JP2011024499A - Method for producing nucleic acid, tag nucleic acid for delivery which can organ-specifically deliver delivery object, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid specifically accumulating in an organ. <P>SOLUTION: After a nucleic acid candidate is administered to an animal, a nucleic acid candidate specifically accumulated in an organ is collected from the organ of the animal as an objective nucleic acid. Using this nucleic acid with a tag nucleic acid for delivery, administration of a complex between the tag nucleic acid and a delivery object allows the delivery object to be specifically delivered to an organ similar to the organ in which the nucleic acid has been collected. For example, an aptamer is preferably used as the nucleic acid candidate. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to, for example, a method for producing a nucleic acid capable of organ-specific delivery of a substance to be delivered, a tag nucleic acid for delivery, and a use thereof.

  In order to treat or prevent various diseases, drugs according to various diseases such as anticancer agents and immunosuppressive agents are administered into the body. The drug administration method generally includes oral administration, intravenous administration, direct administration to the affected area, and the like. However, in the case of oral administration or intravenous administration, the drug is delivered not only to the target affected area but also to other parts of the body. For this reason, it is necessary to administer a larger amount of medicine than is necessary for the target affected part, and there is a problem that the cost is high and other normal parts are damaged by the medicine. Also, in the case of direct administration to the affected area, there is a possibility that the drug diffuses from the affected area to other sites, resulting in the same problem.

  Therefore, it has been attempted to construct a drug delivery system that selectively delivers a necessary minimum amount of a drug to a necessary affected part. In particular, a method for labeling a drug has been proposed as an effective method. For example, cancer cells usually have specific receptors on their surface. Therefore, when targeting the cancer cell, it is considered that the drug can be efficiently delivered to the target cancer cell by labeling the drug with a ligand that binds to the receptor (patent) Reference 1). However, although this system improves the efficiency of delivery to target cancer cells, it is still difficult to prevent delivery itself to a site other than the affected area when intravenous administration as described above is performed. That is, in the above system, when delivered to the target organ, it is possible to cause the drug to act specifically on the target cancer cells and the like instead of normal cells, but intravenous administration as described above is performed. In this case, delivery to other than the target organ is difficult to avoid and the same problem is feared.

Japanese Patent Publication No. 2006-502966 Japanese Patent Publication No. 2007-511513

Dhruba J Bharali, Marianne Khalil, Mujgan Gurbuz, Tessa M Simone, and Shaker A Mousa, Nanoparticulates and Cancer therapeutics: Int J Nanomedicine. 2009; 4: 1-7. Published online 2009 April 1. Ke-Tai Guo, Angela Paul, Christian Schichor, Gerhard Ziemer, and Hans P. et al. Wendel. CELL-SELEX: Novell Perspectives of Aptamer-Based Therapeutics. Int J Mol Sci. 2008 April; 9 (4): 668-678. Published online 2008 April 24.

  Accordingly, an object of the present invention is to provide, for example, a method for producing a nucleic acid capable of delivering a substance to be delivered in an organ-specific manner, a tag nucleic acid for delivery, and a use thereof.

  The production method of the present invention includes an administration step of administering a candidate nucleic acid to an animal, and a collection step of collecting, from the organ of the animal, the candidate nucleic acid specifically accumulated in the organ as a target nucleic acid. It is a manufacturing method.

  The nucleic acid of the present invention is a nucleic acid obtained by the production method of the present invention.

  The tag nucleic acid for delivery of the present invention is a tag nucleic acid for delivery capable of organ-specific delivery of a substance to be delivered, including the nucleic acid of the present invention.

  The composition of the present invention is a composition capable of organ-specific delivery of the delivery target substance, comprising a complex comprising the delivery tag nucleic acid of the present invention and the delivery target substance.

  According to the present invention, a nucleic acid that specifically accumulates in a desired organ can be obtained. Such a nucleic acid, for example, exhibits the property of being specifically accumulated in the same type of organ as the organ from which the nucleic acid was collected when administered into the body. Therefore, by using the obtained nucleic acid as a tag nucleic acid for delivering the delivery target substance, the delivery target substance is specifically delivered to the same type of organ as the organ from which the nucleic acid was collected. It becomes possible to do. For this reason, according to the present invention, efficient drug delivery is possible, so it can be said that this technique is extremely useful particularly in the medical field.

