JP4368865B2 - Gene transfer method - Google Patents

Description

  The present invention relates to a method for improving the efficiency of gene transfer into a target cell in the fields of medicine, cell engineering, genetic engineering, developmental engineering, etc., and enabling efficient transformation of the target cell and a series of related series. Regarding technology.

  The mechanism of many human diseases has been elucidated, and recombinant DNA technology and gene transfer technology into cells have rapidly advanced, so that somatic gene therapy methods for treating severe genetic diseases in recent years Protocol development is underway. Recently, attempts have been made to apply gene therapy not only to genetic diseases but also to the treatment of viral infections such as AIDS and cancer.

  Many gene therapies that have been studied for clinical application in humans so far involve gene transfer into cells using recombinant retroviral vectors. A retrovirus vector is a preferable gene introduction means for gene therapy in which gene expression over a long period of time is desired because it efficiently introduces a target foreign gene into a cell and stably integrates it into its chromosomal DNA. The vector is devised in various ways so as not to adversely affect the organism into which the gene has been introduced. For example, the replication function in the vector is deleted so that the vector itself used for gene transfer replicates in the cell and does not repeat unlimited infection (gene transfer).

  Since these vectors (replication-defective retrovirus vectors) cannot self-replicate, retrovirus vectors wrapped in virus particles are generally prepared using retrovirus-producing cells (packaging cells). The simplest method for introducing a gene into a target cell with high efficiency is to co-culture the retrovirus-producing cell and the target cell. However, in this method, there is a possibility that retrovirus-producing cells are mixed into the gene-introduced target cells that are transplanted into the living body.

  Recently, it has been reported that the efficiency of gene transfer into cells increases when fibronectin and its fragments, components of the extracellular matrix, coexist during retrovirus gene transfer [Journal of Clinical Investigation. (J. Clin. Invest.), 93, 1451-1457 (1994), Blood, 88, 855-862 (1996)]. In addition, fibronectin fragments produced by genetic engineering have the same properties, and it has been shown that foreign genes can be efficiently introduced into hematopoietic stem cells using this (WO95 / 26200). issue). It has been suggested that the improvement of gene transfer efficiency by fibronectin involves the binding of heparin-binding region in fibronectin and retrovirus.

  WO97 / 18318 discloses that functional substances other than fibronectin such as fibroblast growth factor improve gene transfer efficiency. Furthermore, the same publication shows that when a functional substance having retrovirus binding activity is mixed with another functional substance having cell binding activity, the same improvement in gene transfer efficiency can be seen. Has been.

  Such a gene introduction method using a functional substance enables highly efficient gene introduction without co-culture of retrovirus-producing cells and target cells. The improvement in gene transfer efficiency by this method is thought to be due to the fact that the functional substance co-arranges the retrovirus and the target cell in close proximity and the opportunity for interaction between the two increases.

  Thus, in gene transfer using retroviruses, gene transfer occurs as a result of infection of retrovirus target cells. However, the efficiency of gene transfer by retrovirus is still not satisfactory when considering practical clinical application, and further improvement in infection efficiency is strongly desired.

  As a means of improving infection efficiency, that is, gene transfer efficiency, it is conceivable to increase the concentration (titer) of retrovirus in the virus solution (virus supernatant) to be used, but usually has a high titer virus production ability. Construction and establishment of virus-producing cells requires a great deal of effort. Pseudo-type virus vector using the envelope protein of VSV virus [Proc. Natl. Acad. Sci. USA], Volume 90, Proceedings of the National Academy of Sciences of the USA 8033-8037 (1993)] can be concentrated by centrifugation, but this is a method that can be used only for this vector and is versatile.

  In addition, it is considered that high gene transfer efficiency can be obtained even if the purity of the target cell is low if the retroviral infection can occur specifically in the target cell during gene transfer. There is no known efficient and efficient method.

  In view of such circumstances, the main object of the present invention is to provide an improved method for improving the gene transfer efficiency and efficiently transforming the target cell in the gene transfer to the target cell by retrovirus.

  Other objects and advantages of the present invention will be made clear below with reference to the accompanying drawings.

  The present inventors contact a retrovirus with a functional substance having a virus binding activity immobilized on a carrier before infecting a target cell, and then subject the carrier to a washing operation. We found that gene transfer efficiency was improved.

  In addition, when target cells are infected with retroviruses, specific target cells can be obtained by coexisting functional substances such as target cell-specific antibodies, laminin, laminin-derived sugar chains, and high-mannose sugar chains. And / or found that gene transfer with high efficiency is possible.

  Furthermore, it has been found that gene introduction efficiency can be improved by subjecting target cells before being subjected to gene introduction to an appropriate pretreatment.

  Furthermore, it has been found that the effect on gene transfer can be improved by chemically modifying a functional substance having virus binding activity to increase its basicity.

  The present invention has been completed based on these new findings of the present inventors.

That is, the first aspect of the present invention is characterized by including the following steps in a gene introduction method into a target cell by a retrovirus.
(1) a step of bringing a retrovirus-containing solution into contact with a functional substance having a retrovirus-binding activity immobilized on a carrier;
(2) Washing the retrovirus-bound carrier, and (3) Incubating the retrovirus-bound carrier in contact with the target cells.

  The step (1) is not particularly limited. For example, the step (1) is performed for 1 hour or longer, preferably 3 hours or longer. It is also carried out by physically increasing the frequency of contact between the retrovirus and the retrovirus-binding substance.

  The functional substance having retrovirus binding activity used in the present invention is not particularly limited. For example, fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran, a fragment thereof, or the equivalent thereof Substances having retrovirus binding activity can be used. Moreover, what has target cell binding activity as said functional substance may be used, or you may use said functional substance in combination with the other functional substance which has target cell binding activity. The functional substance having the target cell binding activity is not particularly limited, and for example, a cell adhesive protein, hormone, cytokine, antibody, sugar chain, carbohydrate, metabolite, or the like can be used.

  As the retrovirus used for gene transfer in the present invention, for example, a culture supernatant of a retrovirus-producing cell can be used. The culture supernatant may be obtained in the presence of a substance that promotes retrovirus production, such as sodium butyrate.

A second aspect of the present invention is characterized in that a retrovirus is infected to a target cell in the presence of the following two functional substances in a method for gene transfer into a target cell by a retrovirus.
(1) a functional substance having retrovirus binding activity, and (2) a target cell-specific antibody.

  The target cell-specific antibody used in the present invention is not particularly limited, and examples thereof include those that recognize biological substances present on the target cell surface.

A third aspect of the present invention is characterized in that a retrovirus is infected to a target cell in the presence of the following two functional substances in a method for gene transfer into a target cell by a retrovirus.
(1) a functional substance having retrovirus binding activity; and (2) laminin, laminin fragment, laminin-derived sugar chain or high mannose sugar chain.

  The functional substance having retrovirus binding activity used in the second and third aspects of the present invention is not particularly limited, and examples thereof include fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE- Dextran, a fragment thereof, or a substance having retrovirus binding activity equivalent to these can be used. The functional substance may have a target cell binding activity. Moreover, the functional substance used may be used in a state of being immobilized on a suitable carrier.

  According to a fourth aspect of the present invention, in the method for introducing a gene into a target cell by a retrovirus, the target cell is cultured in a medium having a reduced Fe concentration prior to contact with the retrovirus. There is no particular limitation on the medium used in the present invention. For example, a medium containing deferoxamine can be used, and it is preferably performed in the presence of a functional substance.

  The fifth aspect of the present invention relates to a method for improving the retrovirus binding activity of a peptide or protein, characterized by chemically modifying the peptide or protein. The chemical modification is not particularly limited, and examples thereof include activation of amino acid residues of peptides or proteins and introduction of basic residues. The method used for activating the amino acid residue is not particularly limited, and for example, treatment with water-soluble carbodiimide and treatment with water-soluble carbodiimide and a diamino compound are preferable. The chemically modified peptide or protein obtained by this method can be suitably used for gene transfer into a target cell by a retrovirus.

  Provided is an improved method for improving the efficiency of gene transfer and efficiently transforming target cells in gene transfer into target cells by retroviruses.

  In the gene introduction method of the present invention, a recombinant retroviral vector is usually used, and a replication-defective recombinant retroviral vector is particularly preferable. The vector is non-pathogenic, deficient in replication so that it cannot replicate in infected cells. These vectors can invade a host cell such as a vertebrate cell, particularly a mammalian cell, and stably incorporate a foreign gene inserted into the vector into its chromosomal DNA.

  In the present invention, a foreign gene to be introduced into a cell is used by being inserted into a recombinant retroviral vector under the control of an appropriate promoter, for example, an LTR promoter or a foreign promoter present in a retroviral vector. be able to. In addition, in order to achieve efficient transcription of a foreign gene, other regulatory elements cooperating with a promoter and transcription initiation site, for example, an enhancer sequence and a terminator sequence may be present in the vector. The foreign gene to be introduced may be a natural gene, an artificially produced gene, or a DNA molecule having a different origin bound by a known means such as ligation.

  As the foreign gene inserted into the retroviral vector, any gene desired to be introduced into the cell can be selected. For example, foreign genes include enzymes encoding proteins and proteins related to the disease to be treated, intracellular antibodies (see, for example, WO94 / 02610), growth factors, antisense nucleic acids, ribozymes, false primers (For example, refer to WO90 / 13641) or the like can be used.

