenovirus major late promoter (Ad MLP); he ⁇ e
  • promoter elements from yeast and other fungi may be used such as Gal 4 promoter and the alcohol dehyrodenase (ADH) promoter. These promoters are available commercially from various sources such as Stratagene (La Jolla, CA). It is to be understood that the scope of the present invention is not to be limited to a specific promoter.
  • Preferred promoters include LTR promoters such as the 5' LTR promoter of MoMLV, MSCV and HIV, and CMV promoters.
  • other expression control sequences may be inco ⁇ orated into the nucleic acid constructs used for identifying genetically modified cells according to the invention.
  • RNA polymerase binding sequences sequences conferring inducibility of transcription and other expression control elements such as scaffold attachment regions (SARs).
  • SARs scaffold attachment regions
  • Vectors containing both a promoter and a cloning site into which a polynucleotide sequence can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
  • vectors include vectors derived from viruses, such as baculovirus, retroviruses, adenoviruses, adeno-associated viruses, and he ⁇ es simplex viruses; bacteriophages; cosmids; plasmid vectors; fungal vectors; synthetic vectors; and other recombination vehicles typically used in the art. These vectors have been described for expression in a variety of eukaryotic and prokaryotic hosts and may be used for simple protein expression.
  • vectors include pSG, pSV2CAT, and pXtl from Stratagene and pMSG, pSVL, pBPV and pSVK3 from Pharamacia.
  • Other exemplary vectors include the pCMV mammalian expression vectors, such as pCMV6b and pCMV ⁇ c (Chiron Co ⁇ oration), pSFFV-Neo, and pBluescript-SK-
  • pCMV mammalian expression vectors such as pCMV6b and pCMV ⁇ c (Chiron Co ⁇ oration), pSFFV-Neo, and pBluescript-SK-
  • consensus ribosome binding sites can be inserted 5' or 3' to the selective marker to enhance expression.
  • Retroviral vectors are retroviral vectors and reference is made to Coffin et al., "Retroviruses", ( 1997) Chapter 9 pp; 437-473 Cold Springs Harbor Laboratory Press. Retroviral vectors useful in the invention are produced recombinantly by procedures already taught in the art. W094/29438, W097/21824 and WO97/21825 describe the construction of retroviral packaging plasmids and packing cell lines. Common retroviral vectors are those derived from murine, avian or primate retroviruses. The most common retroviral vectors are those based on the Moloney murine leukemia virus (MoMLV) and mouse stem cell virus (MSCV).
  • MoMLV Moloney murine leukemia virus
  • MSCV mouse stem cell virus
  • Vectors derived from MoMLV include, Lmily, LINGFER, MINGFR, MND and MINT (Bender et al., J. Virol. 61 : 1639 - 1649 (1987); Miller et al., Biotechniques 1: 980 - 990 (1989); Robbin et al. J.Virol., 71 :9466-9474 (1997); and U.S. Pat. No. 5,707,865).
  • Vectors derived from MSCV include MSCV-MiLy (Agarwal et al., J. of Virology 72:3720).
  • vectors include those based on Gibbon ape leukemia virus (GALV), Moloney murine sacroma virus (MoMSV), myeloproliferative sarcoma virus (MPSV); murine embryonic stem cell virus (MESV), spleen focus forming virus (SFFV), and the lentiviruses, such as human immunodeficiency virus (HIV-1 and HIV-2).
  • GALV Gibbon ape leukemia virus
  • MoMSV Moloney murine sacroma virus
  • MPSV myeloproliferative sarcoma virus
  • MMV murine embryonic stem cell virus
  • SFFV spleen focus forming virus
  • New vector systems are continually being developed to take advantage of particular properties of parent retroviruses such as host range, usage of alternative cell surface receptors and the like (C. Baum et al , Chap 4 in Gene Therapy of Cancer Cells eds. Lattime and Gerson (1998)).
  • the present invention is not limited to particular retroviral vectors, but may include any retroviral vector.
  • Particularly preferred vectors include DNA from a murine virus corresponding to two long terminal repeats, and a packaging signal.
  • the vector is a MoMLV or MSCV derived vector and particularly MND (U.S. Pat. No. 5,707,865 and Norris et al., J. Virol. Methods, 75:161 - 167 (1998)).
  • the viral gag, pol and env sequence will generally be removed from the virus, creating room for insertion of foreign DNA sequences.
  • Genes encoded by foreign DNA are usually expressed under the control a strong viral promoter in the long terminal repeat (LTR). While the LTR promoter is preferred, as mentioned above numerous promoters are known.
  • LTR long terminal repeat
  • Non-limiting preferred vector constructs according to the present invention include the general structure as outlined below in the 5' to 3' direction:
  • CMV-X-pmMuSKR-LTR wherein LTR is a long terminal repeat, X is a heterologous gene for a desired protein, mMuSKR is a selectable marker, p is a second promoter; I is an internal ribosomal binding site, SAR is a scaffold attachment region, and CMV is a cytomegalovirus promoter.
  • Such a construct can be packaged into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Therefore when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences. Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced. Alternatively the packaging cell line harbors a provirus.
  • RNA produced from the recombinant virus is packaged instead. Therefore, the virus stock released from the packaging cells contains only recombinant virus.
  • retroviral packaging lines include PA12, PA317, FLYA13, PE501 , PG13, ⁇ CRIP, RD1 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67. Reference is made to Miller et al., Mol. Cell Biol.
  • Retroviral vector DNA can be introduced into packaging cells either by stable or transient transfection to produce vector particles.
  • vectors include adenoviral vectors (Frey et al., Blood 91 :2781 (1998) and WO95/27071 ) and adeno-associated viral vectors (Chatterjee et al., Current Topics in Microbiol. and Immunol. 218:61 (1996). Reference is also made to Shenk, Chapter 6, 161 - 178, Breakefield et al., Chapter 8 201-235; Kroner-Lux et al., Chapter 9, 235 - 256 in Stem Cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998 and U.S. Pat. Nos. 5,693,531 and 5,691,176.