  The present invention is a technique for efficiently finding nucleic acids that accumulate in an organ-specific or disease-affected area-specific manner using a living body, and a nucleic acid thus obtained. Aptamers that are selected using an in vitro evolution method are known as examples of functional nucleic acids that bind to molecules, but in the present invention, in vitro selection is not performed. The present invention is desirable for a specific organ, diseased part, transplanted tissue piece, various tumor cells, iPS cells or ES cells before or after induction of differentiation, for example, using a living body such as a nude mouse. Applies to nucleic acids that specifically accumulate in human-derived transplanted cells and tissues, and methods for obtaining them (manufacturing methods). The target molecule of the nucleic acid of the present invention may be known or unknown. In addition, such newly-obtained nucleic acids alone have new functionalities that neutralize, inhibit, stimulate, or enhance the activity of enzymes, adhesion molecules, receptors, and functional molecules of specific organs. Also includes nucleic acids.

  The nucleic acid of the present invention is a functional nucleic acid that specifically accumulates in a specific organ, tumor, graft, or differentiated iPS cell or ES cell-derived organ. For this reason, such a nucleic acid alone or in a specific organ or disease site, or a specific site in vivo, preferably a drug, a toxin, or siRNA, or any molecule that affects physiological functions It can be delivered in multiple or encased in aqueous or fat soluble membranes or encapsulated in nanoparticles or nanostructures. The present invention embodies a new molecule delivery method that increases a drug effect by reducing a side effect by delivering a molecule or drug having a generally strong side effect to a specific site or a disease site. An industrial advantage of the present invention is that it can solve a problem that other normal sites are damaged by a drug.

<Nucleic acid production method>
As described above, the production method of the present invention includes an administration step of administering a candidate nucleic acid to an animal, and a collection step of collecting candidate nucleic acids specifically accumulated in the organ from the organ of the animal as a target nucleic acid. including. The nucleic acid of the present invention is a nucleic acid obtained by the production method of the present invention. The nucleic acid of the present invention obtained by such a method is preferably used as a tag nucleic acid for delivery because the substance to be delivered can be delivered specifically to the target organ as described above. . In the following, the nucleic acid and the production method of the present invention will be described, but these descriptions are the same for the tag nucleic acid for delivery of the present invention.

  The type of candidate nucleic acid to be administered to the animal is not particularly limited, and for example, single-stranded nucleic acid such as single-stranded RNA and single-stranded DNA, and double-stranded such as double-stranded RNA and double-stranded DNA. Examples include nucleic acids. In the present invention, any one kind of candidate nucleic acids may be used, or two or more kinds may be used. When the nucleic acid is the latter double-stranded nucleic acid, for example, prior to introduction into an animal, the nucleic acid is preferably made into a single strand by denaturation or the like. The nucleic acid may have, for example, a secondary structure by self-annealing, and examples of the secondary structure include a stem-loop structure.

  The candidate nucleic acid may be, for example, a naturally-occurring nucleic acid sequence or a synthesized nucleic acid sequence. The synthesis method is not limited at all, and examples thereof include a method of chemically synthesizing from a terminal base using dNTP or the like as a material with a DNA synthesizer or an RNA synthesizer. Examples of the nucleic acid include DNA and RNA as described above, and may include a peptide nucleic acid such as PNA. The nucleic acid may include, for example, natural nucleic acids (non-artificial nucleic acids) such as A, C, G, T and U, 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, 2 -It may contain artificial nucleic acids such as thiouracil.

  The length of the candidate nucleic acid is not particularly limited. The lower limit of the length is not particularly limited, but is, for example, 10 bases or more, preferably 20 bases or more, more preferably 25 bases, and the upper limit is not particularly limited, for example, 150 bases. , Preferably 100 bases, more preferably 70 bases. The range of length is, for example, 10 to 150 bases, preferably 20 to 100 bases, and more preferably 25 to 70 bases.

