WO2011013698A1 - キャリアペプチドフラグメント及びその利用 - Google Patents
キャリアペプチドフラグメント及びその利用 Download PDFInfo
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- WO2011013698A1 WO2011013698A1 PCT/JP2010/062691 JP2010062691W WO2011013698A1 WO 2011013698 A1 WO2011013698 A1 WO 2011013698A1 JP 2010062691 W JP2010062691 W JP 2010062691W WO 2011013698 A1 WO2011013698 A1 WO 2011013698A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Definitions
- the present invention relates to a method for introducing (transferring) a foreign substance from the outside of a eukaryotic cell to the inside of the cell, and a carrier peptide fragment used in the method. Note that this application claims priority based on Japanese Patent Application No. 2009-177101 filed on July 29, 2009, the entire contents of which are incorporated herein by reference. ing.
- a foreign substance such as a polypeptide, especially a physiologically active substance is introduced into a cell (eukaryotic cell) of a human or other mammal, and the trait of the cell (or tissue or organ comprising the cell) is transformed. Or the function of the cell is improved or improved.
- Patent Document 1 describes a cell-permeable carrier peptide for introducing a foreign substance such as a polypeptide or DNA into a cell.
- a physiologically active substance such as a polypeptide or DNA can be introduced into a cell with high efficiency by using a carrier peptide conjugate in which a cell-permeable carrier peptide and a heterologous polypeptide, DNA or the like are linked. It is stated that it can be done.
- the entire polypeptide having a relatively large molecular weight as a foreign substance (physiologically active substance) to be introduced can be easily introduced into the target cell without using a special device to transform the cell or improve its function ( (Or improvement) is required.
- Patent Document 2 describes a part of a peptide chain (amino acid sequence) constituting SOCS proteins and their family proteins (hereinafter collectively referred to as “SOCS proteins”), and is considered to bind to the Elongin BC complex.
- the amino acid sequence constituting all or part of the specified region “BC-box” is a motif having a high neuronal differentiation-inducing activity against somatic stem cells. It has been disclosed that introduction into stem cells can induce differentiation of the introduced cells into nerve cells.
- conventional cell-permeable carrier peptides for example, cell-permeable carrier peptides derived from HIV and Drosophila
- a peptide (amino acid sequence) for introducing a foreign substance that can be efficiently synthesized and used with a short chain length composed of the number of residues is desired.
- the present invention is an invention created to meet such a demand, and is a peptide fragment having a short amino acid sequence composed of 10 or less amino acid residues, and foreign substances are expressed in cells (typically eukaryotic cells). It is an object of the present invention to provide a carrier peptide fragment having a short chain length that is used for introduction into a cell from the outside (in particular, an animal cell such as a mammalian cell having no cell wall). Another object of the present invention is to provide a method for introducing various foreign substances from the outside of the cell through the cell membrane into the target cell using the carrier peptide fragment. The present invention also provides a construct for introducing the foreign substance prepared so as to contain the carrier peptide fragment disclosed herein and the foreign substance. The present invention also provides cells, organs and other living tissues in which a construct having the carrier peptide fragment disclosed herein and a foreign substance is introduced into the cytoplasm (including in the nucleus).
- the present inventor has studied various peptides (or amino acid sequences constituting a part of the peptide (that is, a motif whose function is specified)) whose amino acid sequence has already been identified as a peptide having some function in the cell, In spite of the shorter chain length than before, the present inventors have found an amino acid sequence that can be preferably used as the carrier peptide (fragment) and completed the present invention.
- One of the methods provided by the present invention is that the cytoplasm of eukaryotic cells (particularly various animal cells represented by humans and other mammals having no cell wall) (ie outside the cell membrane) This is a method of introducing (transferring) a desired foreign substance into the inside (more preferably a nucleus).
- the method for introducing a foreign substance disclosed herein comprises an amino acid sequence consisting of WRRQARFK (SEQ ID NO: 1), or one or two or three amino acid residues of the amino acid sequence are substituted, deleted, and / or Alternatively, introduction of a foreign substance having a carrier peptide fragment consisting of any of the amino acid sequences formed by addition (insertion) and a target foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment Preparing a structure for use, Supplying the foreign substance introduction construct into a sample containing a target eukaryotic cell (typically a culture containing the cell); Incubate the sample supplied with the foreign substance introduction construct (that is, hold the sample for a certain period of time under the condition that the target cell can survive) and introduce the construct into the eukaryotic cell in the sample.
- a target eukaryotic cell typically a culture containing the cell
- the “foreign substance” is an inorganic compound or an organic compound that can be directly or indirectly bonded to the N-terminal side or the C-terminal side of the carrier peptide fragment via an appropriate linker, and is contained in a eukaryotic cell. It has a molecular size and chemical properties that can be introduced.
- N protein nucleocapsid protein
- IBV avian infectious bronchitis virus
- a peptide fragment composed of a partial amino acid sequence “WRRQARFK (SEQ ID NO: 1)” consisting of a single amino acid residue can easily pass through the cell membrane of a target cell, and the passage through the cell membrane
- WRRQARFK SEQ ID NO: 1
- the target foreign substance (typically, an organic compound such as a peptide, nucleic acid, dye, drug, etc.) is converted to the N-terminal side of the carrier peptide (fragment) and / or C.
- a foreign substance introduction construct constructed by binding directly or indirectly via a suitable linker to a terminal side is put into a sample containing eukaryotic cells of interest (typically a culture containing the cells).
- the target foreign substance is allowed to pass through the cell membrane from the outside of the eukaryotic cell (outside the cell membrane) and into the cytoplasm (preferably further through the nuclear membrane). It can be transferred with high efficiency into the nucleus).
- the foreign substance is any organic compound selected from the group consisting of peptides, nucleic acids, dyes, and drugs.
- peptide refers to an organic compound having a structure in which two or more amino acids are linked by peptide bonds, and is a polypeptide (typically 10 to less than 300 amino acid residues) or protein (typically A polymer compound having a larger number (300 or more) of amino acid residues than the above polypeptide).
- the “nucleic acid” herein refers to a polymer of nucleotides, and includes DNA and RNA. A construct prepared so as to contain this kind of organic compound can be efficiently introduced into a target cell.
- the foreign substance is a peptide
- the foreign substance introduction construct comprises a fragment comprising a peptide as the foreign substance, the carrier peptide fragment, Is a synthetic peptide.
- the target peptide that is, the amino acid sequence constituting the peptide
- the target peptide can be introduced into a target cell as one peptide motif in the state of the synthetic peptide.
- the eukaryotic cell into which the foreign substance introduction construct is introduced is a stem cell derived from a human or non-human mammal (including induced pluripotent stem cells).
- a target foreign substance having a predetermined function can be introduced into human or other mammalian stem cells (eg, somatic stem cells or induced pluripotent stem cells). This makes it possible to realize transformation of the stem cells, for example, differentiation into specific cells (neural cells, bone cells, muscle cells, skin cells, etc.) according to the foreign substance to be introduced (peptides or the like).
- the present invention provides the cytoplasm (preferably further in the nucleus) from the outside of eukaryotic cells (particularly various animal cells represented by humans and other mammals having no cell wall).
- An artificially prepared construct for introducing a foreign substance of interest is provided. That is, the foreign substance introduction construct disclosed herein has an amino acid sequence consisting of WRRQARFK (SEQ ID NO: 1), or one or two or three amino acid residues in the amino acid sequence substituted, deleted, and And / or a carrier peptide fragment composed of any of the amino acid sequences formed by addition (insertion) and a target foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.
- WRRQARFK SEQ ID NO: 1
- the target foreign substance can be effectively introduced into the target cells.
- cells into which the foreign substance has been introduced and organs and other biological tissues containing cells containing the foreign substance can be obtained.
- the foreign substance is any organic compound selected from the group consisting of peptides, nucleic acids, dyes and drugs.