  • adenovirus derived vectors may be advantageous under certain situations because they are capable of infecting non-dividing cells, and unlike retroviral DNA, the adenoviral DNA is not integrated into the genome of the target cell. Further the capacity to carry foreign DNA is much larger in adenoviral vectors than retroviral vectors.
  • the adeno-associated viral vectors are another useful delivery system. The DNA of these viruses may be integrated into non-dividing cells, and a number of polynucleotides have been successfully introduced into different cell types using adeno-associated viral vectors. These vectors are capable of transducing several cell types including hematopoietic cells and epithelial cells.
  • the construct or vector will include not only a nucleic acid sequence encoding a MuSK-R or mMuSK-R as a selective marker but also a second nucleic acid sequence encoding a protein of interest to be introduced into a target cell.
  • the nucleic acid molecules are DNA.
  • a protein of interest is broadly defined and includes for example, a therapeutic protein, a structural gene, a ribozyme, or an antisense sequence.
  • the structural protein or gene may be the entire protein or only the functionally active fragment thereof.
  • the protein may include for example one that regulates cell differentiation or a therapeutic gene capable of compensating for a deficiency in a patient that arises from a defective endogenous gene.
  • Gene means a nucleic acid molecule the sequence which includes all the information required for the normal regulated production of a particular protein including the structural coding sequence.
  • a therapeutic protein or gene may be one that antagonizes production or function of an infectious agent, antagonizes pathological processes, improves a host's genetic makeup, or facilitates engraftment.
  • a therapeutic gene or gene sequences are ones effective in the treatment of adenosine deaminase deficiency (ADA); sickle cell anemia; recombinase deficiency; recombinase regulatory gene deficiency; HIV such as an antisense or trans- dominant REV gene or a gene carrying a he ⁇ es simplex virus thymidine kinase (HSV-tk)).
  • the second nucleic acid sequence may encode new antigens; drug resistant genes; a toxin; an apoptosis inducer effective to specifically kill cancerous cells; or a specific suicide gene.
  • the therapeutic gene may be a non-human gene, for example a yeast gene (Seo et al., Proc. Natl. Acad. Sci. 95:9167 (1998)).
  • the vector or construct may also comprise, besides the second nucleic acid sequence encoding a protein of interest, a further DNA sequence. More than one gene may be necessary for the treatment of a particular disease. Alternatively more than one gene can be delivered using several compatible vectors. Depending on the genetic defect, the therapeutic gene can include regulatory and untranslated sequences. For human patients the therapeutic gene will generally be of human origin although genes of closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used if the gene does not produce an adverse immune reaction in the recipient.
  • Nucleotide sequences for the protein of interest or a further DNA sequence will generally be known in the art or can be obtained from various sequence databases such as GeneBank.
  • GeneBank One skilled in the art will readily recognize that any structural gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the structural gene in target cells.
  • the target cells of the invention are mammalian cells that do not normally express ta MuSK-R.
  • Mammalian cells include but are not limited to humans, mice, monkeys, farm animals, sport animals, pets, and other laboratory rodents and animals.
  • the target cells are human cells.
  • Preferred human cells include liver, hematopoietic, neural, endothelial vascular cells, tumor cells and epithelial cells. Hematopoietic cells are particularly preferred, and these cells encompass hematopoietic stem cells, erythrocytes, neutrophils, monocytes, platelets, mast cells, eosinophils and basophils, B and T lymphocytes and NK cells as well as the respective lineage progenitor cells.
  • Hematopoietic stem cells and T-cells are especially preferred.
  • Hematopoietic stem cells are defined as a population of hematopoietic cells containing long term mutlilineage repopulating potential.
  • T-cells are defined as a type of lymphocyte and are thought to develop from hematopoietic stem cells. Methods of obtaining target cells and particularly hematopoietic cells are known in the art and not repeated herein.
  • Non-limiting sources of hematopoietic cells, including hematopoietic stem cells are bone marrow, embryonic yolk sac, fetal liver tissue, adult spleen, and blood such as adult peripheral blood and umbilical cord blood. (To et al., Blood 89:2233 (1997)).
  • Bone marrow cells may be obtained from ilium, sternum, tibiae, femora, spine and other bone cavities.
  • target cells may be separated from other cells.
  • Various procedures may be employed and include physical separation, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody.
  • FACS fluorescence activated cell sorters
  • cell separation is not critical to the invention, and specific cell types may be separated either prior to genetic modification with a MuSK-R or mMuSK-R or after genetic modification. Preferably cells are initially separated by a coarse separation followed by using positive and/or negative selection. In humans the surface antigen expression profile of an enriched hematopoietic stem cell population may be identified by CD34 + Thy- 1 + Lin " .
  • Nonlimiting enriched phenotypes may include: CD2 " , CD3 “ , CD4 “ , CD8 ⁇ CD10 “ , CD14 ' , CD15 “ , CD19 “ , CD20 “ , CD33 “ , CD34 “ , CD38 lo -, CD45RA-, CD59 +/” , CD71 “ , CDW109 + , glycophorin " , AC133 + , HLA-DR +/” , and EM + .
  • Lin ' refers to a cell population selected on the basis of lack of expression of at least one lineage specific marker, such as, CD2, CD3, CD14, CD15 and CD56.
  • Murine HSCs may be identified preferably by kit 4 Thy-l .l ,0 Lin “ ⁇ o Sca-l + (KTLS). Other phenotypes are well known. (U.S. Patent No. 5,061 ,620).
  • T cell markers include CD54RA and T cell antigen receptor (TCR), ⁇ , ⁇ -TCR and ⁇ , ⁇ -TCR.
  • B cells may be selected, for example, by expression of CD19 and CD20.
  • Myeloid cells may be selected for example, by expression of CD14, CD15 and CD16.
  • NK cells may be selected based on expression of CD56 and CD16.
  • Erythrocytes may be identified by expression of glycophorin A.