  The retroviral vector used in the present invention may contain an appropriate marker gene that enables selection of the transfected cell. As the marker gene, for example, a drug resistance gene that confers resistance to antibiotics on a cell, a reporter gene that can distinguish cells introduced by enzymatic activity, and the like can be used.

  Examples of the vector that can be used in the present invention include MFG vector (ATCC No. 68754), α-SGC vector (ATCC No. 68755), LXSN vector [BioTechniques, Vol. 7, 980-990 pages ( (1989)) and the like. In addition to the PM5neo vector [Experimental Hematology (Exp. Hematol.), Vol. 23, 630-638 (1995)], the retroviral vector used in the examples of the present specification is a marker gene. As containing the neomycin phosphotransferase gene. Therefore, cells transfected with the vector can be confirmed using its resistance to G418 as an index.

  These vectors are also known packaging cell lines such as PG13 (ATCC CRL-10686), PG13 / LNc8 (ATCC CRL-10785), PA317 (ATCC CRL-9078), GP + E-86 (ATCC CRL-9642). And GP + envAm12 (ATCC CRL-9641), Proceedings of the National Academy of Sciences of the USA, Vol. 85, pages 6460-6464 (1988), etc. By using a cell line, the vector can be prepared as a packaged virus particle.

  Known culture media such as Dulbecco's modified Eagle's medium, Iscob's modified Dulbecco's medium, etc. can be used for culturing virus-producing cells and target cells produced by introducing retroviral vectors into packaging cells. It can be obtained as a commercial product from Gibco. Various components can be added to these media depending on the type of cells targeted for gene transfer and other purposes. For example, serum and various cytokines can be added and used for the purpose of promoting or suppressing the growth and differentiation of target cells. As the serum, for example, calf serum (CS) or fetal calf serum (FCS) can be used. Cytokines include interleukins (IL-3, IL-6, etc.), colony stimulating factors (G-CSF, GM-CSF, etc.), stem cell factor (SCF), erythropoietin and various cell growth factors. There are many of them that are commercially available. In using these cytokines, those having an action according to the purpose may be selected, and these may be used in combination as necessary.

  The gene introduction method of the present invention uses a retrovirus-containing sample, for example, a culture supernatant of virus-producing cells, and the preparation method is not particularly limited. For example, it is known that the addition of sodium butyrate during the cultivation of virus-producing cells increases the number of virus particles produced in the supernatant [Human Gene Therapy, Vol. 1195-1202 (1995)], by using the gene transfer method of the present invention, the high-titer virus supernatant thus prepared can be used without any problem.

  The present invention is characterized in that a retrovirus is infected with a target cell in the presence of a functional substance having a retrovirus binding site. By infecting cells with a retrovirus in the presence of an effective amount of these functional substances, transgenic cells can be obtained with high efficiency. It is also possible to easily remove a virus infection inhibitory substance present in the virus supernatant using the functional substance. Furthermore, gene introduction can be performed with higher specificity and / or efficiency by allowing a functional substance having target cell binding activity to coexist.

  In the present specification, the effective amount is an amount effective for the transformation of the target cell by gene introduction into the target cell by retrovirus, and an appropriate amount is selected according to the functional substance used and the type of the target cell. . This amount can be determined, for example, by measuring gene transfer efficiency by the method described herein. In addition, the target cell binding activity in the present specification includes not only an activity that substantially binds to cells but also an activity that can maintain a state in contact with the target cells in a solution. This activity can also be measured from its contribution to gene transfer efficiency as described above. Furthermore, gene transfer efficiency means transformation efficiency.

  The functional substance can be used in a state dissolved in a solution or in a state immobilized on a suitable carrier. The carrier for immobilizing the functional substance is not particularly limited, but usually a cell culture container or a bead-like carrier is used.

  When a functional substance having virus binding activity immobilized on a carrier is used, the efficiency of gene introduction can be further improved by the steps exemplified below.

  First, a liquid sample containing a retrovirus, for example, a virus supernatant is brought into contact with a carrier on which a functional substance having a retrovirus binding activity is immobilized. After washing this carrier, the carrier can be brought into contact with the target cell as it is, or by adding virus particles eluted from the carrier by an appropriate method to the target cell, gene transfer can be carried out with high efficiency. . The functional substance having a retrovirus binding activity used herein may have a target cell binding activity, a functional substance having a retrovirus binding activity and a functional substance having a target cell binding activity; May be used in combination.

  The step of bringing a retrovirus-containing liquid sample into contact with a carrier on which a functional substance having a retrovirus binding activity is immobilized is not particularly limited, but for example, for 1 hour or more, preferably 3 hours or more. To be implemented. There are no particular limitations on the conditions such as temperature, and for example, it can be performed at room temperature or 37 ° C. Depending on the stability of the virus, the temperature may be as low as about 4 ° C. A carrier for immobilizing a functional substance may be appropriately selected according to the purpose, but if a cell culture vessel is used, the gene introduction step can be started simply by adding target cells. For washing the carrier, for example, a phosphate-buffered physiological saline or Hanks' physiological saline, or a liquid medium used for culturing target cells can be used.

  Furthermore, by physically increasing the frequency of contact between the retrovirus and the functional substance having retrovirus binding activity, the retrovirus can be efficiently bound to the functional substance. Such physical means is not particularly limited, and for example, shaking, filtration or centrifugal force can be used. Specifically, the centrifugal force is applied by adding a retrovirus-containing liquid sample to a centrifuge tube in which a functional substance having retrovirus binding activity is immobilized at the bottom, and then centrifuging the centrifuge tube. The method used for operation is mentioned. During this centrifugation, the retrovirus settles at the bottom of the centrifuge tube by centrifugal force, so that the frequency of contact between the retrovirus and the functional substance having the retrovirus binding activity increases, and the efficiency of the binding is improved. The above method is a method that can obtain higher gene transfer efficiency without applying physical stress to the cells as in the method of precipitating the virus on the cells by centrifugal force to infect the cells (WO95 / 10619). is there.

  By the above-described operation, the gene introduction can be carried out after removing the substances whose presence in the retrovirus-containing sample is undesirable for gene introduction. Examples of the substance removed by the method of the present invention include, for example, a packaging cell-derived retrovirus infection inhibitor contained in the virus supernatant [Human Gene Therapy, Vol. 8, pages 1459 to 1467 (1997). ), Journal of Virology (Vol. 70, 6468-6473 (1996)) and substances added for the purpose of promoting the production of retroviruses when culturing retrovirus-producing cells. Examples include phorbol 12-myristate 13-acetate (TPA) and dexamethasone [Gene Therapy, Vol. 2, pp. 547-551 (1995)], and sodium butyrate as described above. .

  The functional substance having a retrovirus binding activity used in the present invention is not particularly limited, and examples thereof include fibronectin heparin-II binding region, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran and the like. In addition, substances functionally equivalent to these functional substances, for example, functional substances having a heparin-binding site can also be used. In addition, a mixture of the functional substance, a polypeptide containing the functional substance, a polymer of the functional substance, a derivative of the functional substance, and the like can be used.

  It is possible to enhance the virus binding activity by chemically modifying these functional substances. Examples of the chemical modification method include a method of activating an amino acid residue on a functional substance to be used and a method of introducing a basic residue into the substance. For example, by modifying a free carboxyl group in a functional substance consisting of a peptide or protein with a water-soluble carbodiimide (for example, 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride), the carboxyl group is activated, Retrovirus binding activity can be increased. Furthermore, retrovirus-binding activity can be enhanced by introducing a basic residue, such as an amino group, onto the functional substance using the thus activated carboxyl group.

  In addition, the functional substance having target cell binding activity used in the present invention is not particularly limited. For example, it is a substance having a ligand that binds to a target cell. Examples of the ligand include cell adhesion proteins and hormones. And cytokines, antibodies to cell surface antigens, polysaccharides, glycoproteins, glycolipids, sugar chains derived from glycoproteins and glycolipids, and metabolites of target cells. In addition, a polypeptide containing the functional substance, a polymer of the functional substance, a derivative of the functional substance, a functional equivalent of the functional substance, and the like can also be used.

  An antibody that specifically binds to a target cell is particularly useful for introducing a gene specifically into a specific cell and with high efficiency. The antibody that can be used in the present invention is not particularly limited, and an antibody against an antigen expressed in a target cell into which a gene is to be introduced can be appropriately selected and used. The antibody can be prepared by a known method, but many antibodies are currently commercially available, and these can also be used. These antibodies may be either polyclonal antibodies or monoclonal antibodies as long as they have desired properties such as cell specificity. Furthermore, antibodies or antibody derivatives modified by known techniques such as humanized antibodies, Fab fragments, single chain antibodies and the like can also be used.

  The leukocyte antigen known as the CD antigen has been examined in detail for the expression of various antigens in various cells. Therefore, by selecting an antibody that recognizes the CD antigen expressed in the target cell of interest and using it in the gene introduction method of the present invention, the gene can be introduced into the target cell with high specificity. For example, gene transfer can be directed to helper T cells when anti-CD4 antibodies are used, and to hematopoietic stem cells when anti-CD34 antibodies are used.