  Examples of the candidate nucleic acid include a delivery functional nucleic acid. The delivery functional nucleic acid is a nucleic acid having a function of delivering a substance to a target location. Examples of the delivery functional nucleic acid include an aptamer. The aptamer generally means a nucleic acid molecule that can specifically bind to a specific target substance. In the present invention, the aptamer may be, for example, an unknown target substance that specifically binds, or an unknown aptamer. Examples of the latter aptamer include an aptamer that can bind to a specific target substance present in a target cell on which a delivery target substance acts. As specific examples, when the target cell is, for example, a cancer cell, a receptor present on the surface of the cancer cell, all molecules that enter a proliferation signal into the cell, an enzyme, an adhesion molecule, a specific sugar chain, and An aptamer that binds to a target substance such as a lipid molecule present on the cell surface is preferable. Such aptamers are administered to animals as candidate nucleic acids, and candidate aptamers that accumulate organ-specifically are collected as target nucleic acids, which are used as delivery tag nucleic acids to introduce substances to be delivered such as drugs into the body. For example, in addition to realizing organ-specific delivery, the delivered drug can also act on target cells with high affinity.

  As described above, the aptamer may be DNA or RNA, for example, single-stranded nucleic acid such as single-stranded RNA and single-stranded DNA, or double-stranded nucleic acid such as double-stranded RNA and double-stranded DNA. . The aptamer may be, for example, a naturally-derived nucleic acid sequence, a synthesized nucleic acid sequence, a peptide nucleic acid such as PNA, or the natural nucleic acid (non-artificial nucleic acid). The artificial nucleic acid may also be included.

  The method for producing the aptamer is not particularly limited, and can be produced, for example, by the above-described known synthesis method. As described above, in the present invention, for example, when aptamers capable of binding to a target substance are introduced into an animal, the aptamers can be produced by, for example, the well-known SELEX (Systematic Evolution of Ligand Exponential Enrichment) method, etc. Can be adopted.

  The preparation of the aptamer by the SELEX method is not particularly limited, and can be performed, for example, as follows. First, a nucleic acid pool containing a plurality of nucleic acids is prepared, and the nucleic acid library and the target substance are bound (associated) to form a complex of the nucleic acid pool and the target substance. A nucleic acid aptamer that can specifically bind to the secondary reagent can be prepared by recovering only the nucleic acid pool involved in the formation of the complex from the complex. As an example, a method for preparing a nucleic acid aptamer that can specifically bind to a target substance using the SELEX method will be described below, but the present invention is not limited thereto.

The nucleic acid pool is, for example, a library (mixture) of nucleic acids having random regions. Examples of the nucleic acid in the library include polynucleotides such as RNA and DNA. The random region is, for example, a region in which bases of A, G, C, and U or T are randomly connected, and the length thereof is, for example, 20 to 120 mer. The nucleic acid pool preferably includes, for example, 4 ²⁰ to 4 ¹²⁰ kinds (about 10 ¹² to 10 ⁷² ) kinds of nucleic acids, and more preferably 4 ³⁰ to 4 ⁶⁰ (about 10 ¹⁸ to 10 ³⁶ ) kinds. .

  The polynucleotide included in the nucleic acid pool may have, for example, the random region, and other structures are not particularly limited. In addition to the random region, the polynucleotide has, for example, at least one of the 5 ′ end and the 3 ′ end of the random region, a primer region used for nucleic acid amplification described later, a polymerase recognition region recognized by a polymerase, and the like. It is preferable. The polymerase recognition region can be appropriately determined according to, for example, the type of polymerase used in nucleic acid amplification described later in aptamer preparation. When the nucleic acid pool is an RNA pool, the polymerase recognition region is preferably, for example, a DNA-dependent RNA polymerase recognition region (hereinafter also referred to as “RNA polymerase recognition region”), and specific examples include T7 RNA polymerase recognition region. Examples of the region include the T7 promoter. As a specific example of the RNA pool, for example, from the 5 ′ end side, the RNA polymerase recognition region and the primer region (hereinafter also referred to as “5 ′ end side primer region”) are linked in this order, The random region was linked to the 3 ′ end side of the 5 ′ end side primer region, and the primer region (hereinafter also referred to as “3 ′ end side primer region”) was linked to the 3 ′ end side of the random region. Structure. The 5 ′ terminal primer region in the RNA is, for example, a sequence complementary to the 3 ′ end of a DNA antisense strand synthesized using the RNA as a template, that is, bound to the 3 ′ end of the antisense strand. The sequence is preferably the same as possible primers. In addition, the RNA pool may further include a region that assists in binding to a secondary reagent that is a target substance, for example. In addition, the polynucleotides contained in the nucleic acid pool may have different random regions, or may have random regions that share some sequences. In the polynucleotide, the regions may be directly adjacent to each other, for example, or may be indirectly adjacent via an intervening sequence.