  • Neuronal cells may be identified by NCAM and LNGFR (Baldwin et al., J. Cell Biochem., 15:502 (1996)).
  • Vascular endothelial cells may be identified by VEGFR2, CD34, P-Selectin, VCAM-1, ELAM-1 and ICAM-1 (Horvathova et al., Biol. Trace Elem. Res., 69:15-26 (1999).
  • VEGFR2 vascular endothelial cells
  • CD34 CD34
  • P-Selectin P-Selectin
  • VCAM-1 VCAM-1
  • ELAM-1 ELAM-1
  • ICAM-1 ICAM-1
  • the target cells are cultured in a suitable medium comprising a combination of growth factors that are sufficient to maintain growth.
  • suitable medium comprising a combination of growth factors that are sufficient to maintain growth.
  • Methods for culturing target cells are well known to those skilled in the art., and reference is made to Freshney, R.I. "Culture of Animal Cells, A Manual of Basic Techniques", Wiley-Liss, Inc (1994).
  • Various culture media are commercially available and non -limiting examples include DMEM, IMDM, X-vivo 15 and RPMI-1640.
  • the formulations may be supplemented with a variety of different nutrients and growth factors.
  • Non-limiting examples of supplemental compounds which may be used are TPO, FL, KL, IL-1 , IL-2, IL-3, IL-6, IL-12, IL-1 1, stem cell factor, G-CSF, GM- CSF, Stl factor, MCGF, LIF MlP-l ⁇ and EPO. These compounds may be used alone or in any combination, and preferred concentration ranges may be readily determined from the published art.
  • the medium can be serum free or supplemented with suitable amounts of serum such as fetal calf serum, autologous serum or plasma. If cells or cellular products are to be used in humans, the medium will preferably be serum free or supplemented with autologous serum or plasma (Lansdo ⁇ et al., J. Exp. Med.
  • a preferred non-limiting medium includes mIL-3, mIL-6 and mSCF.
  • Other molecules can be added to the culture media, for instance, adhesion molecules, such as fibronection or RetroNectinTM (Takara Shuzo Co., Otsu Shigi, Japan).
  • the seeding level is not critical and will depend on the type of cells used, but in general the seeding level will be at least 10 cells per ml, more usually at least about 100 cells per ml and generally not more than 10 cells per ml when the cells express CD34.
  • LTCIC long- term culture initiating cell assay
  • CAFC cobblestone-area-forming cell
  • This assay gives frequency readouts that correlate with LTCIC and are predictive of engraftment in in vivo assays and patients.
  • a particularly preferred CAFC assay is described in Young et al., Blood 88:1619 (1996).
  • Flow cytometry can be used to subset hematopoietic cells from various tissue sources by the surface antigens they express.
  • a combination of these assays may be used to test for target cells that are genetically modified according to the invention.
  • the invention concerns a method of identifying genetically modified mammalian cells, particularly human cells comprising introducing a polynucleotide sequence encoding a MuSK-R or mMuSK-R as a selectable marker operatively linked to a promoter into the target cell to form a genetically modified cell; allowing expression of the MuSK-R or mMuSK-R in the genetically modified cell; and identifying said genetically modified cell expressing the MuSK-R or mMuSK-R.
  • the polynucleotide sequence encodes mMuSK-RI, mMuSK-RII or a mMuSK-R derived from the MuSK-R set forth in SEQ ED NO. 1 or a sequence substantially similar to said sequence with minor changes.
  • a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art it can be transcribed and/or translated to reproduce a polypeptide or fragment thereof.
  • a construct or vector including the MuSK-R or mutant thereof may be inco ⁇ orated into the target population by any means of genetic transfer or modification known in the art.
  • genetic modification refers to any addition, deletion or disruption to a cells normal nucleotides and the methods of genetic modification are intended to encompass any genetic modification method of exogenous or foreign gene transfer or nucleic acid transfer into mammalian cells (particularly human hematopoietic cells).
  • the term includes but is not limited to transduction (viral mediated transfer of host DNA from a host or donor to a recipient, either in vivo or ex vivo) and transfection (transformation of cells with isolated DNA genomes), including liposome medicated transfer, electroporation, calcium phosphate coprecipitation and others.
  • transduction viral mediated transfer of host DNA from a host or donor to a recipient, either in vivo or ex vivo
  • transfection transformation of cells with isolated DNA genomes
  • Methods of transduction include direct co-culture of cells with producer cells (Bregni et al., Blood 80:1418 - 1422 (1992)) or culturing with viral supernatant alone with or without appropriate growth factors and polycations (Xu et al., Exp. Hemat. 22:223 - 230 (1994)).
  • the target cells are transduced with a retroviral vector as previously described.
  • the host cell range that may be infected is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable inco ⁇ oration of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodents' cells
  • amphotropic env allows infection of rodent, avian and some primate cells including human cells.
  • VSV- G G-glycoprotein from vesicular stomatitis virus
  • Xenotropic vector systems also exist which allow infection of human cells.
  • the modified cells expressing the MuSK-R or mMuSK-R may be identified by numerous techniques known in the art.
  • Methods of identifying the target cells expressing MuSK-R or mMuSK-Rs include well known techniques such as antibody selection, particularly immunoselection; nucleotide selection by northern blots or by southern blots; PCR amplification of genomic DNA; protein detection by western blots; reverse transcription of mRNA and amplification with PCR; and FISH wherein chromosomes are analyzed by fluorescence in situ hybridization with a liquid phase DNA (Lawrence et al., Science, 249: 928 -932 (1990)).
  • the method of identifying mammalian cells includes exposing the target cells to an antibody wherein the antibody specifically recognizes and binds to the cells expressing the mMuSK-R and does not bind to the cells lacking expression of mMuSK-R. The bound cells are then separated from cells that do not bind to the antibody.
  • Antibodies may be obtained by methods well known in the art and reference is made to Harlow et. Al., "Antibodies: A Laboratory Manual: (1988), Biosupplynet Source Book (1999) Cold Spring Harbor Laboratory Press. Polyclonal antibodies that are reactive to the antigen of interest may be used or monoclonal antibody producing cell clones may be generated.