  Furthermore, by using laminin, which is a glycoprotein, as a functional substance having target cell binding activity, genes can be efficiently introduced into various target cells, for example, blood cells. The laminin that can be used in the present invention may be mouse-derived or human-derived as long as it has binding activity to the target cell, and may be a fragment thereof. As shown in the following examples, the sugar chain plays an important role in gene transfer using laminin. Therefore, sugar chains cut out from laminin by a known method can also be used in the method of the present invention. In addition, glycoproteins having a high mannose N-linked sugar chain similar to laminin, sugar chains cut out from this, and chemically synthesized sugar chains can also be used in the present invention. Furthermore, those obtained by binding the above sugar chain to a substance such as a protein can be used. For example, those bound to a functional substance having a retrovirus binding activity can be suitably used for gene introduction.

  The above-described high mannose-type sugar chain is not particularly limited as long as it has 1 to 20 residues of mannose in the molecule, but those having a mannose residue at the non-reducing end are included in the method of the present invention. preferable. The sugar chain is used in combination with other appropriate molecules such as biomolecules such as monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, proteins, lipids, and artificial substances such as synthetic polymers. You can also

A representative example of a high mannose sugar chain derived from a living body is one having a structure of (Mannose) _n- (GlucNAc) ₂ [Protein / Nucleic Acid / Enzyme, Vol. 43, pp. 2631 to 2639 ( 1998)]. Although not particularly limited, for example, it has the above structure, the sugar chain containing nine mannose residues in the molecule (Mannose) ₉ - a (GlucNAc) ₂ (structure of the sugar chain in FIG. 1 Can be suitably used for the gene introduction method of the present invention.

  The functional materials as described above can be obtained from naturally occurring materials, and can be artificially produced (for example, by recombinant DNA technology or chemical synthesis technology). And a combination of artificially prepared materials. Further, as described in WO97 / 18318, when gene transfer is carried out using these functional substances, functional substances having a retrovirus binding site and other functionalities having a target cell binding site. It is possible to use a mixture of substances or a functional substance having a retrovirus binding site and a target cell binding site on the same molecule. In addition, as these functional substances, those that do not substantially contain other proteins in which they coexist in nature are used. Furthermore, these functional substances or combinations of functional substances can be combined with a medium used for culturing target cells, cell growth factors, or the like to form a kit for gene transfer.

  Fibronectin and fragments thereof used in the method of the present invention are described in, for example, Journal of Biochemistry, Vol. 256, page 7277 (1981), Journal of Cell Biology. (J. Cell. Biol.), 102, 449 (1986), Journal of Cell Biology, 105, 489 (1987). It can be produced in substantially pure form from the material. Moreover, it can also be produced using recombinant DNA technology by the method described in US Pat. No. 5,198,423. In particular, fibronectin fragments comprising the retroviral binding site heparin-II region, eg, CH-296 and recombinant polypeptides such as H-271, H-296, CH-271 used in the examples below, and The method for obtaining these is described in detail in this patent. As described in the above publication, these fragments were transferred to FERM P-10721 (H-296) (original deposit date: Tsukuba City, Ibaraki Pref. May 12, 1991), FERM BP-2799 (CH-271) (original deposit date: May 12, 1991), FERM BP-2800 (CH-296) (original deposit date: 1991) And FERM BP-2264 (H-271) (original deposit date: January 30, 1991) can be obtained by culturing the deposited Escherichia coli. Further, a fragment that can be routinely derived from these fragments can be prepared by modifying the plasmid held in the above-mentioned Escherichia coli by a known gene recombination technique. Of the above fibronectin fragments, H-296 has a VLA-4 binding domain polypeptide, CH-271 has a VLA-5 binding domain peptide, and CH-296 has both. [Nature Medicine, Vol. 2, pp. 876-882 (1996)].

  By infecting a target cell with a retrovirus in the presence of the functional substance, a cell into which a gene has been introduced can be efficiently obtained. Retrovirus infection can be performed by a conventional method, for example, incubation at 37 ° C. and a carbon dioxide gas concentration of 5%. This condition and incubation time may be appropriately changed according to the target cell and purpose.

Since the retrovirus does not infect when the target cell is a G ₀ phase cell, it is preferably induced into the cell cycle by pre-stimulation. For this purpose, prior to infection with the retrovirus, the target cell is treated with an appropriate target. Culture in the presence of cell growth factor. For example, various cytokines, such as interleukin-3, interleukin-6, and stem cell factor, are used for pre-stimulation when gene transfer is performed to bone marrow cells or hematopoietic stem cells.

  It is known that receptors present on the cell surface are involved in retroviral infection of cells. Basic amino acid transporters and phosphate transporters are known to function as receptors for ecotropic and amphotropic viruses, respectively [Proceedings of the National Academy of Sciences of The USA, Vol. 93, 11407-11413 (1996)], in order to activate the expression and turnover of these transporters, basic amino acids or phosphoric acids and their salts and precursors are added. By pretreating the target cells in a reduced medium, the cells can be easily infected with the virus.

  Surprisingly, the present inventors have found that the efficiency of retrovirus infection, that is, gene transfer efficiency is increased even when a transferrin receptor that has not been known to be associated with viral infection is activated. The activation of transferrin receptor is not particularly limited, but can be performed by treating the target cells in a medium with limited Fe concentration. For example, deferoxamine was added to chelate Fe in the medium. Medium can be used.

  Preferably, gene transfer by transferrin activation is also performed in the presence of the functional substance.

  There are no particular limitations on the cells targeted for gene transfer according to the method of the present invention. For example, stem cells, hematopoietic cells, non-adherent low density mononuclear cells, adherent cells, bone marrow cells, hematopoietic stem cells, peripheral blood Stem cells, umbilical cord blood cells, fetal hematopoietic stem cells, embryogenic stem cells, embryonic cells, primordial germ cells, oocytes, oocytes, ovaries, spermatocytes, spermatozoa, CD34 + cells, C-kit + cell, pluripotent hematopoietic progenitor cell, unipotent hematopoietic progenitor cell, erythroid progenitor cell, lymphocyte mother cell, mature blood cell, lymphocyte, B cell, T cell, fibroblast, neuroblast, nerve Cells, endothelial cells, vascular endothelial cells, hepatocytes, myoblasts, skeletal muscle cells, smooth muscle cells, cancer cells, myeloma cells, leukemia cells and the like can be used. Hematopoietic cells obtained from blood or bone marrow are relatively easy to obtain, and since their culture and maintenance techniques have been established, they are suitable for utilizing the method of the present invention. Particularly when long-term expression of the introduced gene is intended, hematopoietic stem cells, CD34 positive cells, C-kit positive cells, pluripotent hematopoietic progenitor cells and the like are suitable as target cells.

  For example, gene therapy using hematopoietic stem cells as target cells can be performed by the following procedure.

  First, a material containing hematopoietic stem cells such as bone marrow tissue, peripheral blood, umbilical cord blood and the like is collected from a donor. These materials can be used for gene transfer as they are, but usually, a mononuclear cell fraction containing hematopoietic stem cells is prepared by a method such as density gradient centrifugation, or CD34 and / or C -Purification of hematopoietic stem cells using cell surface marker molecules such as -kit. These materials containing hematopoietic stem cells are prestimulated with an appropriate cell growth factor, if necessary, and then infected with a recombinant retroviral vector into which the target gene has been inserted by the method of the present invention. Let The gene-transferred cells thus obtained can be transplanted into a recipient by intravenous administration, for example. The recipient is preferably the donor himself, but it is also possible to perform allogeneic transplantation. For example, when umbilical cord blood is used as the material, allogeneic transplantation is performed.

  As gene therapy targeting hematopoietic stem cells, there are those that complement a gene that is deficient or abnormal in a patient, for example, gene therapy for ADA deficiency or Gaucher disease. In addition, for example, a drug resistance gene may be introduced into hematopoietic stem cells in order to alleviate the damage of hematopoietic cells caused by chemotherapeutic agents used for the treatment of cancer and leukemia.

  In addition, as a cancer gene therapy method, tumor vaccine therapy is being studied that introduces cytokine genes into cancer cells and then deprives them of their growth ability and returns them to the patient's body to enhance tumor immunity [Human Gene • Therapy, Vol. 5, pp. 153-164 (1994)]. Furthermore, attempts have been made to treat AIDS by gene therapy. In this case, a gene encoding a nucleic acid molecule (antisense nucleic acid, ribozyme, etc.) that interferes with HIV replication and gene expression is introduced into T cells infected with HIV (human immunodeficiency virus) that causes AIDS. [For example, Journal of Willology, 69, 4045-4052 (1995)].

  As explained in detail above, by using the present invention, it is possible to carry out gene introduction with high efficiency and high specificity for target cells. In addition, the method of the present invention does not require special equipment and equipment, and is effective for various retroviral vectors and target cells.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the scope of the following examples.

Example 1
Preparation of Fibronectin Derived Polypeptide Human fibronectin derived polypeptide, H-271 is a recombinant plasmid containing DNA encoding the polypeptide, Escherichia coli HB101 / pHD101 (FERM BP-2264), US It was prepared by the method described in Japanese Patent No. 5,198,423.

  Human fibronectin-derived polypeptide, H-296 is a recombinant plasmid containing DNA encoding the polypeptide, E. coli containing pHD102, Escherichia coli HB102 / pHD102 (FERM P-10721), the method described in the above publication Prepared according to

Polypeptide CH-271 was prepared by the method shown below.
That is, Escherichia coli HB101 / pCH101 (FERM BP-2799) was used and cultured by the method described in the above publication, and CH-271 was obtained from the culture.