- the foreign substance is a peptide
- the foreign substance introduction construct is a synthetic peptide having a peptide fragment as the foreign substance and the carrier peptide fragment.
- FIG. 1 shows a sample (cell) in which human neonatal foreskin fibroblasts supplied with a foreign substance introduction construct (sample 1) according to an example were cultured for 1 hour and then fixed with methanol using a confocal laser microscope. It is a micrograph. The scale in the photograph is 100 ⁇ m.
- FIG. 2 shows a sample (cell) in which human neonatal foreskin fibroblasts supplied with a foreign substance introduction construct (sample 1) according to one example were cultured for 4 hours and then fixed with methanol using a confocal laser microscope. It is a micrograph. The scale in the photograph is 100 ⁇ m.
- FIG. 1 shows a sample (cell) in which human neonatal foreskin fibroblasts supplied with a foreign substance introduction construct (sample 1) according to one example were cultured for 4 hours and then fixed with methanol using a confocal laser microscope. It is a micrograph. The scale in the photograph is 100 ⁇ m.
- FIG. 3 is a micrograph obtained by observing a sample (cell) fixed with methanol after culturing a human iPS cell supplied with a foreign substance introduction construct (sample 1) according to an example for 1 hour using a confocal laser microscope. is there. The scale in the photograph is 100 ⁇ m.
- FIG. 4 is a micrograph obtained by observing a sample (cell) fixed with methanol after culturing a human iPS cell supplied with a foreign substance introduction construct (sample 2) according to an example for 1 hour using a confocal laser microscope. is there. The scale in the photograph is 100 ⁇ m.
- the “carrier peptide fragment” disclosed herein is a sequence defined (obtained) by the amino acid sequence of SEQ ID NO: 1, and is eukaryotic cell membrane permeability (more preferably nuclear translocation (nuclear membrane permeability). )).
- the specific amino acid sequence described in SEQ ID NO: 1 is a sequence portion consisting of a total of 8 amino acid residues from the 71st to the 78th amino acid residues of the IBV nucleocapsid protein (N protein) (that is, Nucleolar localization signal (Nucleolar localization sequence) corresponding to the motif) is a sequence newly found by the present inventors to exhibit excellent cell membrane permeability.
- a foreign substance can be introduced from the outside of the cell into the cytoplasm even though it is a peptide fragment having an extremely short chain length consisting of 10 or less amino acid residues (here, 8 amino acid residues).
- the “carrier peptide fragment” disclosed herein is typically the same sequence as the amino acid sequence set forth in SEQ ID NO: 1. However, in addition to the same sequence, one or a number can be used without impairing cell membrane permeability. It includes an amino acid sequence formed by substitution, deletion and / or addition (insertion) of individual (typically 2 or 3) amino acid residues.
- such a minor modified sequence is easily utilized by those skilled in the art based on the information disclosed herein, and is included in the “carrier peptide fragment” as the technical idea disclosed herein. This is because that. As a typical example, it was caused by so-called conservative amino acid replacement in which one or several (typically 2 or 3) amino acid residues in the amino acid sequence of SEQ ID NO: 1 were conservatively substituted.
- a sequence eg, a sequence in which a basic amino acid residue is replaced with another basic amino acid residue
- one or several (typically 2 or 3) amino acid residues for a given amino acid sequence Examples include added (inserted) or deleted sequences.
- the construct for introducing a foreign substance disclosed herein binds (links) a desired foreign substance directly or indirectly via an appropriate linker to the N-terminal side and / or C-terminal side of the carrier peptide fragment described above. It is a structure that can be designed and built by doing For example, when the foreign substance is a peptide, the peptide chain is designed to include the amino acid sequence that constitutes the peptide and the amino acid sequence that constitutes the carrier peptide fragment, and the peptide chain is synthesized.
- a foreign substance introduction construct can be prepared.
- nucleic acids such as DNA or RNA
- dyes for example, fluorescent dye compounds such as FITC
- drugs for example, nucleic acid anticancer agents such as 5-fluorouracil (5FU), and antiviral agents such as azidothymidine (AZT)
- nucleic acid anticancer agents such as 5-fluorouracil (5FU)
- antiviral agents such as azidothymidine (AZT)
- the organic compound that functions as a) is bound directly or indirectly to the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment by various known chemical methods to construct a foreign substance introduction construct. be able to.
- a foreign substance when a foreign substance is a peptide, it does not specifically limit as a peptide (amino acid sequence) employ
- a polypeptide or protein having a relatively large number of amino acid residues for example, about 100 to 1000 amino acid residues can also be employed as the foreign substance.
- the total number of amino acid residues constituting a synthetic peptide produced as a foreign substance introduction construct is suitably 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly 300 or less ( Further, 100 or less, for example, 10 to 50) is preferable.
- Such relatively short peptides are easy to synthesize and easy to use.
- the foreign substance to be adopted examples include peptides (polypeptides and proteins) involved in functions such as development, differentiation, proliferation, canceration, homeostasis (homeostasis), and metabolism of various cells and tissues (organs). ) Mature type or precursor (including pro-type and pre-pro type).
- the present invention can also be carried out to elucidate the function of the peptide in the cell (in a living tissue) by introducing a peptide whose function has not been conventionally known into the cell.
- the eukaryotic cells to be introduced are human or other mammalian stem cells (including somatic stem cells, embryonic stem cells, induced pluripotent stem cells (hereinafter referred to as iPS cells)).
- the eukaryotic cell to be introduced is a cancer cell (tumor cell)
- a plurality of genes for example, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin28
- a predetermined cell for example, a human or other mammalian skin cell or other somatic cell.
- a product (peptide) of at least one of these genes may be introduced by the introduction method of the present invention. This makes it possible to produce iPS cells by introducing the gene product (ie, peptide) into the cell (preferably into the nucleus) instead of directly introducing the gene.
- a peptide for example, SOX2 protein
- at least one gene for example, Sox2
- An iPS cell preparation method characterized by preparing a foreign substance introduction construct and introducing the construct into a predetermined eukaryotic cell (human skin fibroblast, etc.).
- sequence motifs can be used.
- eukaryotic cells to be introduced are human or other mammalian stem cells (including somatic stem cells, embryonic stem cells, and iPS cells)
- use of various peptide motifs involved in the differentiation induction of the stem cells is possible.
- the eukaryotic cell to be introduced is a cancer cell (tumor cell)
- Elongin BC complex specifically, a part of Elongin C
- SOCS proteins A part of amino acid sequences constituting various SOCS proteins (suppressors of cytokine signal transduction) having a SOCS-box which is a region to be obtained (amino acid sequence) and their family proteins (hereinafter collectively referred to as “SOCS proteins”)
- amino acid sequence amino acid sequence
- BC-box family proteins
- SEQ ID NOs: 2 to 19 are amino acid sequences contained in BC-boxes of various proteins identified as SOCS proteins (see Non-Patent Documents 3 to 6). Specifically, mSOCS-1 (SEQ ID NO: 2), mSOCS-2 (SEQ ID NO: 3), mSOCS-3 (SEQ ID NO: 4), mSOCS-4 (SEQ ID NO: 5), mSOCS-5 (SEQ ID NO: 6), hSOCS-6 (SEQ ID NO: 7), hSOCS-7 (SEQ ID NO: 8), hRAR-1 (SEQ ID NO: 9), hRAR-like (SEQ ID NO: 10), mWSB-1 (SEQ ID NO: 11), mWSB-2 (sequence) No.
- SEQ ID NOs: 20 to 80 are amino acid sequences contained in BC-boxes of various SOCS proteins identified from viruses (HIV, AdV, SIV, etc.) and mammals, A peptide consisting of a sequence is shown.
- SEQ ID NO: 75 and SEQ ID NO: 79 are amino acid sequences contained in the BC-box of the SOCS protein (MUF1) identified from human.
- SEQ ID NO: 80 is an amino acid sequence contained in the BC-box of the SOCS protein mCIS (cytokine-inducible SH2-containing protein) identified from the mouse.