  • the antibody must recognize the extracellular domain of the MuSK-R or mMuSK-R selectable marker. More particularly if parts of the extracellular domain are modified, for example by deletion, the antibody should recognize an epitope of the remaining amino acid sequence of a mMuSK-R.
  • Particularly preferred antibodies are monoclonal antibodies that specifically recognize and bind to a mMuSK-R derived from or substantial similar to the MuSK-R sequence as set forth in SEQ ID NO.2. These antibodies are referred to as " ⁇ -MuSK-R" and the term encompasses any antibody or fragment thereof, either native or recombinant, synthetic or naturally derived which retains significant specificity to bind to a mMuSK-R derived from or substantially similar to the sequences set forth in SEQ ID NO. and 2.
  • Exemplary of a ⁇ -MuSKR are the monoclonal antibodies referred to as HI, H2 and H4 described in the Example section G and produced by the deposited hybridomas.
  • Hyridomas producing antibodies to mMuSK-RI and mMuSK-RII designated HI , H2 and H4 have been deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard., Manassas, VA 20110 on March 22, 2000 and have been given ATCC Accession Nos. PTA-1547, PTA-1548, and PTA-1549, respectively.
  • the HI monoclonal antibody is most preferred for identifying and further selecting target cells expressing the selective markers.
  • an antibody may be used in the methods according to the invention wherein the antibody binds specifically to an epitope in the extracellular domain as recognized by the antibody HI.
  • the ⁇ -MuSKR may be identified and assayed in vitro by a range of methods known in the art including gel diffusion, immunoassay, immunoelectrophoresis and immunofluorescence. Once the target cells are labeled they can be incubated with the ⁇ - MuSKR.
  • a secondary antibody may also be used to further identify or select antibody coated cells, if the secondary antibody is coupled to either a fluorophore or immuno-magnetic beads.
  • the genetically modified cells expressing the selectable marker may then be selected by flow cytometry including FACS or by using a magnet to select bead-coated cells (U.S. Pat. No. 5,011,912).
  • a primary ⁇ -MuSKR can be conjugated to a fluorophore, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), cy-chrome (CyC), allophycocyanine (APC), tricolor (TC) or Texas Red (TX).
  • a secondary antibody that is conjugated to a fluorophore may be introduced into the cell sample containing the cells that express mMuSK-R and which is recognized by the primary antibody.
  • the primary antibody is attached to the mMuSK-R. Separation may be achieved by the fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • FACS can also be used to separate cells expressing a tag sequence.
  • a tag is a small amino acid sequence of approximately 10 - 20 amino acid which can be recognized by an antibody.
  • tags include, HA (hemagglutinin), myc tag, his tag, and FLAG® (Kunz et al., J. Biol. Chem 267: 91091 (1992)) which may be bound to a primary antibody specific to the tag.
  • Tag products are available commercially. For example, from Eastman Kodak Company, New York.
  • the target cells will be genetically modified with a construct including the mMuSK-R and a polynucleotide sequence encoding a tag polypeptide.
  • the modified cell will express the tagged selective marker at the cell surface.
  • Anti-tag monoclonal antibodies can be used to identify the cells expressing tagged MuSK-R at the cell surface.
  • Anti-FLAG® is described in U.S. Pat. No. 5,01 1 ,912. Reference is also made to U.S. Pat. Nos. 4,703,004, 4,782,137 and 4,851,341 and Brizzard et al., Biotechniques 16:730 (1994).
  • the genetically modified cells identified according to the methods of the invention may be expanded, either prior to or after identification or selection by culturing the cells for days or weeks in appropriate culture media, with or without supplements by means well known in the art.
  • the genetically modified cells identified according to the invention may further be used in an autologous or allogeneic setting wherein the modified target cells, preferably hematopoietic cells, most preferably stem cells or T-cells are expanded and then used in gene therapy for example in bone marrow transplantation, graft facilitation, or immune reconstitution.
  • the expanded cells including the mMuSK-R may be infused into a subject. Samples may be taken and then retested for the MuSK-R or mMuSK-R selectable markers by FACS analysis, PCR or FISH as described above to determine the persistence of the genetically modified cells and further to assess efficiency of transformation, particularly efficiency of transduction.
  • MuSK-R is isolated by PCR from fetal skeletal muscle cDNA (Marathon cDNA,
  • MuSK21FN CGT CCT GCGTGAGCCTGG ATT AAT C SEQ ID NO: 3
  • MuSK34FN GCC TGG ATT AAT CAT GAG AGA
  • MuSK2666RN CGA GGC CTGTCTTCAACCTTAGAC ACT CAC AGTTCC CTCTGC SEQ ID NO: 5
  • the 5' primer MuSK21FN covers 25nucleotide (nt) before the start codon
  • the second 5' primer MuSK34FN covers the start codon (aa 1) of MuSK-R and surrounding sequence.
  • the 3'-primer MuSK2666RN covers the stop codon of MuSK-R and surrounding sequence.
  • 2.5 ⁇ mol dATP 2.5 ⁇ mol dCTP
  • 2.5 ⁇ mol dGTP 2.5 ⁇ mol dGTP
  • 2.5 ⁇ mol TTP 2.5 ⁇ mol TTP
  • 1 ⁇ g primer MuSK21FN 1
  • the PCR is performed as follows: Cycle 1 : 94°C for 5 min, Cycle 2-11 : 94°C for 0.5 min, 63°C for 1 min, 68°C for 6 min, and Cycle 12: 68°C for 10 min.
  • the reaction is cooled to 4°C in the PCR machine, and the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate.
  • the pellet is washed once with 70% ethanol, dried and resuspended in 100 ⁇ l H 2 O.
  • 10 ⁇ l of the above PCR reaction is then reamplified.