Polypeptide CH-296 was prepared by the method shown below.
That is, Escherichia coli HB101 / pCH102 (FERM BP-2800) was used and cultured by the method described in the above publication, and CH-296 was obtained from the culture.

Polypeptide C-274 was prepared by the method shown below.
That is, Escherichia coli JM109 / pTF7221 (FERM BP-1915) was used and cultured by the method described in US Pat. No. 5,102,988, and C-274 was obtained from the culture.

  ColV, which is a polypeptide having retrovirus binding activity derived from type V collagen, was prepared according to the method described in International Publication No. WO97 / 18318.

Example 2
Retroviral vector construction and retroviral supernatant preparation A retroviral plasmid containing the neomycin phosphotransferase gene, PM5neo vector [Experimental Hematology, 23, 630-638, 1995] was introduced. GP + E-86 cells (ATCC CRL-9642) are 10% fetal bovine serum (FCS, Gibco) and Dulbecco's modified Eagle's medium containing 50 units / ml penicillin and 50 μg / ml streptomycin (both Gibco). The culture was performed in DMEM (Bio Whittaker). In addition, all the DMEM used for subsequent operation contains the said antibiotic. The supernatant containing PM5neo virus was prepared by adding 4 ml of DMEM containing 10% FCS to a plate (10 cm diameter gelatin-coated cell culture dish, manufactured by Iwaki Glass Co., Ltd.) on which the production cells were grown semiconfluent. It was collected after culture and prepared. The collected medium supernatant was filtered through a 0.45 micron filter (Millipore) to obtain a virus supernatant stock and stored at −80 ° C. until use.

  In addition, an ecotropic packaging cell BOSC23 [Proceedings of of Retroviral Plasmid pLEIN (manufactured by Clontech) containing a neomycin phosphotransferase gene and an enhanced green fluorescent protein (EGFP) gene is introduced. The National Academy of Sciences of the USA, 90, 8392-8396 (1993)], and amphotropic packaging cells ψCRIP [Proceedings of the National -Academic of Sciences of the USA, Vol. 85, 6460-6464 (1988)], a virus supernatant was prepared by the same operation as above. Hereinafter, a virus prepared from BOSC23 cells is referred to as Eco-EGFP, and a virus prepared from ψCRIP cells is referred to as Ampho-EGFP.

  Furthermore, a retroviral plasmid containing a neomycin phosphotransferase gene, a TKNeo vector [Journal of Experimental Medicine (J. Exp. Med.), 178, 529-536, (1993)] A virus supernatant was prepared from the contained GP + EnvAm12 cells (ATCC CRL-9641) in the same manner as described above. DMEM containing 10% calf serum (CS, manufactured by Gibco) was used instead of FCS.

  The titer of the virus supernatant is determined by a standard method using the introduction of neomycin phosphotransferase gene into NIH / 3T3 cells (ATCC CRL-1658) [Journal of Willology, Vol. 62, No. 1120. ˜1124, (1988)]. The number of infectious particles contained per ml of supernatant (cfu / ml) was calculated, and this was used as the titer of the supernatant to determine the amount of virus supernatant added in subsequent experiments.

Example 3
Preparation of retrovirus-binding functional substance and measurement of its activity In a 96-well microplate for surface untreated cell culture (Falcon), 80 μg / ml H-271, H-296, C-274, CH-271 each CH-296, ColV, human basic fibroblast growth factor (bFGF, manufactured by Progen), tenascin (Tenascin, manufactured by Gibco), epidermal cell growth factor (EGF, manufactured by Takara Shuzo) or 2% bovine serum albumin ( 50 μl of BSA (manufactured by Sigma) was added per well and allowed to stand at 4 ° C. overnight, and then the plate was washed twice with phosphate buffered saline (PBS, manufactured by Roman Kogyo). After the same treatment as above, 0.1 ml of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride solution (manufactured by Sigma) adjusted to 4 mg / ml with sterile pure water was dispensed at 37 ° C. Then, the plate was sufficiently washed with pure water to prepare a carbodiimide-treated plate. These plates were stored at 4 ° C. until virus infection experiments were performed.

10 ⁴ mouse leukemia cells L1210 (ATCC CCL-219) grown in RPMI 1640 medium (BioWitaker) supplemented with 10% FCS, 50 units / ml penicillin and 50 μg / ml streptomycin, and on PM5neo virus 50 μl of the clear solution (10 ⁴ cfu / ml) was added to one well of the microplate. After culturing for 24 hours, the medium was replaced with a medium containing G418 (manufactured by Gibco) having a final concentration of 0.75 mg / ml, and further culturing was performed for 48 hours. G418-resistant cells were assayed by partially modifying the method of S. Kim et al. [Gene Therapy, Vol. 3, pp. 1018-1020 (1996)] to develop the color of Premix WST-1 reagent (Takara Shuzo). The measurement was performed by measuring the absorbance at 450 nm. After culturing, 10 μl of WST-1 reagent per 100 μl of culture medium was added and further cultured at 37 ° C. for 4 hours, and then the absorbance at 450 nm and 650 nm was measured with a microplate reader to calculate the difference (450 nm−650 nm). did. The value obtained in the 2% BSA coat and carbodiimide untreated group was used as the background. The results of the three tests are summarized and the results are shown in Table 1.

  As shown in Table 1, an increase in gene transfer efficiency was observed with H-271, H-296, CH-271, CH-296, ColV, and bFGF, which are functional substances having known virus binding activity. . Furthermore, when C-274, tenascin, EGF, and BSA, which do not have virus binding activity, were treated with carbodiimide, the incidence of G418-resistant cells increased.

  Next, the following experiment was performed using CH-296 as a functional substance.

To a 24-well microplate for surface untreated cell culture (manufactured by Falcon), 40 μg / ml of CH-296 was added at 0.5 ml per well and incubated at 4 ° C. overnight. After this plate was washed with PBS (pH 5.8), ethylenediamine [NH ₂ (CH ₂ ) ₂ NH ₂ , manufactured by Nakarai] or trimethylenediamine [NH ₂ (CH ₂ ) ₃ NH ₂ , manufactured by Nacalai ] Or putrescine [NH ₂ (CH ₂ ) ₄ NH ₂ , manufactured by Nacalai Co., Ltd.] PBS of 10 mg / ml 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Sigma) at various concentrations (pH 5. 8) 625 μl of the solution was added and incubated at 37 ° C. for 2 hours. By this operation, an amino group was introduced into the carboxyl group on the CH-296 molecule via carbodiimide. The plate was washed 3 times with PBS and then sequentially blocked with 2% glycine / PBS and 2% BSA / PBS.

GP + E86 cells introduced with the retroviral vector plasmid pLEIN were cultured in DMEM containing 10% CS, and the supernatant was collected. The virus solution diluted to 1 × 10 ⁵ cfu / ml of this supernatant was added at 0.5 ml per well of the above plate, incubated for 4 hours, and further 1 × 10 ⁴ NIH / 3T3 cells. And cultured for 2 days. After completion of the culture, the cells were collected and washed with a cell detachment buffer (BioWitaker), and then flow cytometry (excitation wavelength 488 nm, fluorescence wavelength 515 to 545 nm) using FACSvantage (Becton Dickinson). EGFP-expressing cells were analyzed, and the ability of the virus to bind to the plate was expressed by the efficiency of gene transfer into the cells. The result is shown in FIG.

  As shown in FIG. 2, the virus binding ability increased as the concentration of the diamino compound used for the amino group introduction reaction increased. When putrescine, trimethylenediamine, and ethylenediamine were used in the reaction at 2 mM, the virus-binding ability was about twice that of untreated CH-296.

Example 4
Gene transfer efficiency enhancing effect of laminin Gene transfer was carried out by combining mouse laminin (Gibco) or human laminin (Takara Shuzo) and a functional substance having virus binding activity. A 24-well microplate for surface untreated cell culture (manufactured by Falcon) used in the experiment was coated with these functional substances by the following two methods.
Cocktail method: A mixture of two functional substances was added to the plate and allowed to stand at 4 ° C. overnight, followed by blocking with 2% BSA (37 ° C., 20 minutes), and then the plate was washed with PBS. .
Pre-coating method: A functional substance solution having virus-binding activity is added to a plate and allowed to stand at 4 ° C. overnight, and then the solution is removed. Next, laminin solution is added and incubated at 37 ° C. for 2 hours. After further blocking with 2% BSA, the plate is washed with PBS.

  The functional substance solution used for the coating operation was added at 0.5 ml per well.

10 ⁵ L1210 cells and 0.5 ml of Eco-EGFP virus supernatant (10 ⁵ cfu / ml) were added, and the culture was continued for 24 hours. After completion of the culture, the cells were collected and washed with a cell detachment buffer (BioWitaker), and then flow cytometry (excitation wavelength 488 nm, fluorescence wavelength 515 to 545 nm) using FACSVantage (Becton Dickinson). EGFP-expressing cells were analyzed, and gene transfer efficiency (ratio of EGFP-expressing cells to total cells) was calculated. Experimental results are shown in Tables 2-5.