- mCIS cytokine-inducible SH2-containing protein
- an amino acid sequence derived from any of the above-mentioned BC-boxes (typically, any one of SEQ ID NOs: 2 to 80) is used as a peptide motif involved in induction of neural differentiation.
- a synthetic peptide that can be used as a (sequence motif) and introduced into target eukaryotic cells can be constructed. Therefore, as is apparent from the above description, the present invention provides a method for inducing differentiation of at least one eukaryotic cell into a nerve cell.
- the amino acid sequence derived from any BC-box typically SEQ ID NO: 2 to Synthesizing a peptide chain comprising at least 10 (for example, an amino acid sequence consisting of at least 10 consecutive amino acid residues) selected from the amino acid sequences shown in any of 80, and the synthesis Including supplying a peptide (ie, an artificial peptide that is a construct for introducing a foreign substance) to a target eukaryotic cell or a sample containing a tissue having the cell (typically, a culture containing the cell). .
- the method further includes incubating a sample supplied with the synthetic peptide.
- an artificial peptide used in such a method for inducing differentiation into nerve cells and a method for producing the same. That is, an artificial peptide (neural differentiation-inducing peptide) having such a structure is provided on the N-terminal side or C-terminal side of the carrier peptide fragment as an amino acid sequence derived from any BC-box (hereinafter referred to as “BC” -It is also referred to as “box-related sequence.” Typically, it consists of at least 10 (for example, at least 10 from the N-terminal) consecutive amino acid residues selected from the amino acid sequences shown in any of SEQ ID NOs: 2 to 80 Amino acid sequence).
- the 15 consecutive amino acid sequences shown in SEQ ID NO: 81 can also be used for the same purpose as the BC-box related sequence. That is, the amino acid sequence shown in SEQ ID NO: 81, as described in Patent Document 3, is from the 157th to the 157th amino acid sequence of the VHL (von Hippel Lind) protein, which is known to exhibit neuronal differentiation-inducing properties. This is a partial amino acid sequence (VHL-related peptide motif) consisting of 15 consecutive amino acid residues up to the 171st.
- VHL von Hippel Lind
- a modified amino acid sequence formed by substituting, deleting and / or adding (inserting) amino acid residues can also be used as a peptide motif (foreign substance) for induction of neural differentiation.
- the construct for introducing a foreign substance having the above-described configuration has a high nerve differentiation-inducing activity against at least one kind of cell (typically a stem cell) as a nerve differentiation-inducing peptide. For this reason, it can be suitably used as an active ingredient of a neuronal differentiation inducer.
- the neuronal differentiation-inducing peptide contained in the neuronal differentiation-inducing agent may be in the form of a salt as long as the neuronal differentiation-inducing activity is not impaired.
- an acid addition salt of the peptide that can be obtained by addition reaction of an inorganic acid or an organic acid usually used according to a conventional method can be used.
- other salts for example, metal salts
- the nerve differentiation-inducing agent can contain various carriers that are pharmaceutically (pharmaceutical) acceptable depending on the form of use, in addition to the neuronal differentiation-inducing peptide having the above-described structure as an active ingredient.
- Carriers generally used in peptide medicine as diluents, excipients and the like are preferred.
- water, a physiological buffer solution, and various organic solvents can be mentioned, although it may vary depending on the use and form of the neuronal differentiation inducer.
- It can be a non-drying oil such as an aqueous solution of alcohol (such as ethanol) of a suitable concentration, glycerol, olive oil. Or a liposome may be sufficient.
- a secondary component which can be contained in a nerve differentiation-inducing agent various fillers, extenders, binders, moisturizers, surfactants, pigments, fragrances and the like can be mentioned.
- the form of the neuronal differentiation inducer include solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules and ointments.
- it since it uses for injection etc., it can also be set as the freeze-dried material and granulated material for melt
- compositions using a neuronal differentiation-inducing peptide (main component) and various carriers (subcomponents) as materials may be in accordance with a conventionally known method. As such, it does not characterize the present invention and will not be described in detail.
- main component a neuronal differentiation-inducing peptide
- subcomponents various carriers
- the nerve differentiation-inducing agent provided by the present invention can be used in a method or dosage depending on its form and purpose.
- a neuronal differentiation-inducing peptide that is, a neuronal differentiation-inducing agent containing the synthetic peptide
- synthesized so as to contain a BC-box-related sequence or VHL peptide motif disclosed herein and a carrier peptide fragment is used as a solution.
- the desired amount can be administered to a patient (ie, a living body) by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection.
- solid forms such as tablets can be administered orally.
- nerve cells can be generated (produced) from somatic stem cells that are typically present in or around the affected area in vivo. For this reason, it becomes possible to effectively treat various neurological diseases for which nerve regeneration is an effective therapeutic method.
- regenerative medical approaches to treat neurological diseases such as Parkinson's disease, cerebral infarction, Alzheimer's disease, body paralysis due to spinal cord injury, brain contusion, amyotrophic lateral sclerosis, Huntington's disease, brain tumor, retinal degeneration Is realized.
- neuronal differentiation-inducing agent neural differentiation-inducing peptide
- cell material temporarily or permanently removed from a living body that is, living tissue or cell mass (eg, culture of somatic stem cells).
- the target peptide motif BC-box-related sequence or the like
- the desired nerve cells can be produced in large quantities in the cell material.
- nerve cells produced in large quantities or cell materials (living tissue or cell mass) containing the produced nerve cells are returned to the living body (typically, the affected area where nerve regeneration is required).
- the present invention induces differentiation into nerve cells, which is useful for the treatment of neurological diseases, by using any of the above-described neuronal differentiation-inducing peptides disclosed herein.
- Cell, cell mass or living tissue can be provided.
- the polynucleotide encoding the neuronal differentiation-inducing peptide of the present invention can be used as a material used for so-called gene therapy.
- a gene encoding a neuronal differentiation-inducing peptide (typically a DNA segment or an RNA segment) is incorporated into an appropriate vector and introduced into a target site, so that the present invention is always in vivo (cell).
- the foreign substance introduction construct in which the foreign substance provided by the present invention is a peptide, such as the above-described neuronal differentiation-inducing peptide described as a typical example, has at least one amino acid residue. May be amidated. Amidation of the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of the peptide chain) can improve the structural stability (eg, protease resistance) in the cytoplasm and nucleus of the peptide.
- the artificial peptide desirably has a total number of amino acid residues constituting the peptide chain of 1000 or less (preferably 600 or less, particularly preferably 300 or less, for example 50 or less).
- Such a short-chain peptide can be easily constructed by a chemical synthesis method, and thus can be easily supplied to a sample containing a target eukaryotic cell.
- the conformation (steric structure) of the peptide is not particularly limited, but is preferably a linear or helical form from the viewpoint that it is difficult to become an immunogen (antigen).
- the artificial peptide those in which all amino acid residues are L-type amino acids are preferable. However, as long as the desired functions of the underlying carrier peptide fragment and peptide motif are not lost, part or all of the amino acid residues are It may be substituted with a D-type amino acid.
- sequences that cannot be included in these sequences may be partially included as long as they do not lose the desired function of the endogenous carrier peptide fragment and the peptide motif that is a foreign substance.
- any conventionally known solid phase synthesis method or liquid phase synthesis method may be employed.
- a solid phase synthesis method in which Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) is applied as an amino-protecting group is preferred. That is, it has a desired amino acid sequence and a modified (C-terminal amidation, etc.) portion by a solid phase synthesis method using a commercially available peptide synthesizer (available from PerSeptive Biosystems, Applied Biosystems, etc.).
- Peptide chains can be synthesized.