  • the reaction mix contains for the second round of amplification step in addition to 10 ⁇ l of the above PCR reaction: Pfu buffer (20mM Tris-HCl (pH8.8), 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH ⁇ SO ⁇ 0.1 % Triton X- 100, 0.1 mg/ml BSA), 2.5 ⁇ mol of each dNTP (dATP, dCTP, dGTP, dTTP), 1 ⁇ g primer MuSK34FN, 1 ⁇ g primer MuSK2666RN, 5 U Pfu Turbo Polymerase (from Pyrococcus furiosus) and water in a final volume of 50 ⁇ l.
  • the PCR is performed as follows: Cycle 13: 94°C for 5 min, Cycle 14-43: 94°C for 0.5 min, 62°C for 1 min, 72°C 6 min, and Cycle 44: 72°C for 10
  • the reaction is cooled to 4°C in the PCR machine and the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate. The pellet is washed once with 70% ethanol, dried and resuspended in 20 ⁇ l H 2 O.
  • the PCR reaction is loaded on a l xTAE gel. A band with the size of ⁇ 2600 bp is isolated from the gel and cloned into the Srfl restriction site of pPCR-Script Amp vector (Stratagene, CA) according to the manufacturer's protocol. The resulting vector is called pPCR-Script MuSK-R-wt. The correctness or the subcloned PCR product is confirmed by restriction analysis and sequencing by methods well known in the art. (The nucleotide sequence is illustrated in SEQ ID NO.1)
  • the primers MuSK1380F, MuSK1657R, and 1747R are used to generate intracellular deletion mutants of MuSK-R from the plasmid pPCRScriptMuSK-R.
  • the primer sequences are as follows wherein p means phosphorylated: Primer 1380F: 5' pCG GCC TGT GCC AGA CTG CCA CAT CTA G (SEQ ID NO:
  • Primer 1657R 5' pCG TCT AGG TGA GGG TTA CTG CTG CTG ATT CTC (SEQ ID NO: 7);
  • Primer 1747R 5' pGG TTA ACC CTA TTC AAT GTT ATT CCT TGA ATA CTC CAG (SEQ ID NO: 8).
  • MuSK1380F and 1657R results in the deletion of amino acid residues 538 - 879 of MuSK-R
  • primer pair MuSK1380F and 1747R results in the deletion of amino acid residues 577 - 879.
  • the two mutant forms of MuSK-R are designated MuSK-R ⁇ 538-879 (MuSK-RI) and MuSK-R ⁇ 577-879 (MuSK-R ⁇ ).
  • MuSK-RI and MuSK-RII most of the intracellular domain of MuSK-R as shown in Figure 2 is deleted. While not meant to limit the invention in any manner, it is believed that both truncations result in a deletion of the kinase domain and most of the substrate binding motifs of the wt MuSK-R illustrated in Figure 2.
  • the 5' primer MuSK1380F covers the nucleotide sequence 1333-1410 of the
  • MuSK-R The 3'-primers MuSK1657R and 1747R contain stop codons in place of amino acid 538 and 577 of MuSK-R.
  • Using primer MuSK1380F with MuSK1657R or MuSK1747R results in the amplification of MuSK-R nucleotide sequence 1333 to 1614 that has a stop codon in the position of amino acid 538 or nucleotide sequence 1333-1728 that has a stop codon in the position of amino acid 577, respectively.
  • the PCR reaction includes -10 ng hMuSK-R wt DNA, 1 x Pfu buffer, 1 ⁇ g of primer MuSK1380F and either 1 ⁇ g primer MuSK1657R or MuSK1747R, 2.5 ⁇ mol of each dNTP, 5 U Pfu polymerase and H 2 0 in a final volume of 50 ⁇ l.
  • the PCR reaction is performed as follows: Cycle 1: 95°C for 5 min, Cycle 2-31: 95°C 0.5 min, 60°C for 1 min, 72°C for 4 min, Cycle 32: 72°C for 10 min.
  • the PCR reaction is cooled to 4°C in the PCR machine and then loaded on a 1 x TAE gel.
  • MuSK-RI nt 1380-1614
  • MuSK-RII nt 1380 - 1728
  • Srfl site Srfl site of pPCR-ScriptAmp (Stratagene)
  • this sequence is excised from the plasmid pPCR-Script MuSK-wt using restriction sites Nael and Aatll.
  • the two pPCR-Script vectors containing the modified MuSK sequence nt 1 - 1614 and 1 - 1726 are called pPCR-Script-MuSK-RI and pPCR- Script MuSK-RII, respectively.
  • the correctness of the vectors are confirmed by restriction analysis and sequencing by methods well known in the art.
  • Wild-type and mutant MuSK-R are excised from pPCRScriptMuSK-Rwt, pPCRScriptMuSK-RI and pPCRScriptMuSK-RII using the Notl and Xhol site and are cloned into the multiple cloning site of the Moloney Murine Leukemia Virus (MoMLV) based retroviral vector pGla (GTI, Maryland) which is cut with Notl and Xhol.
  • the retroviral vectors are designated pGlaMuSK-R, pGlaMuSK-RI and pGlaMuSK-RII.
  • the constructs pGlaMuSK-R, pGl aMuSK-RI and pGlaMuSK-RII are cotransfected into human embryonic kidney cells 293T (293T cells) (Gary Nolan, Stanford) with an envelope construct pCiGL that permits expression of the Vesicular Stomatitis Virus G-Protein (VSV- G envelope) under the control of the cytomegalovirus (CMV) promoter.
  • VSV- G envelope Vesicular Stomatitis Virus G-Protein
  • CMV cytomegalovirus
  • packaging construct pCiGP encoding MoMLV gag-pol under the control of the CMV promoter
  • CaCl technique CaCl technique
  • Viral supernatants are collected 24, 48, and 72 hours after transfection.
  • Supernatants are centrifuged at 1200 ⁇ in a Beckman GS-6KR centrifuge to remove particulate material, and either used immediately to transduce cells or frozen in a dry ice/methanol bath.
  • the viral supernatants are used to transduce the packaging cell line ProPak-A-6 (PPA-6) (Systemix, Inc.).