  As shown in Tables 2 and 3, when mouse and human laminin is used for gene transfer by retrovirus in combination with a functional substance having virus-binding activity, gene transfer into the target cell is performed regardless of the immobilization method. It has been shown that it occurs with very high efficiency. Further, as shown in Tables 4 and 5, when the gene transfer efficiency was examined using CH-296 and CH-271 and a plate coated with these and laminin by the cocktail method, the optimal concentration ratio was determined as CH- 296 / mouse laminin was found to be 8: 1 (eg, 80 μg / ml: 10 μg / ml) and CH-271 / mouse laminin was found to be 16: 1 (eg, 80 μg / ml: 5 μg / ml). The gene transfer efficiency at that time was 2.6 times higher with CH-296 and 5.1 times higher with CH-271 than when no laminin was added.

Example 5
Gene transfer into mouse C-kit positive bone marrow cells using laminin Mouse C-kit positive bone marrow cells were prepared as follows. Bone marrow cells were collected from the femurs of 6-8 week old C3H / He female mice (Japan SLC, Inc.), and the resulting cells were treated with Ficoll-Hypac (1.0875 g / ml, Pharmacia). A low density mononuclear cell fraction was prepared by density gradient centrifugation used. The obtained cells were washed with PBS, red blood cells were lysed with Ery-Lysis buffer (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM EDTA, pH 7.4), and further washed with PBS. To the bone marrow cells thus obtained, 1 μg of anti-mouse CD117 antibody (Pharmingen) was added per 10 ⁷ cells and reacted on ice for 30 minutes. The cells were washed with PBS containing 5 mM EDTA and 0.5% BSA and then suspended in the same buffer. Next, a microbead-bound secondary antibody (manufactured by Miltenyi Biotech) was added at 20 μl per 10 ⁷ cells, reacted at 4 ° C. for 30 minutes, washed with the above buffer and resuspended. Cells bound to the microbeads were collected by a MACS system (Miltenyi Biotech) to obtain C-kit positive cells.

  Prior to viral infection, mouse C-kit positive bone marrow cells were pre-stimulated based on the method of Lusky et al. [Blood, 80, 396-402 (1992)]. That is, 20% FCS, 20 ng / ml recombinant mouse interleukin-3 (Genzyme), 50 ng / ml recombinant human interleukin-6 (Genzyme), 100 ng / ml recombinant mouse stem cell factor (Genzyme) ), 50 units / ml penicillin and 50 μg / ml streptomycin in α-MEM (manufactured by BioWitaker) in the presence of 5% carbon dioxide at 37 ° C. for 2 days.

A 24-well microplate for surface untreated cell culture was coated by a cocktail method using a mixture of murine laminin at various concentrations and 80 μg / ml of CH-271, then blocked with 2% BSA for 30 minutes, and further PBS Washed with As a control, a plate using 2% BSA instead of CH-271 was also prepared. It was performed viral infection by the addition of Eco-EGFP virus supernatant of the 10 ⁵ cells per well in microplate C-kit-positive bone marrow cells and ^{0.5ml (10 5 cfu / ml)} . After culturing this for 48 hours, 0.5 ml of fresh medium was added and the culture was continued for another 24 hours. After completion of the culture, the cells were collected and washed with a cell detachment buffer, and the gene transfer efficiency was calculated by the method described in Example 4. The results of two experiments are shown in Tables 6 and 7.

As shown in Tables 6 and 7, even when C-kit positive bone marrow cells were retrovirally infected on a plate coated with murine laminin and a functional substance CH-271 having virus binding activity by the cocktail method. It was revealed that a very high effect of enhancing gene transfer efficiency was observed. The gene transfer efficiency when laminin was combined increased by a maximum of 5.7 times compared to the case of using CH-271 alone.
Further, the same operation as described above was performed using an Eco-EGFP virus supernatant liquid having a titer of 10 ⁷ cfu / ml. Table 8 shows the average of gene transfer efficiencies obtained in three experiments. Also in this case, it was shown that the gene transfer efficiency by the functional substance having retrovirus binding activity is improved by the combined use of laminin.

Example 6
Gene transfer to mouse spleen cell-derived CD3 positive T cells using laminin Preparation of mouse spleen cell-derived CD3 positive T cells was performed as follows. Cells were collected from the spleen of 6-8 week old C3H / He female mice and passed through a 100 μm mesh (Falcon) to remove residues. The obtained cells were washed with Hanks balanced salt solution containing 10% FCS (HBSS, manufactured by BioWitaker), erythrocytes were lysed with Ery-Lysis buffer, and further washed with HBSS. The obtained cells were passed through a 30 μm mesh (Miltenyi Biotech) to remove the residue, and then purified with a CD3-positive T cell concentration column (R & D Systems). Mouse CD3-positive T cells used in the virus infection experiment were 10% FCS, 50 units / ml penicillin and anti-mouse CD3 and CD28 antibodies (both 1 μg / ml, manufactured by Pharmingen). Pre-stimulation was performed by culturing at 37 ° C. for 2 days in the presence of 5% carbon dioxide in RPMI1640 medium (Bio Whitaker) containing 50 μg / ml streptomycin.

A 24-well microplate was coated in the same manner as in Example 5 using a mixture of 20 μg / ml mouse laminin and 80 μg / ml CH-296. To this microplate, 10 ⁵ CD3 positive T cells and 0.5 ml of Eco-EGFP virus supernatant (10 ⁵ cfu / ml) were added per well to carry out virus infection for 3 hours. Thereafter, 10% FCS, 500 units / ml recombinant mouse interleukin-1α (Genzyme), 10 ng / ml recombinant mouse interleukin-2 (Genzyme), 50 units / ml penicillin and 50 μg / ml RPMI 1640 medium containing ml of streptomycin was added, and the culture was further continued for 48 hours. After completion of the culture, the cells were collected and washed with a cell detachment buffer, and the gene transfer efficiency was calculated by the method described in Example 4. The results are shown in Table 9.

  As shown in Table 9, it was revealed that the gene transfer efficiency into mouse CD3-positive T cells was increased by the coexistence of laminin.

Example 7
Involvement of sugar chain in laminin molecule for gene transfer Using a mixed solution of 50 μl of 5 μg / ml mouse laminin and 80 μg / ml CH-271 per well, a 96-well microplate was prepared in the same manner as in Example 5. After coating, the plate was treated with various enzymes having glycosylation activity, and the influence on gene transfer efficiency was examined.

  The plate was subjected to enzyme treatment under the following conditions: O-glycanase (Endo-α-N-acetylgalactosaminidase, manufactured by Seikagaku Corporation), endoglycosidase H (Endo-β-N-acetylglucosaminidase H, manufactured by Seikagaku Corporation) ), Endo-β-galactosidase (Endo-β-galactosidase, manufactured by Seikagaku Corporation) and α-mannosidase (α-Mannosidase, manufactured by Seikagaku Corporation) are 50 mM citrate-phosphate buffer (pH 5.0). The enzyme solutions of 500 mU / ml, 500 mU / ml, 250 mU / ml and 2 mU / ml were prepared respectively. For glycopeptidase F (Peptide: N-glycosidase F, manufactured by Takara Shuzo Co., Ltd.), a 250 mM U / ml enzyme solution was prepared with 100 mM Tris-HCl buffer (pH 8.6). 50 μl of each enzyme solution was dispensed per well and reacted at 37 ° C. for 20 hours. Then, after washing 3 times with PBS, it was used for virus infection experiments.

10 ⁴ mouse leukemia cells L1210 grown in RPMI 1640 medium supplemented with 10% FCS and 50 units / ml penicillin and 50 μg / ml streptomycin and 50 μl (10 ⁴ cfu / ml) PM5neo virus supernatant. Added to 1 well of the microplate. After culturing for 24 hours, the medium was replaced with a medium containing G418 (manufactured by Gibco) having a final concentration of 0.75 mg / ml, and further culturing was performed for 48 hours. G418 resistant cells were measured by the method described in Example 3, and the results are shown in Table 10. Table 10 summarizes the results of three experiments.

As shown in Table 10, when laminin is used in combination, the appearance rate of G418 resistant cells is increased as compared with CH-271 alone. When the plate coated with laminin was treated with enzyme, the effect of promoting gene introduction by laminin was completely lost by endoglycosidase H treatment. Further, even when α-mannosidase or glycopeptidase F was treated, a slight decrease in gene transfer efficiency was observed. According to a report on sugar chains on laminin molecules [Biochim. Biophysi. Acta, 883, 112-126 (1986)], the carbohydrates on laminin molecules Most are N-linked sugar chains that bind to asparagine, and 43 molecules of N-linked sugar chains are bound to each laminin molecule. Among these sugar chains, the sugar chain cut out by endoglycosidase H treatment is a high mannose-type asparagine-N-linked sugar chain. Further, from the reduction in the gene transfer efficiency by α- mannosidase treatment was observed, in laminin _{(Mannose) 9 - (GlucNAc)} 2 -Asn and / or (Mannose) ₆ - (GlucNAc) of ₂ -Asn It is considered that the sugar chain having a mannose structure of α1-2 and / or α1-6 bonds cleaved by α-mannosidase treatment plays an important role. Thus, it has been clarified that the effect of promoting gene transfer by laminin is due to the sugar chain in the laminin molecule, particularly a high mannose type sugar chain.

Above (Mannose) ₉ - involvement in (GlucNAc) of ₂ -Asn transgenic was confirmed by the following experiments.