- an artificial peptide construct for introducing a foreign substance
- an artificial peptide may be biosynthesized based on a genetic engineering technique. This approach is preferred when producing polypeptides with relatively long peptide chains. That is, DNA having a nucleotide sequence (including the ATG start codon) encoding the amino acid sequence of a desired artificial peptide is synthesized. An expression comprising this DNA and various regulatory elements (including a promoter, a ribosome binding site, a terminator, an enhancer, and various cis elements that control the expression level) for expressing the amino acid sequence in a host cell. A recombinant vector having the gene construct for use is constructed according to the host cell.
- This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, animal (mammalian) cell) by a general technique, and the host cell or a tissue or an individual containing the cell under a predetermined condition Is cultured. Thereby, the target polypeptide can be expressed and produced in the cell. Then, by isolating and purifying the polypeptide from the host cell (in the medium if secreted), a peptide having the desired amino acid sequence can be obtained.
- a predetermined host cell for example, yeast, insect cell, plant cell, animal (mammalian) cell
- This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, mammalian cell) by a general technique, and the host cell or a tissue or an individual containing the cell is cultured under a predetermined condition. .
- a predetermined host cell for example, yeast, insect cell, plant cell, mammalian cell
- the target polypeptide can be expressed and produced in the cell.
- the target peptide that is, the foreign substance introduction construct
- a method for constructing a recombinant vector and a method for introducing the constructed recombinant vector into a host cell a method conventionally used in the field may be employed as it is, and such method itself particularly characterizes the present invention. Since it is not a thing, detailed explanation is omitted.
- a fusion protein expression system can be used for efficient mass production in a host cell. That is, a gene (DNA) encoding the amino acid sequence of the target peptide is chemically synthesized, and the synthesized gene is provided by an appropriate fusion protein expression vector (for example, pET series provided by Novagen and Amersham Bioscience). It is introduced into a suitable site of a GST (Glutathione S-transferase) fusion protein expression vector) such as the pGEX series. A host cell (typically E. coli) is transformed with the vector. The obtained transformant is cultured to prepare the desired fusion protein. The protein is then extracted and purified.
- the obtained purified fusion protein is cleaved with a predetermined enzyme (protease), and the released target peptide fragment (ie, designed artificial peptide) is recovered by a method such as affinity chromatography.
- a predetermined enzyme protease
- the released target peptide fragment ie, designed artificial peptide
- affinity chromatography a method such as affinity chromatography.
- a template DNA for a cell-free protein synthesis system that is, a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the target artificial peptide
- various compounds ATP, RNA polymerase
- the target polypeptide can be synthesized in vitro using a so-called cell-free protein synthesis system using amino acids and the like.
- cell-free protein synthesis systems for example, Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)), Madin et al. (Madin et al., Proc. Natl. Acad. Sci. USA, 97 (2), 559-564 (2000)) is helpful.
- PROTEIOS (trademark) Wheat germ cell-free protein synthesis kit) is commercially available. Therefore, the amino acid sequence (for example, the BC-box related sequence described above) corresponding to the peptide motif to be introduced into the cytoplasm (preferably in the nucleus) is determined, and the cell membrane-permeable carrier peptide fragment shown in SEQ ID NO: 1 is determined.
- the desired artificial peptide can be easily synthesized and produced by the cell-free protein synthesis system according to the amino acid sequence.
- peptides can be easily produced based on the Pure System (registered trademark) of Post Genome Research Institute, Japan.
- Example 1 Preparation of a foreign substance introduction structure> A total of two types of peptides (Samples 1 and 2) were produced using the peptide synthesizer described below. Table 1 lists the amino acid sequences of these synthetic peptides.
- peptide 1 is a peptide consisting of the carrier peptide fragment of SEQ ID NO: 1 according to the present invention.
- Peptide 2 has one glycine residue as a linker on the C-terminal side of the carrier peptide fragment shown in SEQ ID NO: 1 and the above VHL peptide motif (SEQ ID NO: 81) as a peptide fragment as a foreign substance on the C-terminal side.
- SEQ ID NO: 81 VHL peptide motif
- the carboxyl group (—COOH) of the C-terminal amino acid is amidated (—CONH 2 ).
- a fluorescent dye was further linked as a foreign substance to the N-terminal side of the peptides 1 and 2 obtained above to prepare two foreign substance introduction constructs according to this example.
- a general FAM that is, C 21 H 12 O 7 : 5 (6) -Carboxyfluorescein, molecular weight 376.3
- FAM fluorescein, molecular weight 376.3
- FITC Fluorescein isothiocyanate, molecular weight 389.4
- linker ie, Acp, ie 6-aminohexanoic acid (molecular weight). 131.2
- sample 2 a foreign substance introduction construct “(FITC)-(Acp) -WRRQARFKGTLKERCLQVVRSLVK” based on the peptide 2 Produced.
- sample 2 a foreign substance introduction construct “(FITC)-(Acp) -WRRQARFKGTLKERCLQVVRSLVK”
- Example 2 Cell membrane permeation function evaluation of sample 1 and sample 2 (1)> Human neonatal foreskin fibroblasts (ATCC, catalog number CRL-2097) were used as eukaryotic cells, and the cell membrane permeation function of the two types of samples (constructs for introduction of foreign substances) obtained in Example 1 was examined. That is, 90% Eagle MEM medium (containing 0.1% non-essential amino acid, 2 mM L-glutamine, 1 mM sodium pyruvate, 1.5 g / L sodium bicarbonate) and 10% serum (FBS). The fibroblasts were cultured using the mixed solution as a medium. The cultured cells were trypsinized with a 0.25% trypsin solution at 37 ° C. for 1 minute.
- ATCC catalog number CRL-2097
- trypsin is inactivated in a medium containing FBS, and a cell suspension (sample for introducing foreign substance) adjusted to have a cell concentration of about 5 ⁇ 10 4 cells / mL with the medium is prepared. did.
- 0.5 mL of the cell suspension was placed in a predetermined cell culture vessel (culture slide coated with 0.1% gelatin) and incubated overnight at 37 ° C. under 5% CO 2 . Thereafter, the medium was replaced with a fresh one, and any 1 mM sample solution prepared in Example 1 was added to the cell culture container so that the final sample concentration (peptide concentration) was 1 ⁇ M.
- 0.5 ⁇ L of the sample solution was added to the container. Then, 5% CO 2 conditions, and incubated for 1 hour or 4 hours at 37 ° C.. Cells cultured for 1 hour and cells cultured for 4 hours were each washed with PBS (Phosphate buffered saline), and the cells were fixed with methanol (10 minutes on ice).
- PBS Phosphate buffered saline
- FIGS. 1 and 2 are photomicrographs showing the results for the cell suspension to which the above sample 1 was added.
- FIG. 1 shows the results for the sample after 1 hour of culture and FIG.
- Example 2 shows the results for the sample after 4 hours of culture. As is apparent from these micrographs, it was confirmed that the added sample 1 rapidly permeated the cell membrane from the outside of the cell after the addition and was introduced into the cell. Furthermore, from the result of nuclear staining with DAPI, it was confirmed that a part of sample 1 introduced into the cytoplasm was also transferred into the nucleus (that is, introduced into the nucleus). Moreover, although the micrograph was not attached, the same result was obtained for Sample 2. That is, it was confirmed that the two types of constructs for introducing foreign substances (samples 1 and 2) obtained in Example 1 described above showed excellent cell membrane permeability by containing the carrier peptide fragment.
- Example 3 Cell membrane permeation function evaluation of sample 1 and sample 2 (2)> The two types of samples obtained in Example 1 above (constructs for introducing foreign substances) by changing the target cells into which foreign substances are introduced from human neonatal foreskin fibroblasts to human-derived iPS cells (Human induced pluripotent stem cells) ) Cell membrane permeation function was examined.
- iPS cells cell line: 201B2-082008KU
- mouse embryonic fibroblasts cell line: SNL 76/7, hereinafter referred to as “MEF”
- MEF mouse embryonic fibroblasts
- the obtained MEF was inactivated by mitomycin C treatment (3 hours), and trypsinized with a 0.25% trypsin solution containing 1 mM EDTA. After the above treatment, trypsin was inactivated in an FBS-containing medium, MEF medium (7% FBS (Gibco product), 2 mM L-glutamine (Gibco product), 50 units / mL penicillin and 50 ⁇ g / mL.