  • PPA-6 cell line is a derivative of 293T cells expressing the MLV amphotropic envelope and MLV gag/pol stabley under the control of the CMV promoter (Rigg et al. supra).
  • the positively transduced PPA-6 cells are sorted by bead selection (described in section F).
  • Supernatants from PPA-6 cells are collected on day 2, 3 and 4 after transduction and treated as described for 293T cells.
  • the so generated supernatants of PPA-6 cells contain recombinant viral particles that have the amphotropic envelope and are used to transduce human primary cells and cell lines as described below.
  • the following cell lines and primary cells are used: (a) human T cell line, CEMSS (Frederico et al., S.Biol. Regul. Homeost. Agents, 1: 41-49 (1993)) (b) human embryonic kidney cells 293T (293T) (Pear et al., Proc. Natl. Acad. Sci. USA 90:8392 - 8396 (1993)), and (c) PPA-6 (Rigg et al., supra).
  • a CEMSSMuSK-R cell line is generated by transducing CEMSS cells with PPA-6 supernatants that are made using the pGla-MuSK- Rwt construct.
  • DMEM fetal bovine serum
  • PBS fetal bovine serum
  • sodium pyruvate obtained from JRH Biosciences (CA)
  • FBS FBS from Hyclone (UT)
  • L-glutamine Trypsin from Life Technologies
  • MD Trypsin from Life Technologies
  • ITS insulin/transferrin/sodium selenite
  • PHA phytoreactive protein
  • Interleukin-2 (11-2) from Sigma (Missouri).
  • 293T cells and PPA-6 cells are cultured in DMEM, 10% FBS, 1 % sodium pyruvate, and 1% L-glutamine.
  • CEMSS cells are cultured in RPMI, 10%FBS, 1% L-glutamine, and 1% sodium pyruvate.
  • Hybridoma cells are grown in (hypoxanthine aminopterin thymidine (HAT) media or HT media (Iscove's medium, 10% FBS, 5% hybridoma cloning factor (Igen; MD) plus 0.5 M hypoxanthine, 4 ⁇ M aminopterin, 16 ⁇ M thymidine in HAT medium or 0.5 mM hypoxanthine, 16 ⁇ M thymidine in HT medium).
  • HAT hyperxanthine aminopterin thymidine
  • HT media Iscove's medium, 10% FBS, 5% hybridoma cloning factor (Igen; MD) plus 0.5 M hypoxanthine, 4 ⁇ M aminopterin, 16 ⁇ M thymidine in HAT medium or 0.5 mM hypoxanthine, 16 ⁇ M thymidine in HT medium).
  • adherent cells (293T and PPA-6) cells are washed once with PBS, then trypsinized for 5 min and subsequently split into new tissue culture flasks (VWR; NJ).
  • step (D) 10 6 cells/ml from step (D) are transduced with 1-3 ml of viral supernatant, that had been either generated from 293 T cells or PPA-6 cells, by spinoculation with 8 ⁇ g/ml protamine sulfate (Sigma, Missouri). Using standard techniques, spinoculation is done at 37°C for 3 hrs at 2750 ⁇ m for PPA-6 and CEMSS cells. PPA-6 cells are transduced in 6 well plates, and CEMSS cells in 6-ml tubes.(VWR)
  • FACS analysis is done on a FACScan (Becton Dickinson).
  • the following antibodies and reagents are used for staining.
  • CD4-FITC Caltag
  • propidium iodide PI
  • goat anti- mouse IgG-PE Caltag
  • goat anti-mouse IgG coupled magnetic beads Dynal, Oslo
  • anti- MuSK-R polyclonal serum and anti-MuSK-R hybridoma supernatant (see section G). All antibodies are titrated and optimal concentrations are used. 1x10 cells are stained in 50 ⁇ l of PBS/2% FBS for 20 to 60 minutes at 4°C.
  • the cells are washed once with 2 ml of PBS/2%FBS, then again incubated in 50 ⁇ l PBS/2%FBS and the secondary antibody is added. Before the FACSanalysis the cells are again washed once with PBS/2%FBS, centrifuged and resuspended in 500 ⁇ l PBS/2%FCS containing l ⁇ g/ml PI. FACSanalysis is performed on a FACSscan (Becton-Dickinson Immunocytometry Group, CA) according to manufacturer's instructions.
  • FACSscan Becton-Dickinson Immunocytometry Group, CA
  • the cells are stained with an anti-MUSK- R antibody.
  • the 10 7 cells/ml are incubated with 1-3 ml anti-MuSK-R hybridoma supernantant in PBS/2%FBS for 1 hr on ice with occasional shaking.
  • the cells are washed 3 times with PBS/2%FCS and then anti-IgG antibody coupled magnetic beads, that can recognize anti-MuSK-R antibodies, are added ( ⁇ 5 beads per positive cell).
  • the cells are incubated for 1 hr on ice.
  • Cells that express MuSK-R are selected by positive selection with a Dynal magnet (Dynal, Oslo) for 10 min. The unbound cells are removed and the MuSK-R expressing cells are put into culture as described in section D.
  • MuSK-R XC is amplified by PCR and cloned into the expression construct pSecTag2b (Invitrogen). Cloning the XC domain of MuSK-R into the multiple cloning site (MCS) of the plasmid pSecTag2b allows for the expression of the XC under the control of the CMV promoter.
  • MCS multiple cloning site
  • the plasmid contains the sequence of a myc and (His) 6 -tag after the multiple cloning site, which allows to fuse the protein of interest (MuSK-RXC) to the myc and (His) 6 -tag.
  • the signal peptide of MuSK-R is replaced by the Ig ⁇ leader.
  • Figure 3 The extracellular domain of MuSK-R without the signal peptide is amplified by PCR using the following primers wherein p means phosphorylated:
  • MuSK 1 16FPC 5'pCT TCC AAA AGC TCC TGT CAT CAC C SEQ ID NO: 9 and
  • MuSK 1532RC 5' pCC AGT CAT GGA GTA TGT AGG TGA GAC SEQ ID NO: 10
  • Primer MuSKl 16FPC starts with the sequence after the signal peptide (nt 69 - 93).