1 ml of soybean agglutinin prepared from defatted soybean flour (manufactured by Sigma) using Sepharose CL-2B (manufactured by Pharmacia) to which lactose is immobilized is heat-denatured and then 20 ml of 50 mM Tris-HCl buffer containing 10 mM calcium chloride. In the solution (pH 7.2), digestion was performed at 37 ° C. for 2 days using 20 mg of actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.). After heating to inactivate the enzyme, subjected to chromatography using Sephadex G-15 (50ml), and Sephadex G-25 (150ml) column, (Mannose) ₉ - was purified (GlucNAc) ₂ -Asn . Figure _{1 (Mannose) 9 - (GlucNAc} ) shows a structure obtained by removing the asparagine residues of ₂ -Asn.

And CH-271, the above (Mannose) ₉ - created a microplate (GlucNAc) and a ₂ -Asn were immobilized by covalent bonding. That is, a 96-well carboplate (manufactured by Sumitomo Bakelite Co., Ltd.) was activated with a 4 mg / ml water-soluble carbodiimide solution at 37 ° C. for 2 hours, and then washed three times with sterilized water. 2% BSA or 80 [mu] g / ml of CH-271 and various concentrations _{(Mannose) 9, - (GlucNAc} ) 2 solution was prepared containing the -Asn, one by its 50μl in the well of the activated 96-well carbonitrile plate After the addition, an immobilization reaction was performed at 37 ° C. for 2 hours. Subsequently, blocking with a 0.2% glycine solution was performed at 4 ° C. for 15 hours, and used for the following gene transfer experiment.

10 ³ L1210 cells and 0.1 ml of Eco-EGFP virus supernatant (10 ⁶ cfu / ml) were added to each well of the microplate and cultured for 48 hours, and then fresh RPMI 1640 medium (FCS , Containing penicillin and streptomycin), and further cultured for 24 hours. After collecting and washing the cells, gene transfer efficiency was calculated by the method described in Example 4. Table 11 shows the average of the results of two experiments.

As shown in Table _{11, (Mannose) 9 - (} GlucNAc) a ₂ -Asn immobilized wells with CH-271 is dose-dependent manner the gene transfer efficiency of the sugar chain used was increased. That is, it was confirmed that a sugar chain having the same structure as that present on the laminin molecule contributes to improvement of gene transfer efficiency.

Example 8
Gene introduction specific for CD4-positive cells using anti-CD4 monoclonal antibody 1 μg / ml of anti-mouse CD4 and anti-mouse CD44 monoclonal antibodies (both from Pharmingen), 80 μg / ml of H-271, CH- 271 and CH-296 were combined, and a 24-well microplate for surface untreated cell culture was coated according to the method described in Example 4. Note that the precoat method was used for H-271, and the cocktail method was used for CH-271 and CH-296.

0.5 ml of Eco-EGFP virus supernatant (10 ⁷ cfu / ml) was added per well of the microplate and incubated at 32 ° C. for 3 hours, followed by 10% FCS, 50 units / ml penicillin and 50 μg. Plates were washed with RPMI 1640 medium containing / ml streptomycin. Subsequently, 10 ⁵ per mouse spleen cell-derived CD3-positive T cells prepared and pre-stimulated according to the method described in Example 6 were added for 3 hours of virus infection. Thereafter, RPMI 1640 medium containing 10% FCS, 500 units of recombinant mouse interleukin-1α, 10 ng / ml recombinant mouse interleukin-2, 50 units / ml penicillin and 50 μg / ml streptomycin was further added. The culture was continued for 48 hours. After completion of the culture, the cells were collected and washed with a cell detachment buffer, and the cells were labeled with phycoerythrin (PE, Pharmingen) labeled anti-mouse CD4 monoclonal antibody (Pharmingen) and propinium iodide (PI, Sigma). The cells were subjected to flow cytometry using FACSVantage (excitation wavelength 488 nm, fluorescence wavelengths 515 to 545 nm and 562 to 588 nm), and two-dimensional analysis of CD4 antigen expression and EGFP expression in living cells was performed, and CD4 positive cells The gene transfer efficiency into each CD4 negative cell was calculated. The results are shown in Table 12. Table 12 summarizes the results of the four experiments.

  As shown in Table 12, when retrovirus infection was performed on a plate coated with both a monoclonal antibody and a fibronectin fragment, an effect of enhancing gene transfer efficiency was observed in mouse spleen cell-derived CD3 positive T cells.

  Particularly noteworthy is that when virus infection is performed by combining an anti-CD4 monoclonal antibody and a functional substance having a retrovirus binding activity, the efficiency of gene transfer into CD4 positive cells is compared with the efficiency of CD4 negative cells. It was very expensive. For example, in the combination with H-271, the gene transfer efficiency to CD4 positive cells was as high as about 60%, whereas the gene transfer efficiency to CD4 negative cells was only about 20%. Similar results were obtained when CH-271 and CH-296 were used as fibronectin fragments.

  On the other hand, CD44 antigen is expressed in 98% or more in both CD4 positive cells and CD4 negative cells, and when anti-CD44 monoclonal antibody is used for retrovirus infection similar to the above, regardless of the expression of CD4 antigen in the cells. The gene transfer efficiency was thought to increase, but the results in Table 12 confirmed this.

Example 9
Gene introduction specific to CD8 positive cells using anti-CD8a monoclonal antibody H-271 was used as a functional substance having retrovirus binding activity, and anti-mouse CD8a (Pharmingen) and anti-mouse CD44 monoclonal antibody were used as antibodies. The others were performed in the same manner as in Example 8. For detection of CD8 positive and negative cells, anti-mouse CD8a monoclonal antibody (Pharmingen) labeled with phycoerythrin (PE, Pharmingen) was used. The obtained results are shown in Table 13. Table 13 summarizes the results of two experiments.

  As shown in Table 13, the effect of enhancing gene transfer efficiency into mouse spleen cell-derived CD3-positive T cells was observed by the combined use of the anti-CD8a monoclonal antibody and the fibronectin fragment.

  In the case of the anti-CD8a monoclonal antibody, similar to the result of Example 8, the gene transfer efficiency was high for cells expressing the CD8 antigen recognized by the antibody. Further, when the anti-CD44 monoclonal antibody expressed in 98% or more of both CD8 positive and negative cells was used, there was no difference in gene transfer efficiency between CD8 positive and negative cells.

  The significance of the experimental results of Examples 8 and 9 above is very great, and the incubator is coated with a mixture of a target cell-specific antibody and a virus-binding functional substance (cocktail). It was proved that a target gene can be introduced in a target cell-specific manner by infecting a group of cells including the target cell with a retrovirus incorporating the target gene.

Example 10
Cell-selective gene transfer using antibodies 80 μg / ml of CH-271 and monoclonal antibodies against various cell surface antigens (anti-CD4, anti-CD8, anti-CD44, anti-CD49c, anti-CD49d and anti-CD49e antibodies Each of which was manufactured by Pharmingen Co., Ltd.) 1 μg / ml each was used, and a 24-well microplate for surface untreated cell culture was coated by the cocktail method according to the method described in Example 4.

  Target cells include K562 (human chronic myeloid leukemia cell, ATCC CCL-243), HSB-2 (human acute lymphocytic leukemia cell, CCRF-HSB-2, ATCC CCL-120.1), MOLT-3 ( Human acute lymphocytic leukemia cells, ATCC CRL-1552), TF-1 (human erythroleukemia cells, ATCC CRL-2003) were used. These cells were subjected to FACS analysis using the labeled various monoclonal antibodies, and the expression rate of the antigen corresponding to the antibodies was measured.

0.5 ml of Ampho-EGFP virus supernatant (1 × 10 ⁶ cfu / ml) was added per well of the above microplate and incubated at 32 ° C. for 3 hours, followed by 10% FCS, 50 units / ml Was washed with RPMI 1640 medium containing 50 μg / ml streptomycin. The above-mentioned various cells were suspended in 1 ml of the above medium so as to be 1 × 10 ⁵ cells and added to wells, and virus infection was performed. After further culturing for 3 days, the cells were collected and washed with a cell detachment buffer, and the introduction efficiency of the EGFP gene was calculated by the flow cytometry method described in Example 4.

  The above results are shown in Table 14. This result is an average of three experimental results.

遺伝子導入効率は、各々の細胞における抗体無添加での遺伝子導入効率を
１００％とした場合の相対値（％）で表した。
ＣＤ抗原発現率は、ＦＡＣＳ測定における陽性率（％）をそれぞれ以下のよ
うに示した。
−：１０％以下、 ＋／−：１０−３０％、 ＋：３０−６０％、
＋＋：６０−９０％、＋＋＋：９０％以上

The gene transfer efficiency was expressed as a relative value (%) when the gene transfer efficiency without addition of antibody in each cell was taken as 100%.
As for the CD antigen expression rate, the positive rate (%) in FACS measurement was shown as follows.
-: 10% or less, +/-: 10-30%, +: 30-60%,
++: 60-90%, ++: 90% or more

As shown in Table 14, in the gene transfer by cocktail method using CH-271 as a virus-binding substance and antibodies against antigens on various cells as a cell-binding substance, there is a correlation between the antigen expression rate and the gene transfer efficiency. The relationship was recognized.
Furthermore, a gene introduction experiment was conducted using 80 μg / ml polylysine instead of CH-271 as a functional molecule having a retrovirus binding ability. The monoclonal antibodies, cells, and other experimental conditions used were the same as described above. The results obtained are shown in Table 15. In addition, a result shows the experimental result of 3 times by the average value.