- MEF medium 7 FBS (Gibco product), 2 mM L-glutamine (Gibco product), 50 units / mL penicillin and 50 ⁇ g / mL.
- the MEF was seeded on a slide shape or a plate shape.
- the cell density was about 1.25 ⁇ 10 5 cells / mL.
- the vessel was then placed in an incubator and incubated overnight at 37 ° C. under 5% CO 2 . Thereafter, the MEF medium was removed and washed with PBS to prepare feeder cells.
- hESC medium ie human ES cell medium, here 20% KSR (Gibco product), 2 mM L-glutamine (Gibco product), 0.1% non-essential amino acid (Gibco product), DMEM / F12 medium (Gibco) containing 0.1 mM 2-mercaptoethanol (Gibco), 50 units / mL penicillin and 50 ⁇ g / mL streptomycin (Invitrogen, 4 ng / mL bFGF (Basic Fibroblast Growth Factor)) Product) was added, the iPS cells were peeled off using a cell scraper, and the colonies were broken by light pipetting. The suspension of iPS cells thus obtained was seeded on feeder cells in the culture vessel prepared as described above. Then was added the hESC medium, put the culture vessel in an incubator, 5% CO 2 conditions, and incubated overnight at 37 ° C..
- hESC medium ie human ES cell medium, here 20% KSR (Gi
- the medium was removed from the culture vessel, and the hESC medium supplemented with any 1 mM sample solution prepared in Example 1 so that the final sample concentration (peptide concentration) was 1 ⁇ M was cultured. Added to the container. Then, 5% CO 2 conditions, and incubated for 1 hour or 4 hours at 37 ° C.. Cells cultured for 1 hour and cells cultured for 4 hours were each washed with PBS (Phosphate buffered saline), and the cells were fixed with methanol (10 minutes on ice).
- PBS Phosphate buffered saline
- Example 2 Next, the same treatment as in Example 2 was performed, and the localization of the peptide bound with the fluorescent dye (ie, labeled with the fluorescent dye) in the iPS cells was confirmed using a confocal scanning laser microscope.
- 3 is a photomicrograph showing the results after 1 hour of culturing the cell suspension to which the sample 1 was added
- FIG. 4 is the results after 1 hour of culturing of the cell suspension to which the sample 2 was added. It is a microscope picture shown. As is clear from these micrographs, it was confirmed that both of the added sample 1 and sample 2 were rapidly introduced from the cell through the cell membrane and introduced into the cell after the addition.
- the present invention provides stem cells derived from human or non-human mammals (especially ES cells, iPS cells, or somatic cells) as a particularly preferred embodiment of the foreign substance introduction method disclosed herein.
- Stem cell from the outside of the cell cytoplasm (more preferably further into the nucleus), the method comprising introducing the desired foreign substance into the amino acid sequence of SEQ ID NO: 1 as the carrier peptide fragment, A carrier peptide fragment consisting of a modified amino acid sequence formed by substitution, deletion and / or addition (insertion) of one, two or three amino acid residues is provided. .
- the carrier peptide fragment consisting of the amino acid sequence of the above SEQ ID NO is a protein (typically about 300 to 1000 amino acids (for example, about 300 to 600)) or amino acid residues, particularly in stem cells such as iPS cells and ES cells. It is suitable for the purpose of introducing a polypeptide having a number of less than 300 or a peptide motif having a number of amino acid residues of 100 or less (particularly 50 or less).
- a target foreign substance having a predetermined function can be introduced into human or other mammalian stem cells (eg, somatic stem cells or induced pluripotent stem cells) and other target cells.
- mammalian stem cells eg, somatic stem cells or induced pluripotent stem cells
- transformation of the target cell for example, differentiation into a specific cell (neural cell, bone cell, muscle cell, skin cell, etc.) according to the foreign substance (peptide or the like) to be introduced. it can.