  The method for preparing the nucleic acid pool is not particularly limited, and a known method can be adopted. When the nucleic acid pool is an RNA pool, for example, an initial pool containing DNA can be used and the DNA can be prepared as a template. Hereinafter, a DNA strand that serves as a template for RNA in a nucleic acid pool is also referred to as an antisense strand, and a DNA strand having a sequence in which U of the RNA is replaced with T is also referred to as a sense strand. The initial pool containing the DNA has, for example, DNA (antisense strand) in which the U of the random region in the RNA pool is replaced with T (antisense strand), and a sequence in which the U in the random region is replaced with T It is preferable that any one of DNA (sense strand) is included. After performing nucleic acid amplification using DNA of this initial pool as a template and using DNA-dependent DNA polymerase, transcription reaction using DNA-dependent RNA polymerase is performed using the obtained DNA amplification product as a template. Nucleic acid pools can be prepared.

  In addition, an initial pool containing DNA in which U in the random region is replaced with T is prepared, and using this as a template, a primer containing the RNA polymerase recognition region and a sequence complementary to the 5′-end primer region A nucleic acid pool of RNA can also be prepared by annealing and nucleic acid amplification.

  Then, the nucleic acid pool and the target substance are reacted to form a complex of the nucleic acid and the target substance. In the preparation of the aptamer, the target substance to be reacted with the nucleic acid pool may be, for example, the target substance itself or a degradation product of the target substance. The binding form between the nucleic acid pool and the target substance is not particularly limited, and examples thereof include intermolecular forces such as hydrogen bonding. Examples of the binding treatment between the nucleic acid pool and the target substance include a method of incubating the both in a solvent for a predetermined time. The solvent is not particularly limited, but is preferably one that retains the binding between the two, and examples thereof include various buffer solutions.

  Subsequently, the complex of the nucleic acid pool and the target substance is recovered. In addition, in the reaction solution obtained by reacting the two for forming the complex, in addition to the complex, for example, a nucleic acid pool that does not participate in the complex formation (hereinafter referred to as “unreacted nucleic acid pool”). included. Therefore, for example, it is preferable to separate the complex and the unreacted nucleic acid pool from the reaction solution. The separation method is not particularly limited, and examples thereof include a method using a difference in adsorptivity between the target substance and the nucleic acid pool and a method using a difference in molecular weight between the complex and the nucleic acid pool.

  Examples of the method using the former difference in adsorptivity include the following methods. That is, first, a carrier having adsorptivity to the target substance is brought into contact with the reaction solution containing the complex. At this time, the unreacted nucleic acid pool is not adsorbed on the carrier, while the complex of the target substance and the nucleic acid pool is adsorbed on the carrier. Thereby, the unreacted nucleic acid pool and the complex can be separated. Then, after removing the unreacted nucleic acid pool, the complex adsorbed on the carrier may be recovered. Moreover, before recovering the complex from the carrier, for example, it is preferable to wash the carrier in order to sufficiently remove the unreacted nucleic acid pool. The carrier having adsorptivity to the target substance is not particularly limited, and can be appropriately selected according to, for example, the type of the target substance. When the target substance is a protein such as an antibody, examples of the adsorptive carrier include a nitrocellulose membrane.

  Examples of the method using the latter difference in molecular weight include a method using a carrier. Examples of the carrier include a carrier having a pore of a size that allows the nucleic acid pool to pass through and the complex cannot pass through. By using such a carrier, the complex and the unreacted nucleic acid pool can be separated. The separation may be electrical separation using, for example, an agarose gel or a polyacrylamide gel.

In addition to this, for example, a method of using a target substance immobilized on a carrier at the time of forming the complex can be mentioned. That is, the target substance is immobilized on a carrier in advance, the carrier and the nucleic acid pool are brought into contact with each other, and a complex of the immobilized target substance and the nucleic acid pool is formed. Then, after removing the unreacted nucleic acid pool not bound to the immobilized target substance, the complex of the target substance and the nucleic acid pool may be dissociated from the carrier. The method for immobilizing the target substance on the carrier is not limited at all, and a known method can be adopted. Specific examples include a method in which a label is bound to the target substance, and the target substance bound to the label is brought into contact with a carrier having a binding group with the label. Examples of the label include His-tag, and examples of the binding group include metal ions such as nickel ion (Ni ²⁺ ) and cobalt ion (Co ²⁺ ). Specific examples of the carrier include Ni-agarose and Ni-Sepharose based on the metal ions.