遺伝子導入効率は、各々の細胞における抗体無添加での遺伝子導入効率を
１００％とした場合の相対値（％）で表した。
ＣＤ抗原発現率は、ＦＡＣＳ測定における陽性率（％）をそれぞれ以下のよ
うに示した。
−：１０％以下、 ＋／−：１０−３０％、 ＋：３０−６０％、
＋＋：６０−９０％、＋＋＋：９０％以上

The gene transfer efficiency was expressed as a relative value (%) when the gene transfer efficiency without addition of antibody in each cell was taken as 100%.
As for the CD antigen expression rate, the positive rate (%) in FACS measurement was shown as follows.
-: 10% or less, +/-: 10-30%, +: 30-60%,
++: 60-90%, ++: 90% or more

  As shown in Table 15, gene introduction experiments were conducted by cocktail method using polylysine as a virus-binding substance and antibodies against antigens on various cells as cell-binding substances. There was a correlation between efficiency.

  As a result of the above two experiments, gene introduction specific to the desired target cell is achieved by gene introduction by the cocktail method using an antibody that specifically recognizes the antigen expressed on the target cell as a cell binding substance. Was shown to be possible.

Example 11
Gene transfer to target cells pre-cultured in medium containing deferoxamine Human myeloid leukemia cells HL-60 (ATCC CCL) cultured in RPMI 1640 medium containing 10% FCS, 50 units / ml penicillin and 50 μg / ml streptomycin -240) was transferred to the above medium containing various concentrations of deferoxamine (deferoxamin, manufactured by Sigma) from the day before the infection experiment, and precultured at 37 ° C. for 20 hours in the presence of 5% carbon dioxide gas. At the time of use, the cells were washed with a fresh medium not containing deferoxamine, adjusted to 2 × 10 ⁵ cells / ml, and used for the following infection experiments.

0.5 μl / well of 80 μg / ml CH-271 was added to a 24-well microplate for surface untreated cell culture, allowed to stand at 4 ° C. overnight, blocked with 2% BSA for 30 minutes, and further with PBS Washed. 0.5 ml of Ampho-EGFP virus supernatant (10 ⁶ cfu / ml) was added per well of the microplate and incubated at 32 ° C. for 3 hours, followed by 10% FCS, 50 units / ml penicillin and 50 μg. Plates were washed with RPMI 1640 medium containing / ml streptomycin. Subsequently, 10 ⁵ precultured HL-60 cells were added per well and cultured for 48 hours, after which 0.5 ml of RPMI 1640 medium containing 10% FCS, 50 units / ml penicillin and 50 μg / ml streptomycin was added. After the addition, the culture was further continued for 24 hours. Thereafter, gene transfer efficiency was examined by the same operation as in Example 4. The results are shown in Tables 16 and 17.

  As shown in Tables 16 and 17, it was confirmed that even when HL-60 cells having a very low gene transfer efficiency with CH-271 alone were treated with deferoxamine for 20 hours in advance, the gene transfer efficiency was improved.

Example 12
Detection of presence of viral infection inhibitor in culture supernatant DTK, culture supernatant of NIH / 3T3 cells (ATCC CRL-1658), and culture supernatant of ψCRIP cells, respectively, were prepared as the TKNeo virus supernatant prepared in Example 2. It diluted to 312.5 cfu / ml and used for the following operations.

0.5 ml per well of 32 μg / ml CH-296 was added to a 24-well microplate for surface untreated cell culture, left at room temperature for 2 hours, blocked with 2% BSA for 30 minutes, and further washed with PBS did. 1 ml of the above virus solution and 2 × 10 ⁴ NIH / 3T3 cells were added to each well of the plate and incubated at 37 ° C. overnight. Thereafter, the cells were cultured in a selective medium containing 0.75 mg / ml G418 for 10 days, and the number of colonies that appeared was counted. The ratio of the number of G418-resistant colonies to the number of colonies obtained in a medium not containing G418 was taken as the gene transfer efficiency, and the results are shown in Table 18.

  As shown in Table 18, when the virus was diluted with NIH / 3T3 cell culture supernatant or ψCRIP cell culture supernatant as compared with the case of diluting the virus with DMEM, the introduction efficiency decreased to 1/5 or less. NIH / 3T3 cells are the parent strains of many packaging cells such as ψCRIP cells and GP + EmvAm12 cells used for the preparation of TKNeo virus vector-producing cells used in this experiment. The activity of inhibiting retroviral infection was found in the culture supernatant of the cells, indicating that the virus supernatant prepared using similar packaging cells also contained inhibitors. Suggest.

Example 13
Removal of the viral infection inhibitory substance present in the virus solution In order to remove the viral infection inhibitory substance shown in Example 12, the following method was used. As a sample containing a retrovirus, the TKNeo virus supernatant prepared in Example 2 was diluted with a culture supernatant of ψCRIP cells to 5000 cfu / ml, and further diluted 2-fold with DMEM.

1 ml of the above virus solution was added to the well of a plate coated with CH-296 by the method described in Example 11, and incubated for 1 to 5 hours to allow the virus particles to contact and hold on CH-296. The plate was washed 3 times and 1 ml of DMEM containing 2 × 10 ⁴ NIH / 3T3 cells was added. As a control, a suspension of 2 × 10 ⁴ NIH / 3T3 cells in 1 ml of the virus solution was immediately transferred to a plate coated with CH-296. These plates were incubated overnight at 37 ° C. to infect the cells with virus. The cells after infection were cultured in a selective medium containing 0.75 mg / ml G418 for 10 days, and the number of colonies that appeared was counted. The ratio of the number of G418-resistant colonies to the number of colonies obtained with a medium not containing G418 was taken as the gene transfer efficiency, and the results are shown in FIG.

  As shown in FIG. 3, when the virus particles were contacted and held on the plate coated with CH-296, the introduction efficiency was higher than that of the control experimental group after 3 hours. That is, it was shown that the virus infection inhibitory activity present in the virus solution can be removed by the above operation.

Example 14
Removal of sodium butyrate present in virus solution Recombinant retrovirus-producing cells obtained by introducing retrovirus vector plasmid pLEIN into ψCRIP cells were cultured in DMEM containing 10% CS. When grown to semi-confluent on 10 cm diameter plates, the medium is replaced with 7 ml RPMI 1640 containing 10% FCS or 7 ml RPMI 1640 containing 5 mM sodium butyrate (Nacalai Tesque) and 10% FCS. After culturing for 24 hours, the supernatant was filtered through a 0.45 micron filter to obtain a virus supernatant. The titer of the virus supernatant was measured by the method described in Example 2. The titer of the virus solution containing no sodium butyrate was 3.3 × 10 ⁴ cfu / ml, and the titer of the virus solution containing 5 mM sodium butyrate was 2 × 10 ⁶ cfu / ml.

  Sodium butyrate has the effect of stopping the cell cycle to suppress cell growth or inducing differentiation, and may have an adverse effect on infected cells. In order to remove sodium butyrate contained in the virus solution, the following method was used for evaluation.

HL-60 cells were used as target cells. Add 0.5 ml of the above virus solution per well on the plate coated with CH-296 by the method described in Example 12, and incubate at 37 ° C. for 3 hours to bring the virus particles onto CH-296. Held. After the incubation, the plate was washed three times with PBS, and 0.5 ml of RPMI 1640 medium containing 10% FCS containing 5 × 10 ⁴ HL-60 cells was added. As a control, a suspension of 5 × 10 ⁴ HL-60 cells in 0.5 ml of the virus solution was immediately added to a plate coated with CH-296. After overnight incubation at 37 ° C. to infect cells, 1 ml of RPMI 1640 medium containing 10% FCS was added and further cultured for 48 hours, then the number of cells was counted, and the flow site described in Example 4 EGFP-expressing cells were detected by a measurement method, and gene transfer efficiency was analyzed. The results are shown in Table 19.

  As shown in Table 19, the gene transfer efficiency in the control experimental group was higher when the virus solution prepared by adding sodium butyrate was used, and the effect of sodium butyrate on virus preparation was confirmed. However, the number of growing cells in the supernatant using sodium butyrate was 1/4 or less of that not used, and it was confirmed that the growth of cells was suppressed by sodium butyrate. On the other hand, when the virus particles were previously contacted and retained on the plate coated with CH-296, the cell growth inhibition observed in the control experimental group and the supernatant using sodium butyrate was not observed. Moreover, the gene transfer efficiency did not decrease but rather increased. Thus, it was shown that, by bringing the virus into contact with CH-296 and washing it, it is possible to obtain high gene transfer efficiency without being affected by sodium butyrate.

  Next, a similar experiment was performed using DEAE-dextran.

  DEAE-dextran (manufactured by Sigma) was dissolved in PBS to a concentration of 10 mg / ml, sterilized by filtration with a 0.22 micron filter, and used for coating the plate. 1.1 ml of a mixture of 10 volumes of PBS and 1 volume of the above DEAE-dextran solution is added to each well of a surface treatment 6-well plate for cell culture (manufactured by Iwaki Glass Co., Ltd.) at 4 ° C. overnight. Incubated. The DEAE-dextran solution was removed from the plate, 2 ml of 2% BSA solution was added per well and treated for 30 minutes, and then the plate was washed 3 times with 2 ml of PBS per well. As a control, a plate was prepared by performing the same operation with PBS instead of the DEAE-dextran solution.