- a desired foreign substance can be introduced into the cytoplasm (preferably further in the nucleus) from the outside of eukaryotic cells (particularly various animal cells represented by humans and other mammals having no cell wall). Artificially created constructs are provided for introduction. By using such a construct, it is possible to effectively introduce a target foreign substance into a target cell, and to obtain a cell into which the foreign substance has been introduced and an organ or other living tissue containing the cell containing the foreign substance. .
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Abstract
Description
なお、本出願は2009年7月29日に出願された日本国特許出願2009-177101号に基づく優先権を主張しており、当該日本国出願の全内容は本明細書中に参照として援用されている。
例えば、特許文献1には、ポリペプチド、DNA等の外来物質を細胞内に導入するための細胞透過性キャリアペプチドが記載されている。この特許文献には、細胞透過性キャリアペプチドと異種ポリペプチド、DNA等を連結したキャリアペプチドコンジュゲートを用いることによって、ポリペプチド、DNA等の生理活性物質を高効率に細胞内に導入することができると記載されている。
また、導入したい外来物質(生理活性物質)として比較的分子量の大きいポリペプチド全体を特別な装置を用いることなく目的の細胞内に容易に導入して当該細胞の形質を転換したり機能を改善(若しくは向上)させたりする手法が求められている。
例えば、特許文献2には、SOCSタンパク質及びそれらのファミリータンパク質(以下総称して「SOCS系タンパク質」という。)を構成するペプチド鎖(アミノ酸配列)の一部であって、ElonginBCコンプレックスに結合すると考えられている特定領域「BC-ボックス」の全部又は一部を構成するアミノ酸配列が体性幹細胞に対する高い神経分化誘導活性を有するモチーフであることが開示されており、該モチーフを哺乳動物の体性幹細胞に導入することにより該導入細胞を神経細胞に分化誘導し得ることが開示されている。
即ち、ここで開示される外来物質導入方法は、WRRQARFK(配列番号1)からなるアミノ酸配列、若しくは該アミノ酸配列のうちの1個又は2個又は3個のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成されたアミノ酸配列の何れかから成るキャリアペプチドフラグメントと、該キャリアペプチドフラグメントのN末端側及び/又はC末端側に結合した目的の外来物質と、を有する外来物質導入用構築物を用意する工程と、
上記外来物質導入用構築物を、目的とする真核細胞を含む試料(典型的には該細胞を含む培養物)中に供給する工程と、
上記外来物質導入用構築物が供給された上記試料をインキュベートして(即ち対象の細胞が生存可能な条件下に試料を一定期間保持して)、上記試料中の真核細胞内に上記構築物を導入する工程と、を包含する。
ここで「外来物質」は、上記キャリアペプチドフラグメントのN末端側又はC末端側に直接的又は適当なリンカーを介して間接的に結合可能な無機化合物及び有機化合物であって、真核細胞内に導入可能な分子サイズ及び化学的性質を有するものをいう。
即ち、上記構成の本発明の導入方法によると、目的とする外来物質(典型的にはペプチド、核酸、色素、薬剤等の有機化合物)を上記キャリアペプチド(フラグメント)のN末端側及び/又はC末端側に直接的又は適当なリンカーを介して間接的に結合させて構築した外来物質導入用構築物を、対象とする真核細胞を含む試料(典型的には該細胞を含む培養物)中に供給する(即ち生存する真核細胞に添加する)ことによって、当該目的の外来物質を真核細胞の外部(細胞膜の外側)から細胞膜を通過させて細胞質内(好ましくはさらに核膜を通過させて核内)に高効率に移送することができる。
ここで「ペプチド」は、2個以上のアミノ酸がペプチド結合により結合した構造を有する有機化合物をいい、ポリペプチド(典型的にはアミノ酸残基数が10以上300未満)やタンパク質(典型的には上記ポリペプチドよりも多い数(300以上)のアミノ酸残基から成る高分子化合物)を包含する。
また、ここで「核酸」は、ヌクレオチドの重合体をいい、DNAおよびRNAを包含する。
この種の有機化合物を含むように作製された構築物は、効率よく目的の細胞内に導入することができる。
本態様の方法によると、上記合成ペプチドの状態で、目的のペプチド(即ち該ペプチドを構成するアミノ酸配列)を一つのペプチドモチーフとして標的とする細胞内に導入することができる。
本発明によると、ヒトその他哺乳類の幹細胞(例えば体性幹細胞や人工多能性幹細胞)に所定の機能を有する目的の外来物質を導入することができる。このことにより、導入する外来物質(ペプチド等)に応じて当該幹細胞の形質転換、例えば特定の細胞(神経細胞、骨細胞、筋肉細胞、皮膚細胞、等)への分化を実現することができる。
即ち、ここで開示される外来物質導入用構築物は、WRRQARFK(配列番号1)からなるアミノ酸配列、若しくは該アミノ酸配列のうちの1個又は2個又は3個のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成されたアミノ酸配列の何れかから成るキャリアペプチドフラグメントと、該キャリアペプチドフラグメントのN末端側及び/又はC末端側に結合した目的の外来物質とを有する。
かかる構築物を利用して本発明の外来物質導入方法を実施することにより、目的の細胞に目的の外来物質を効果的に導入することができる。また、該外来物質が導入された細胞並びに該外来物質を含む細胞を含む器官その他の生体組織を得ることができる。
また、特に好ましくは、上記外来物質はペプチドであり、上記外来物質導入用構築物は、該外来物質としてのペプチドのフラグメントと上記キャリアペプチドフラグメントとを有する合成ペプチドである。
また、本発明は、本明細書に開示されている内容と当該分野における技術常識とに基づいて実施することができる。なお、以下の説明では、場合に応じてアミノ酸をIUPAC-IUBガイドラインで示されたアミノ酸に関する命名法に準拠した1文字表記(但し配列表では3文字表記)で表す。
ここで配列番号1に記載される具体的なアミノ酸配列は、上記IBVのヌクレオカプシドタンパク質(Nプロテイン)の第71番目~第78番目のアミノ酸残基までの合計8アミノ酸残基から成る配列部分(即ちモチーフ)に相当する核小体局在シグナル(Nucleolar localization sequence)であるところ、本発明者によって新たに優れた細胞膜透過性を示すことが見出された配列である。即ち、10以下のアミノ酸残基(ここでは8アミノ酸残基)から成る極めて短い鎖長のペプチドフラグメントであるにもかかわらず外来物質を細胞外から細胞質内に導入することができる。
ここで開示される「キャリアペプチドフラグメント」は、典型的には配列番号1に記載のアミノ酸配列と同一の配列であるが、当該同一配列の他に、細胞膜透過性を損なうことなく1個または数個(典型的には2個又は3個)のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成されたアミノ酸配列を包含する。即ち、そのような軽微な改変配列は、ここで開示される情報に基づいて当業者にとって容易に利用されるものであり、ここで開示される技術的思想としての「キャリアペプチドフラグメント」に包含されるからである。典型例として、配列番号1のアミノ酸配列のうち1個又は数個(典型的には2個又は3個)のアミノ酸残基が保守的に置換したいわゆる同類置換(conservative amino acid replacement)によって生じた配列(例えば塩基性アミノ酸残基が別の塩基性アミノ酸残基に置換した配列)、或いは、所定のアミノ酸配列について1個又は数個(典型的には2個又は3個)のアミノ酸残基が付加(挿入)した若しくは欠失した配列が挙げられる。
典型的には、外来物質導入用構築物として作製する合成ペプチドを構成する総アミノ酸残基数が1000以下が適当であり、好ましくは600以下であり、さらに好ましくは500以下であり、特に300以下(更には100以下、例えば10~50)が好適である。このような比較的短いペプチドは合成が容易であり、使用しやすい。
例えば、導入対象の真核細胞がヒトその他哺乳動物の幹細胞(体性幹細胞、胚性幹細胞、人工多能性幹細胞(Induced pluripotent stem cells:以下iPS細胞という。)を包含する。)である場合、当該幹細胞の分化誘導に関与する種々の生理活性を有するペプチドの成熟型又はその前駆体の利用が好ましい。また、導入対象の真核細胞が癌細胞(腫瘍細胞)である場合、当該癌細胞(腫瘍細胞)のアポトーシス誘導に関与する種々のペプチドの利用が好ましい。
従って、本発明の好適な一実施形態として、iPS細胞の作製に関与する複数の遺伝子のうちの少なくとも一つの遺伝子(例えばSox2)がコードするペプチド(例えばSOX2タンパク質)を外来物質として本発明に係る外来物質導入用構築物を作製し、該構築物を所定の真核細胞(ヒト皮膚線維芽細胞等)に導入することを特徴とするiPS細胞作製方法が挙げられる。