  • Primer MuSK1532RC covers the sequence before the transmembrane domain starts and the first 2 amino acids of the transmembrane domain corresponding to nucleotide sequence
  • the PCR reaction is cooled to 4°C in the PCR machine and then gel-purified.
  • the PCR fragment is cloned into to the EcoRV restriction site of pSecTag2b.
  • MuSK-R XC is cloned in frame with the Igk leader at the N-terminus and the myc- and (His) 6 -tag at the C-terminus.
  • the resulting plasmid is called pSecTag-hMuSK-R.
  • the plasmid pSecTag-hMuSK-R is transfected into 293T cells by the CaCl 2 technique (as described in section C). 24 hrs after the transfection, the media is replaced with either fresh DMEM/10%FBS or serum-free X-Vivo 15. Supernatants of the transfected cells are collected after 48 and 72 hrs. A total of 400 ml supernatants are collected and are frozen at -80°C until the supernatants are purified.
  • the MuSK-R XC is purified from tissue culture supernatants by immobilized metal affinity chromatography.
  • the metal ion is 0.1 M NiCl 2 .
  • the column is a 1 or 5 ml Pharmacia metal HiTrap chelating sepharose column.
  • the equilibration buffer (Buffer A) consisted of 20 mM Na 2 HP0 4 pH 7.4, IM guanidine hydrochloride, IM NaCl, filtered through 0.2 ⁇ M cellulose acetate filter.
  • the elution buffer (Buffer B) is 20 mM Na 2 HP0 , pH 7.4, 1 M guanidine hydrochloride, IM NaCl, 0.5 M imidazole, filtered through 0.2 ⁇ M cellulose acetate filter.
  • the Pharmacia FPLC chromatography system is used to run columns, with FPLC director program software and a Pharmacia P50 pump. The purification is performed at 4°C.
  • the pump is primed with buffer A before the load is started. Before the column the column is attached, the load is pumped through until the pink color of the tissue culture media is seen at the connection so that the column is not washed with non-equilibration conditions.
  • tissue culture supernatants are adjusted to contain 0.85 M NaCl, IM guanidinium chloride and 40 mM imidazole and the pH is adjusted to 7.4.
  • the column is equilibrated with 8% buffer B. The sample is loaded and the column then washed in above conditions for seven column volumes. MuSK-R is eluted at 30% Buffer B (150 mM imidazole) over eight column volumes. Fractions are collected from start of the run.
  • each fraction is tested in a Dot Blot Assay (see below). Selected positive fractions are tested in Western Blot assays and Elisa (see below). Positive fractions are pooled and dialyzed in 10,000 MWCO membrane (Pierce Snakeskin) against PBS. After dialysis, the optical density is determined at OD 280 . The samples are filtered through 0.2 ⁇ M filters and then concentrated in Centricon Centriprep 30 devices in a refrigerated Sorvall RT6000D according to manufacturer's protocol.
  • the gel is blotted onto 0.45 ⁇ M nitrocellulose for 1.4 hours at 100 volts, using the Biorad wet transfer blotting cassette with tris-glycine-methanol transfer buffer (25 mM trizma Base, 192 mM glycine, 20% methanol). After blotting the gel, the blot is blocked in Pierce TBS superblock for 10 minutes with mild agitation. The blot is washed twice in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for five min per wash on a rotating platform.
  • TBST 50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20
  • the Pierce IndiaTM His-HRP Probe is diluted to 1 :5000 in TBST and the blot is incubated with the probe for 1 hour at room temperature and washed 3 times in TBST. After that, horseradish peroxidase reagent (Sigma Fast HRP Insoluble Substrate D4418) is added to the blot and the blot is developed. The blot is washed in three changes of water to stop development. Alternatively an mouse anti-c-myc antibody, (Santa Cruz Biotechnology; CA) is used to detect recombinant MuSK-R protein. This antibody is diluted in superblock to 1 ⁇ g/ml final concentration.
  • the blot is washed three times with TBST and then a goat anti-mouse IgG-HRP antibody (Sigma) is added at 1 :5000 dilution in superblock.
  • the blot is incubated for 1 hour at room temperature, with gentle agitation and developed as described above with Fast HRP insoluble substrate (Sigma).
  • the recognized protein traveled at about 85 kD on the SDS PAGE, and it is considered to be 19 kD heavier due to glycosylation.
  • the recombinant MuSK-R protein is injected into 3 different Balb/c mice.
  • 25-50 ⁇ g are mixed with 2.25 mg alhydrogel and 100 ⁇ g MDP (muranyl dipeptide; Pierce) in a final volume of 200 ⁇ l and injected 5 times every 14 days subcutaneously.
  • MDP muranyl dipeptide
  • FACSanalysis the 5xl0 5 cells of cell lines CEMSS and CEMSSMuSK-R are used. Both the presera and sera are diluted 1 :100. 1 :300, 1:900 and 1 :2700.
  • a rat anti-mouse IgG-PE antibody is used as a secondary reagent at a 1 :20 dilution.
  • 96 well plates are coated with 50 ⁇ l of 10 ⁇ g/ml anti-mouse IgGF ⁇ (Jackson; Maine) The plates are incubated with various dilutions of sera (1 :100 to 1 :218700), subsequently with MuSK-R protein and with Nickel activated horse radish peroxidase at a 1 :1000 dilution (HRP, Pierce). Nickel activated HRP is binding to the recombinant MuSK-R protein via the (His) 6 tag.
  • the plate is incubated with TMP peroxidase substrate (Zymed; CA).
  • TMP peroxidase substrate Zymed; CA
  • one mouse shows the highest reactivity against native and recombinant MuSK-R.