Using the above-described method in which the retroviral vector plasmid pLEIN is introduced into GP + E86 cells [Journal of Virology, Vol. 62, pages 1120 to 1124 (1988)], and sodium butyrate is added. Viral supernatant was prepared. 1 ml of this virus supernatant diluted with 20 volumes of DMEM containing 10% CS (1.6 × 10 ⁶ cfu / ml) was added at 1 ml per well and incubated at 37 ° C. for 2 hours. The plate was washed 3 times with 2 ml of PBS per well, 5 × 10 ⁴ NIH / 3T3 cells were added, and the cells were cultured at 37 ° C. for 3 days in the presence of 5% carbon dioxide gas. After completion of the culture, the cells were collected by trypsin treatment, and EGFP-expressing cells were analyzed by the flow cytometry method described in Example 4 to examine gene transfer efficiency. The results are shown in Table 20.

遺伝子導入効率は全細胞に対するＥＧＦＰ陽性細胞の割合（％）で示した。

Gene transfer efficiency was expressed as the ratio (%) of EGFP positive cells to all cells.

  As shown in Table 20, DEAE-dextran also has a retrovirus binding activity and was shown to be usable for the gene transfer method of the present invention.

Example 15
Binding of retroviruses and functional substances using centrifugation method Retrovirus plasmid containing neomycin resistance gene, DOL vector [Proceedings of the National Academy of Sciences of the USA, No. 1 84, 2150-2154 (1987)] were cultured in DMEM containing 10% CS and 50 units / ml penicillin and 50 μg / ml streptomycin. As for the DOL virus supernatant, the medium was replaced with 5 ml of DMEM containing 10% CS on a 10 cm diameter plate on which the production cells were grown semi-confluent, and the supernatant collected 24 hours later was 0.45 microns. And filtered through a filter (manufactured by Millipore). The titer of this virus supernatant was 8.7 × 10 ⁵ cfu / ml.

  A centrifuge tube (50 ml polypropylene conical tube, manufactured by Falcon) used for infection of retrovirus cells was coated with CH-296 according to the following procedure. That is, 3 ml of PBS containing 40 μg / ml of CH-296 was gently put into the bottom of the centrifuge tube and incubated at 4 ° C. for 16 hours in an upright state. Next, the CH-296 solution was replaced with 3.5 ml of PBS containing 2% BSA, and further incubated at room temperature for 30 minutes, and then the centrifuge tube was washed with 5 ml of Hanks balanced salt solution (HBSS, Gibco) did.

Binding of DOL retrovirus to the bottom of the CH-296 coated centrifuge tube was performed as follows. That is, 5 ml each of the above DOL virus supernatant stock solution, 10-fold diluted solution, or 100-fold diluted solution is placed in a centrifuge tube and centrifuged at 2900 × g, 25 ° C. for 3 hours, forcing the retrovirus into CH−. 296. For comparison, PBS containing 40 μg / ml CH-296 was used, and this was used at 8 μg / cm ² to coat CH-296. 6-well plate for surface untreated cell culture (Falcon) The same virus supernatant as above was added to the product, and incubated and allowed to bind at 37 ° C. for 4 hours.

Using a CH-296 coated centrifuge tube to which retrovirus was forcibly bound by centrifugation, gene transfer into NIH / 3T3 cells was performed. That is, 1 × 10 ⁵ NIH / 3T3 cells were placed in the CH-296-coated centrifuge tube obtained by centrifuging the virus solution diluted in each stage, and incubated at 37 ° C. for 3 hours (hereinafter referred to as centrifugation). . The above microplate was also incubated under the same conditions (hereinafter referred to as the binding method). Further, as a control, a virus solution and an NIH / 3T3 cell mixture solution added to a microplate coated with CH-296 and incubated at 37 ° C. for 3 hours were compared as a conventional infection method (hereinafter referred to as a supernatant method). ). After completion of the incubation, the gene transfer efficiency into the collected cells was examined by the method described in Example 13. The result is shown in FIG. In the figure, the horizontal axis represents the dilution rate of the virus supernatant, and the vertical axis represents the gene transfer efficiency. In addition, white indicates the results obtained by the supernatant method, shaded indicates the binding method, and black indicates the results obtained by the centrifugal method.

  As shown in FIG. 4, when the centrifugation method is used, it is higher than the conventional supernatant method and the case where the virus is naturally adsorbed on CH-296 prior to infection (binding method). Gene transfer efficiency could be obtained. That is, it was shown that more virus particles bind to CH-296 on the bottom of the container by forcibly sedimenting the virus by centrifugal force. In particular, in the case of a dilute virus solution, the effect of centrifugation was noticeable.

  Table 21 shows the results of titration of the virus supernatant collected after the virus binding operation under centrifugation and in the stationary state.

  As shown in Table 21, in the case of standing, the collected supernatant has a titer of about 80% to 90% before the operation, but when it is forcibly bound under centrifugation. The titer of the supernatant was 10% or less before the operation. This indicates that more virus particles were bound on CH-296 by centrifugal force. In addition, the amount of virus contained in PBS after washing the centrifuge tube after centrifugation is about 2% of the original solution, and the presence or absence of this washing operation has little effect on gene transfer efficiency. In this case, it was suggested that the virus particles were firmly retained on CH-296.

  Furthermore, the gene transfer efficiency by the above centrifugation method was compared with the method of carrying out virus infection to cells under centrifugation.

The virus supernatant prepared using GP + E86 cells described in Example 14 was centrifuged using NIH / 3T3 cell culture supernatant diluted to 1 × 10 ⁵ cfu / ml with ⁵ ml of virus solution, and the centrifugal force The efficiency of gene transfer into NIH / 3T3 cells was compared by each of the methods of precipitating the virus on the cells by the infection method (centrifugal infection method, see WO95 / 10619). That is, the above virus solution was added to a centrifuge tube coated with CH-296, centrifuged at 2900 × g for 4 hours at 30 ° C., then the centrifuge tube was washed with PBS, and then cells were added to infect at 37 ° C. for 4 hours. The cells were added to a centrifuge tube coated with CH-296 and cultured for 2 hours, and then the virus solution was added, followed by further centrifugation at 30 ° C. at 2900 × g for 4 hours for infection. Gene transfer was carried out for each of those (centrifugal infection method). The centrifuge tube was coated with CH-296 by the above method, and 1 × 10 ⁵ NIH / 3T3 cells were used for each gene transfer operation. The infected cells were re-seeded on a 60 mm plate and cultured for 2 days, and then the EGFP gene introduction efficiency was examined by the flow cytometry method described in Example 4. The result is shown in FIG.

  As shown in FIG. 5, it was shown that the gene transfer efficiency in the centrifugal method is higher than that in the centrifugal infection method. This is presumably because the infection inhibitory substance present in the virus solution was removed by the washing operation.

  Provided is an improved method for improving the efficiency of gene transfer and efficiently transforming target cells in gene transfer into target cells by retroviruses.

The structure of a high mannose sugar chain containing 9 mannose residues in the molecule is shown. It is a graph which shows chemically modified CH-296 in Example 3, and the gene transfer efficiency (%) obtained by it. It is a graph which shows the relative gene transfer efficiency (%) in the test of the removal effect of the virus infection inhibitor in Example 13, and the relationship between contact and a joint time. It is a graph which shows the relationship between relative gene transfer efficiency (%) in the test of the binding effect of the retrovirus and functional substance using the centrifugation method in Example 15, and each virus binding operation. It is a graph which shows the gene transfer efficiency (%) obtained by each of the centrifugation method in Example 15, and the centrifugal infection method.

Claims (8)

A method for improving the efficiency of gene transfer into a target cell by a retrovirus comprising the following steps:
(1) A step of bringing a solution containing a retrovirus into contact with a functional substance having a retrovirus binding activity immobilized on a carrier for 3 hours or more;
(2) Washing the retrovirus-bound carrier to remove viral infection inhibitory activity, and (3) Incubating the retrovirus-bound carrier in contact with target cells.   The method according to claim 1, wherein the functional substance having a retrovirus binding activity is selected from fibronectin, fibroblast growth factor, type V collagen, polylysine, DEAE-dextran and fragments thereof.   The method according to claim 1 or 2, wherein the functional substance having a retrovirus binding activity has a target cell binding activity.   The method according to any one of claims 1 to 3, wherein a carrier on which a functional substance having a retrovirus binding activity and another functional substance having a target cell binding activity are immobilized is used.   5. The method according to claim 4, wherein the functional substance having a target cell binding activity is selected from cell adhesive proteins, hormones, cytokines, antibodies, sugar chains and carbohydrates.   The method according to any one of claims 1 to 5, wherein a cell culture vessel or a particulate carrier is used as the carrier.   The method according to any one of claims 1 to 6, wherein the solution containing the retrovirus is a culture supernatant of a retrovirus-producing cell obtained in the presence of a substance that promotes retrovirus production. The method according to claim 7, wherein the solution containing the retrovirus is a culture supernatant obtained in the presence of sodium butyrate.

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