  Next, the nucleic acid pool involved in the complex formation is recovered from the recovered complex. The nucleic acid pool involved in the complex formation can be recovered, for example, by releasing the binding between the target substance and the nucleic acid pool for the complex.

  Subsequently, nucleic acid amplification is performed on the collected nucleic acid pool involved in the complex formation. The nucleic acid amplification method is not particularly limited, and can be performed by a known method, for example, depending on the type of the nucleic acid pool. When the nucleic acid pool is an RNA pool, for example, cDNA is prepared by a reverse transcription reaction using an RNA-dependent DNA polymerase, DNA nucleic acid amplification is performed by PCR or the like using the cDNA as a template, and the obtained amplification product is used as a template. In addition, RNA can be transcribed using a DNA-dependent RNA polymerase. As a result, the RNA pool involved in the complex formation described above can be amplified.

  For example, as described above, when the RNA pool has the RNA polymerase recognition region, the 5′-end primer region, the random region, and the 3′-end primer region, an amplification method using these regions can be used. can give. In the reverse transcription reaction for preparing the cDNA using the RNA as a template, for example, it is preferable to use a polynucleotide containing a sequence complementary to the 3'-end primer region of the RNA pool as a primer. For amplification of DNA using the cDNA as a template, for example, as a primer, a polynucleotide containing the 5′-end primer region and a polynucleotide containing a complementary strand of the 3′-end primer region are used. It is preferable. The former polynucleotide preferably further has, for example, the RNA polymerase recognition region on the 5 'end side and the 5' end side primer region on the 3 'side. For amplification of RNA using the obtained DNA amplification product as a template, the DNA amplification product is used as a template and the 5′-end primer region and the 3′-end primer region contained in the DNA are used. Nucleic acid amplification such as PCR is performed using a sex DNA polymerase. In this case, it is preferable to use, for example, a polynucleotide containing the 5'-end primer region and a polynucleotide containing a complementary strand of the 3'-end primer region. The former polynucleotide preferably further has, for example, the RNA polymerase recognition region on the 5 'end side and the 5' end side primer region on the 3 'side. Then, using the obtained amplification product as a template, using the RNA polymerase recognition region contained in the amplification product, a DNA-dependent RNA polymerase is used to perform an in vitro transcription reaction, thereby forming the complex. Nucleic acid amplification of RNA pools involved in Among the amplification products, for example, the antisense strand DNA has an RNA polymerase recognition region on the 3 ′ end side thereof, so that the DNA-dependent RNA polymerase binds to this region, and the antisense strand as a template, RNA can be synthesized. The RNA-dependent DNA polymerase used for the reverse transcription reaction is not particularly limited, and for example, avian myeloblastosis virus-derived reverse transcriptase (AMV Reverse Transscriptase) can be used.

  The nucleic acid amplification method is not particularly limited, and for example, a PCR method or various isothermal amplification methods can be employed. Also, the conditions are not particularly limited.

  In this way, the nucleic acid pool that has formed a complex with the target substance is recovered, and in the same manner as described above, the formation of the complex using the target substance, the recovery of the complex, and the nucleic acid pool involved in the complex formation The nucleic acid aptamer having the binding property to the target substance can be finally obtained by repeatedly performing the separation of the above and the amplification of the separated nucleic acid pool.

In the production method of the present invention, it is preferable to administer a plurality of nucleic acids having different sequences as candidate nucleic acids. The number of types of candidate nucleic acids to be administered to one animal is not particularly limited, but is preferably 10 ¹² to 10 ³⁶ types, for example.

  In the production method of the present invention, the animal to which the candidate nucleic acid is administered is not particularly limited, but for example, non-human such as mouse, rat, rabbit, guinea pig, hamster, cat, dog, pig, goat, horse, cow, baboon and the like Examples include mammals and birds. The animal may be, for example, an immune response-suppressed animal, and specific examples include nude mice, SCID mice, and the like.