即ち、特許文献2には、エロンジンA(ElonginA)と複合体を形成して転写調節因子として働くことが知られているエロンジンBC(ElonginBC)コンプレックス(具体的にはElonginCの一部)に結合し得る領域(アミノ酸配列)であるSOCS-ボックスを有する種々のSOCSタンパク質(サイトカイン情報伝達のサプレッサー)及びそれらのファミリータンパク質(以下総称して「SOCS系タンパク質」という。)を構成するアミノ酸配列の一部であって、ElonginBCコンプレックスに結合すると考えられている特定領域「BC-ボックス」に包含されるアミノ酸配列が、体性幹細胞に対して高い神経分化誘導活性を有することが記載されている。
具体的には、mSOCS-1(配列番号2),mSOCS-2(配列番号3),mSOCS-3(配列番号4),mSOCS-4(配列番号5),mSOCS-5(配列番号6),hSOCS-6(配列番号7),hSOCS-7(配列番号8),hRAR-1(配列番号9),hRAR-like(配列番号10),mWSB-1(配列番号11),mWSB-2(配列番号12),mASB-1(配列番号13),mASB-2(配列番号14),hASB-3(配列番号15),LRR-1(配列番号16),hASB-7(配列番号17),mASB-10(配列番号18),hASB-14(配列番号19),に含まれるBC-ボックスのN末端から15個の連続するアミノ酸残基から成るアミノ酸配列を示している(非特許文献3~6参照)。
また、特に詳細な説明は省略するが、配列番号20~80は、ウイルス(HIV、AdV、SIV等)や哺乳動物から同定された種々のSOCS系タンパク質のBC-ボックスに含まれるアミノ酸配列及び該配列から成るペプチドを示している。例えば、配列番号75および配列番号79は、ヒトから同定されたSOCS系タンパク質(MUF1)のBC-ボックスに含まれるアミノ酸配列である。また、配列番号80は、マウスから同定されたSOCS系タンパク質mCIS(cytokine-inducible SH2-containing protein)のBC-ボックスに含まれるアミノ酸配列である。
これらは例示であり、BC-ボックスの構成アミノ酸配列(モチーフ)をこれらに限定することを意図したものではない。ここに例示するまでもなく、様々なBC-ボックスの構成アミノ酸配列が本願出願当時に出版されている数々の文献に記載されている。それらアミノ酸配列は一般的な検索手段によって容易に知ることができる。
即ち、かかる構成の人工ペプチド(神経分化誘導ペプチド)は、上記キャリアペプチドフラグメントのN末端側又はC末端側に、神経分化誘導に関するペプチドモチーフとしていずれかのBC-ボックス由来のアミノ酸配列(以下「BC-ボックス関連配列」ともいう。典型的には配列番号2~80のうちのいずれかに示すアミノ酸配列から選択される少なくとも10個(例えばN末端から少なくとも10個)の連続するアミノ酸残基から成るアミノ酸配列)を備えるように合成され得る。
或いはまた、配列番号81に示す15個の連続するアミノ酸配列もまた上記BC-ボックス関連配列と同様の目的に使用することができる。即ち、配列番号81に示すアミノ酸配列は、特許文献3に記載されるように、神経分化誘導性を示すことが知られているVHL(フォン・ヒッペル・リンド)タンパク質のアミノ酸配列の第157番目~第171番目までの15個の連続するアミノ酸残基から成る部分アミノ酸配列(VHL関連ペプチドモチーフ)である。
なお、上述した本発明に係るキャリアペプチドフラグメントの場合と同様、神経分化誘導に関するペプチドモチーフとしての機能を保持する限りにおいて、1個または数個(例えば5個以下典型的には2個又は3個)のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成される改変アミノ酸配列もまた神経分化誘導に関するペプチドモチーフ(外来物質)として使用し得ることは勿論である。
神経分化誘導剤の形態に関して特に限定はない。例えば、典型的な形態として、液剤、懸濁剤、乳剤、エアロゾル、泡沫剤、顆粒剤、粉末剤、錠剤、カプセル、軟膏が挙げられる。また、注射等に用いるため、使用直前に生理食塩水又は適当な緩衝液(例えばPBS)等に溶解して薬液を調製するための凍結乾燥物、造粒物とすることもできる。
なお、神経分化誘導ペプチド(主成分)及び種々の担体(副成分)を材料にして種々の形態の薬剤(組成物)を調製するプロセス自体は従来公知の方法に準じればよく、かかる製剤方法自体は本発明を特徴付けるものでもないため詳細な説明は省略する。処方に関する詳細な情報源として、例えばComprehensive Medicinal Chemistry, Corwin Hansch監修,Pergamon Press刊(1990)が挙げられる。
例えば、ここで開示されるBC-ボックス関連配列或いはVHLペプチドモチーフと、キャリアペプチドフラグメントと、を含むように合成された神経分化誘導ペプチド(即ち該合成ペプチドを含む神経分化誘導剤)は、液剤として、静脈内、筋肉内、皮下、皮内若しくは腹腔内への注射によって患者(即ち生体)に所望する量だけ投与することができる。或いは、錠剤等の固体形態のものは経口投与することができる。これにより、生体内で、典型的には患部又はその周辺に存在する体性幹細胞から、神経細胞を発生(生産)させることができる。このため、神経再生が有力な治療法となる種々の神経疾患を効果的に治療することが可能となる。例えば、パーキンソン病、脳梗塞、アルツハイマー病、脊髄損傷による身体の麻痺、脳挫傷、筋萎縮性側索硬化症、ハンチントン病、脳腫瘍、網膜変性症等の神経疾患を再生医療的アプローチによって治療することが実現される。
以上の説明から明らかなように、本発明は別の側面として、ここで開示される上記構成の神経分化誘導ペプチドのいずれかを利用することによって、神経疾患治療に有用な、神経細胞に分化誘導された細胞、細胞塊又は生組織を提供することができる。
人工ペプチドは、ペプチド鎖を構成する全アミノ酸残基数が1000以下(好適には600以下、特に好ましくは300以下、例えば50以下)であるものが望ましい。このような短鎖長のペプチドは化学合成手法によって容易に構築することができるため、目的とする真核細胞を含む試料に供給し易い。
なお、ペプチドのコンホメーション(立体構造)については特に限定されるものではないが、免疫原(抗原)になり難いという観点から直鎖状又はへリックス状のものが好ましい。
なお、人工ペプチドとしては、全てのアミノ酸残基がL型アミノ酸であるものが好ましいが、内在するキャリアペプチドフラグメントならびにペプチドモチーフの所望する機能を失わない限りにおいて、アミノ酸残基の一部又は全部がD型アミノ酸に置換されているものであってもよい。
また、内在するキャリアペプチドフラグメントならびに外来物質であるペプチドモチーフの所望する機能を失わない限りにおいて、これら配列に含まれ得ない付加的な配列を部分的に含み得る。例えばリンカーとして機能する数個のアミノ酸残基(例えばグリシン残基)をキャリアペプチドフラグメントと外来ペプチドモチーフとの間に配置した構成のアミノ酸配列を構築してもよい。
一般的な技法によって、この組換えベクターを所定の宿主細胞(例えばイースト、昆虫細胞、植物細胞、動物(哺乳類)細胞)に導入し、所定の条件で当該宿主細胞又は該細胞を含む組織や個体を培養する。このことにより、目的とするポリペプチドを細胞内で発現、生産させることができる。そして、宿主細胞(分泌された場合は培地中)からポリペプチドを単離し、精製することによって、目的のアミノ酸配列から成るペプチドを得ることができる。一般的な技法によって、この組換えベクターを所定の宿主細胞(例えばイースト、昆虫細胞、植物細胞、哺乳類細胞)に導入し、所定の条件で当該宿主細胞又は該細胞を含む組織や個体を培養する。このことにより、目的とするポリペプチドを細胞内で発現、生産させることができる。そして、宿主細胞(分泌された場合は培地中)からポリペプチドを単離し、精製することによって、目的のペプチド(即ち外来物質導入用構築物)を得ることができる。
なお、組換えベクターの構築方法及び構築した組換えベクターの宿主細胞への導入方法等は、当該分野で従来から行われている方法をそのまま採用すればよく、かかる方法自体は特に本発明を特徴付けるものではないため、詳細な説明は省略する。
或いは、無細胞タンパク質合成システム用の鋳型DNA(即ち目的とする人工ペプチドのアミノ酸配列をコードするヌクレオチド配列を含む合成遺伝子断片)を構築し、ペプチド合成に必要な種々の化合物(ATP、RNAポリメラーゼ、アミノ酸類等)を使用し、いわゆる無細胞タンパク質合成システムを採用して目的のポリペプチドをインビトロ合成することができる。無細胞タンパク質合成システムについては、例えばShimizuらの論文(Shimizu et al., Nature Biotechnology, 19, 751-755(2001))、Madinらの論文(Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564(2000))が参考になる。これら論文に記載された技術に基づいて、本願出願時点において既に多くの企業がポリペプチドの受託生産を行っており、また、無細胞タンパク質合成用キット(例えば、日本の東洋紡績(株)から入手可能なPROTEIOS(商標)Wheat germ cell-free protein synthesis kit)が市販されている。