  • This mouse is boosted with a 6 th injection of 200 ⁇ g MuSK-R protein in PBS. The injection is done subcutaneously and intravenously. 1 week later the spleen is removed, lymphocytes isolated with lympholite M (Accurate Chemicals) and fused, using 50% polyethylene glycol to the myeloma cell line P3X63AG8.0653 using standard procedures. The resulting hybridomas are grown in bulk in HAT media for one week.
  • Viable cells are recovered using lympholite M and cultured in HAT media plus cloning factor (Igen). After the hybridoma are grown for another week, a batch of the cells are cryopreserved in HAT media plus 10% DMSO. Another batch of the cells is subdivided into individual clones by FACSsorting using the single cell deposit unit. The cells are sorted by forward and side scatter and for PI negative cells. The cells are grown up in HT media for two weeks. The supernatants are tested by Elisa and FACS (as described above) for monoclonal antibodies that can recognize native and recombinant MuSK-R protein.
  • Igen cloning factor
  • the antibodies are isotyped in an Elisa assay by using secondary antibodies that react with IgGl, 2a, 2b, 3, IgM, K, and ⁇ (Caltag, CA). Three monoclonal antibodies are identified HI, H2 and H4. All three can react with
  • MuSK-R expressed on the cell ine CEMSS-MuSK-R in a FACS assay is an IgGl , K; H2 is IgGl , K; H4 is IgM antibody.
  • Figure 4 shows expression of hMuSK-R on CEMSS cells and CEMSS-MuSK-R cells using the antibodies HI , H2 and H$.
  • a secondary PE coupled to anti-mouse IgG is used.
  • Figure 5 illustrates expression of MuSK- on nontransduced CEMSS cells (panel A) and on CEMSS cells that are transduced with PP6-A supernatants so they express hMuSK-R (panel B) or mMuSK-RII (panel D). Both populations were enriched after immuno-magnetic bead selection as illustrated for hMuSK-R (panel C) and mMuSK-RII (panel E). The results of the experiments illustrated in Figure 5 are performed using the monoclonal antibody H2.

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Abstract

Cette invention concerne une méthode permettant d'utiliser une molécule récepteur de tyrosine kinase spécifique des muscles (MuSK-R) ou une MuSK-R mutée (mMuSK-R) de celle-ci en tant que marqueur sélectionnable dans des cellules de mammifère, particulièrement des cellules humaines. Les marqueurs préférés sont les mMuSK-Rs incapables de produire une transduction de signal, et de préférence des molécules dans lesquelles le domaine intracellulaire a été modifié par délétion de la région de signalisation. L'invention concerne également une méthode permettant d'identifier des cellules de mammifère génétiquement modifiées, comprenant l'introduction d'une mMuSK-R dans une cellule cible en tant que marqueur sélectionnable. L'invention concerne en outre une méthode d'immunosélection de cellules de mammifère transduites comportant l'identification des cellules transduites par incubation des cellules à l'aide d'un anticorps qui reconnaît et se lie spécifiquement à une MuSK-R ou à une mMuSK-R correspondante.
PCT/EP2001/003543 2000-03-30 2001-03-28 Genes marqueurs selectionnables WO2001072834A1 (fr)

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JP2001571765A JP2003533183A (ja) 2000-03-30 2001-03-28 選択マーカー遺伝子
EP01940269A EP1272522A1 (fr) 2000-03-30 2001-03-28 Genes marqueurs selectionnables
AU7390601A AU7390601A (en) 2000-03-30 2001-03-28 Selectable marker genes
NZ521634A NZ521634A (en) 2000-03-30 2001-03-28 Identifying genetically modified mammalian cells with a cell surface mutated muscle specific tyrosine kinase receptor (MuSK-R) operatively linked to a promoter
AU2001273906A AU2001273906B2 (en) 2000-03-30 2001-03-28 Selectable marker genes
CA002403852A CA2403852A1 (fr) 2000-03-30 2001-03-28 Genes marqueurs selectionnables
IL15199601A IL151996A0 (en) 2000-03-30 2001-03-28 Selectable marker genes

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CN114619557B (zh) * 2022-04-12 2023-04-07 南京高等职业技术学校（江苏联合职业技术学院南京分院） 一种叠合法生产透光混凝土板的成型装置及成型方法

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1995006723A1 (fr) * 1993-09-01 1995-03-09 Boehringer Mannheim Gmbh Procede de marquage de cellules eucariotes par utilisation d'un recepteur de surface cellulaire en tant que marqueur
WO1999010494A2 (fr) * 1997-08-25 1999-03-04 Genentech, Inc. Anticorps agonistes envers le recepteur de thrombopoietine, et utilisation therapeutique de ces anticorps
US6107477A (en) * 1996-09-26 2000-08-22 Aurora Biosciences Corporation Non-optimal Kozaks sequences

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006723A1 (fr) * 1993-09-01 1995-03-09 Boehringer Mannheim Gmbh Procede de marquage de cellules eucariotes par utilisation d'un recepteur de surface cellulaire en tant que marqueur
US6107477A (en) * 1996-09-26 2000-08-22 Aurora Biosciences Corporation Non-optimal Kozaks sequences
WO1999010494A2 (fr) * 1997-08-25 1999-03-04 Genentech, Inc. Anticorps agonistes envers le recepteur de thrombopoietine, et utilisation therapeutique de ces anticorps

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GLASS, D.J. ET AL.: "Kinase domain of the muscle-specific receptro tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: requried role for the MuSK ectodomain?", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 94, August 1997 (1997-08-01), pages 8848 - 8853, XP002175852 *
HILDINGER M ET AL: "Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking.", GENE THERAPY, vol. 6, no. 7, July 1999 (1999-07-01), pages 1222 - 1230, XP000990645, ISSN: 0969-7128 *
ZHOU, H. ET AL.: "Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations", THE JOURNAL OF CELL BIOLOGY, vol. 146, no. 5, 6 September 1999 (1999-09-06), pages 1133 - 1146, XP002175851 *

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AU7390601A (en) 2001-10-08
IL151996A0 (en) 2003-04-10
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