  Further, the animal may be, for example, an animal into which cells have been transplanted. In this case, it is preferable to collect the target nucleic acid from the organ transplanted with the cells in the collecting step. Cells to be transplanted into the animal are not particularly limited, and examples thereof include normal cells, mutant cells such as cancer cells, differentiated cells and the like. The differentiated cells are not particularly limited. For example, differentiated cells differentiated from iPS cells, cell groups differentiated from ES cells, and various organ specificities, myogenic cells that differentiate into muscle, cancer stem cells, humans Examples include leukemia cells. Examples of the cell include a human cell, or a cell derived from the above-mentioned non-human mammal animal or bird, and the origin of the cell may be the same as or different from the animal into which the cell is transplanted. It may be. The cells to be transplanted are preferably, for example, the same type of organ cells as the transplant destination organ. Thus, when using the animal by which the cell was transplanted, it is preferable to use the immune response suppression animal as mentioned above.

  In the production method of the present invention, the method for administering the candidate nucleic acid to the animal is not particularly limited, and examples thereof include administration to at least one of the blood vessels and lymph vessels of the animal. The blood vessel may be, for example, a vein or an artery, but is preferably a vein. When the nucleic acid is introduced into an animal vein, the site is not particularly limited. For example, an animal having a tail such as a mouse is preferably introduced from the vein of the tail. In addition, the candidate nucleic acid may be locally administered into the body of an animal, for example. In this case, for example, candidate nucleic acids accumulated in a specific region from the administration site may be collected, or candidate nucleic acids remaining in the administration site without spreading around may be collected as accumulated candidate nucleic acids.

  In the production method of the present invention, it is preferable that the nucleic acid specifically accumulated in each organ is collected from a plurality of organs in the collecting step. In this way, by administering the candidate nucleic acid to one animal, nucleic acids that specifically accumulate in each of a plurality of organs can be obtained.

  In the collecting step, it is preferable to remove an organ from the animal and collect a nucleic acid accumulated in the organ.

  The production method of the present invention preferably further includes an amplification step of amplifying the target nucleic acid obtained in the collecting step. Thus, by amplifying the target nucleic acid, a large amount of nucleic acid accumulated in the organ can be obtained.

  In the production method of the present invention, the administration step and the collection step are preferably performed on the amplified nucleic acid obtained in the amplification step. As a result, the nucleic acid that accumulates in the organ can be selected more reliably.

  The production method of the present invention will be described with reference to an example in which a nucleic acid pool containing a plurality of types of nucleic acids as the candidate nucleic acid is administered from a mouse vein.

First, a nucleic acid pool containing a plurality of types of nucleic acids is prepared by the method described in the aptamer production method described above, and this is administered to the tail vein of a mouse. Although the total amount of the nucleic acid pool administered to the mouse is not particularly limited, for example, 10 ¹² to 10 ³⁶ per mouse. The nucleic acid pool is preferably administered, for example, in a state mixed with a solvent, and the solvent is not particularly limited, but for example, those that do not affect the living body are preferable. Specific examples include physiological saline, Examples include buffers such as HEPES and TRIS, serum-free medium for cell culture, and the like.

  Next, various organs are removed from the mouse after a predetermined time has elapsed since the administration of the nucleic acid pool. The predetermined time is not particularly limited, but is, for example, 10 to 180 minutes.

  Then, the accumulated nucleic acid is collected from the various organs removed. The method for collecting nucleic acid from the organ is not particularly limited, and a known method can be adopted. The nucleic acid collected here becomes the nucleic acid of the present invention.

  In addition, when producing a nucleic acid that accumulates in a specific organ more reliably, it is preferable to further perform the following treatment on the collected nucleic acid. That is, first, the collected nucleic acid is amplified. The method for amplifying the nucleic acid is not particularly limited, and can be performed by the same method as the amplification in the above-described aptamer production. In the case where the aptamer is, for example, an RNA aptamer, it is preferable to perform amplification using the obtained cDNA as a template after the reverse transcription reaction, and specific examples include RT-PCR. When the aptamer is, for example, a DNA aptamer, amplification is preferably performed using the DNA aptamer as a template, and a specific example is PCR.