従って、細胞質内(好ましくは核内)への導入対象であるペプチドモチーフに対応するアミノ酸配列(例えば上述したBC-ボックス関連配列)を決定し、上記配列番号1に示す細胞膜透過性のキャリアペプチドフラグメントと合わせてペプチド鎖を設計しさえすれば、そのアミノ酸配列に従って無細胞タンパク質合成システムによって目的の人工ペプチドを容易に合成・生産することができる。例えば、日本の(株)ポストゲノム研究所のピュアシステム(登録商標)に基づいてペプチドを容易に生産することができる。
計2種類のペプチド(サンプル1~2)を後述するペプチド合成機を用いて製造した。表1には、これら合成ペプチドのアミノ酸配列を列挙している。
具体的には、蛍光色素として一般的なFAM(即ち、C21H12O7:5(6)-Carboxyfluorescein、分子量376.3)を常法に基づいて上記ペプチド1のN末端側に直接的に結合させ、ペプチド1に基づく外来物質導入用構築物である「(FAM)-WRRQARFK」を作製した。以下、サンプル1と呼称する。
他方、蛍光色素として一般的なFITC(即ち、C21H11NO5S: Fluorescein isothiocyanate 、分子量389.4)をリンカーとしてよく知られているAcp即ち6-アミノヘキサン酸(6-Aminocaproic acid、分子量131.2)を介在させて常法に基づいて上記ペプチド2のN末端側に間接的に結合させ、ペプチド2に基づく外来物質導入用構築物である「(FITC)-(Acp)-WRRQARFKGTLKERCLQVVRSLVK」を作製した。以下、サンプル2と呼称する。
こうして得られた各サンプルペプチドをそれぞれPBS(Phosphate buffered saline)に希釈し、サンプル(ペプチド)濃度が1mMのサンプル溶液を計2種類作製した。
真核細胞としてヒト新生児包皮線維芽細胞(ATCC、カタログ番号CRL-2097)を使用し、上記実施例1で得られた2種のサンプル(外来物質導入用構築物)の細胞膜透過機能を調べた。
即ち、90%イーグルMEM培地(0.1%の非必須アミノ酸、2mMのL-グルタミン、1mMのピルビン酸ナトリウム、1.5g/Lの炭酸水素ナトリウムを含む。)および10%血清(FBS)の混合液を培地として使用し、上記線維芽細胞を培養した。
培養細胞は、0.25%トリプシン溶液で37℃1分間のトリプシン処理を行った。上記処理後、FBS含有培地にてトリプシンを失活させるとともに、当該培地により細胞濃度が凡そ5×104cells/mLとなるように調整した細胞懸濁液(外来物質導入用の試料)を調製した。
1時間培養後の細胞、4時間培養後の細胞をそれぞれPBS(Phosphate buffered saline)で洗浄し、該細胞をメタノールで固定した(氷上で10分間)。
図1~2は、上記サンプル1を添加した細胞懸濁液についての結果を示す顕微鏡写真であり、図1は1時間培養後、図2は4時間培養後の試料についての結果である。
これら顕微鏡写真から明らかなように、上記添加されたサンプル1は、添加後迅速に細胞外から細胞膜を透過して細胞内に導入されていることが確認された。更にDAPIによる核染色の結果から細胞質内に導入されたサンプル1の一部は核内にも移行していること(即ち核内に導入されたこと)が確認された。また、顕微鏡写真を添付していないが、サンプル2についても同様の結果であった。
即ち、上記実施例1で得られた計2種(サンプル1~2)の外来物質導入用構築物は、上記キャリアペプチドフラグメントを含むことにより優れた細胞膜透過性を示すことが確認された。
外来物質を導入する対象の細胞をヒト新生児包皮線維芽細胞からヒト由来のiPS細胞(Human induced pluripotent stem cells)に変更して上記実施例1で得られた2種のサンプル(外来物質導入用構築物)の細胞膜透過機能を調べた。なお、本実施例で使用するiPS細胞(細胞株:201B2-082008KU)とフィーダー細胞であるマウス胎児線維芽細胞(細胞株:SNL 76/7、以下「MEF」という。)は、京都大学再生医科学研究所山中研究室(山中伸弥教授)から供与されたものを使用した。
先ず、入手したMEFをマイトマイシンC処理(3時間)して不活性化し、1mMのEDTAを含む0.25%トリプシン溶液でトリプシン処理した。上記処理後、FBS含有培地にてトリプシンを失活させるとともに、MEF用培地(7%のFBS(Gibco社製品)、2mMのL-グルタミン(Gibco社製品)、50units/mLのペニシリンと50μg/mLのストレプトマイシン(Gibco社製品)を含むD-MEM培地(Dulbecco's Modified Eagle Medium):Gibco社製品)を用いてMEFを適当な細胞密度に調整し、0.1%ゼラチンで表面コートした培養容器(カルチャースライド形状又はプレート形状)に上記MEFを播種した。ここでは細胞密度は、約1.25×105cells/mLとなるように播種した。次いで、上記容器をインキュベーターに入れ、5%CO2条件下、37℃で一晩インキュベートした。
その後、MEF用培地を除去し、PBSで洗浄することによってフィーダー細胞を作製した。
次いで、1mLのhESC培地(即ちヒトES細胞培地、ここでは20%のKSR(Gibco社製品)、2mMのL-グルタミン(Gibco社製品)、0.1%の非必須アミノ酸(Gibco社製品)、0.1mMの2-メルカプトエタノール(Gibco社製品)、50units/mLのペニシリンと50μg/mLのストレプトマイシン(Invitrogen社製品、4ng/mLのbFGF(Basic Fibroblast Growth Factor)を含むDMEM/F12培地(Gibco社製品))を添加し、セルスクレーパを用いてiPS細胞を剥がし、軽くピペッティングを行ってコロニーを崩した。
こうして得られたiPS細胞の懸濁物を上記のように作製しておいた培養容器中のフィーダー細胞上に播種した。そして上記hESC培地を添加し、培養容器をインキュベーターに入れ、5%CO2条件下、37℃で一晩インキュベートした。
1時間培養後の細胞、4時間培養後の細胞をそれぞれPBS(Phosphate buffered saline)で洗浄し、該細胞をメタノールで固定した(氷上で10分間)。
これら顕微鏡写真から明らかなように、上記添加されたサンプル1およびサンプル2は、いずれも添加後迅速に細胞外から細胞膜を透過して細胞内に導入されていることが確認された。更にDAPIによる核染色の結果から細胞質内に導入されたサンプル1およびサンプル2の一部は核内にも移行していること(即ち核内に導入されたこと)が確認された。即ち、上記実施例1で得られた計2種(サンプル1~2)の外来物質導入用構築物は、上記キャリアペプチドフラグメントを含むことにより優れた細胞膜透過性を示すことが確認され、蛍光色素のみならず、外来物質たるペプチド(ここでは配列番号81に示すVHLペプチド)を効率よく細胞の外部から細胞膜を通して細胞質内、更には核内に導入(移送)し得ることが確かめられた。
また、本発明によって、真核細胞(特に細胞壁を有しないヒトやそれ以外の哺乳動物に代表される種々の動物細胞)の外部から細胞質内(好ましくはさらに核内)に目的とする外来物質を導入するために人為的に作製された構築物が提供される。かかる構築物を利用することにより、目的の細胞に目的の外来物質を効果的に導入させ、該外来物質が導入された細胞並びに該外来物質を含む細胞を含む器官その他の生体組織を得ることができる。
Claims (7)
- 真核細胞の外部から該細胞の細胞質内に目的とする外来物質を導入する方法であって、
以下のアミノ酸配列:
WRRQARFK(配列番号1)
若しくは該アミノ酸配列のうちの1個又は2個又は3個のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成されたアミノ酸配列の何れかから成るキャリアペプチドフラグメントと、該キャリアペプチドフラグメントのN末端側及び/又はC末端側に結合した目的の外来物質と、を有する外来物質導入用構築物を用意する工程と、
前記外来物質導入用構築物を、目的とする真核細胞を含む試料中に供給する工程と、
前記外来物質導入用構築物が供給された前記試料をインキュベートして、前記試料中の真核細胞内に前記構築物を導入する工程と、
を包含する方法。 - 前記外来物質は、ペプチド、核酸、色素及び薬剤から成る群から選択されるいずれかの有機化合物である、請求項1に記載の方法。
- 前記外来物質はペプチドであり、前記外来物質導入用構築物は、該外来物質としてのペプチドのフラグメントと前記キャリアペプチドフラグメントとを有する合成ペプチドである、請求項2に記載の方法。
- 前記外来物質導入用構築物を導入する対象の真核細胞がヒト又はヒト以外の哺乳動物由来の幹細胞である、請求項1~3の何れかに記載の方法。
- 真核細胞の外部から該細胞の細胞質内に目的とする外来物質を導入するために作製された外来物質導入用構築物であって、
以下のアミノ酸配列:
WRRQARFK(配列番号1)
若しくは該アミノ酸配列のうちの1個又は2個又は3個のアミノ酸残基が置換、欠失及び/又は付加(挿入)されて形成されたアミノ酸配列の何れかから成るキャリアペプチドフラグメントと、該キャリアペプチドフラグメントのN末端側及び/又はC末端側に結合した目的の外来物質と、を有する外来物質導入用構築物。 - 前記外来物質は、ペプチド、核酸、色素及び薬剤から成る群から選択されるいずれかの有機化合物である、請求項5に記載の構築物。
- 前記外来物質はペプチドであり、該外来物質としてのペプチドのフラグメントと前記キャリアペプチドフラグメントとを有する合成ペプチドである、請求項6に記載の構築物。
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