  Then, the obtained nucleic acid amplification product is administered from the tail of a new mouse in the same manner as described above, and then various organs are extracted to collect the accumulated nucleic acid. This makes it possible to select nucleic acids that are more reliably organ-specifically accumulated. In the present invention, the number of times of the administration step and the collection step is not particularly limited. For example, the administration step and the collection step are defined as one cycle, and the total number of cycles is preferably 2 cycles or more, more preferably 3 to 15 cycles. is there.

<Tag nucleic acid for delivery>
As described above, the delivery tag nucleic acid of the present invention is a delivery tag nucleic acid capable of organ-specific delivery of a substance to be delivered, including the nucleic acid of the present invention. The tag nucleic acid for delivery of the present invention is the same as the nucleic acid of the present invention described in the production method of the present invention. In addition, the usage method of the tag nucleic acid for delivery of this invention is demonstrated in the composition of this invention.

<Composition>
The composition of the present invention comprises a complex comprising the delivery tag nucleic acid of the present invention capable of delivering a delivery target substance organ-specifically and the delivery target substance, and the delivery target substance is organ-specific. A deliverable composition.

  In the complex, the delivery target substance and the delivery tag nucleic acid may be bound, and the binding may be, for example, direct or indirect. In the latter case, for example, it may be a bond via a linker or the like, or a bond via a carrier as described later. In addition, the coupling | bonding form of both is not restrict | limited at all, For example, chemical coupling | bondings, such as a covalent bond, and physical coupling | bondings, such as adsorption | suction, may be sufficient.

The complex may further include a carrier that assists in the delivery of the substance to be delivered. In this case, the form of the complex is not particularly limited. For example, it is preferable that the carrier tag nucleic acid for delivery and the substance to be delivered are supported on the carrier. The form of loading on the carrier can be appropriately determined according to, for example, the type of carrier, but is not particularly limited, and may be, for example, binding to the carrier or dispersion within the carrier. The bond may be a chemical bond such as a common bond or a physical bond such as adsorption. Examples of the form of the complex include the following forms.
(1) Form in which the delivery tag is carried on the carrier and the delivery target substance is carried on the delivery tag nucleic acid. (2) The delivery target substance is carried on the carrier, and the delivery target substance. (3) The delivery target substance and the delivery tag nucleic acid are respectively carried on the carrier. (4) The delivery target substance and the delivery tag. , Dispersed in the carrier

  The carrier is not particularly limited, and examples thereof include nanoparticles, liposomes, micelles, reverse micelles, polycations, cell membrane permeable peptides, magnetic particles, and calcium phosphate. Any one kind of these carriers may be used, or two or more kinds may be used in combination.

  Although it does not restrict | limit especially as said nanoparticle, For example, nanocarbon, such as carbon nanohorn and a carbon nanotube, can be used. For example, any one kind of nanocarbon may be used, or two or more kinds may be used. Among the nanoparticles, carbon nanohorns are preferable. Carbon nanohorn has the property of being specifically taken up by macrophage cells that are antigen-presenting cells, for example. For this reason, when the delivery tag nucleic acid and the delivery target substance are supported on carbon nanohorn, for example, the delivery target substance is specifically delivered to a desired organ by the delivery tag nucleic acid, and the carbon With the nanohorn, the delivery target substance can be taken into macrophage cells in the organ. For this reason, for example, by carrying a drug that controls the immune reaction as the delivery target substance, it becomes possible to more reliably treat immune-related diseases. The carbon nanohorn may be, for example, a single substance or an aggregate, and the size of the aggregate is not particularly limited. For example, the maximum diameter is 10 to 100 nm.

  The delivery target substance is not particularly limited and includes various substances that are desired to be delivered to an organ. In the medical field, for example, the substance to be delivered includes, for example, a therapeutic drug or a preventive drug for a disease, and the disease is not particularly limited. For example, cancer, allergy, rheumatism, asthma, osteoporosis, Examples include immune related diseases such as inflammation. Specific examples of the therapeutic drug and the preventive drug include, for example, anticancer substances, prednisolone, FK506, NFκB decoy and other immunosuppressive substances, antiviral substances, T cell inducers, gene transcription inhibitory substances, and gene expression Inhibiting substances and the like.

The configuration of the substance to be delivered is not particularly limited, and examples thereof include low molecular compounds, proteins such as antibodies, peptides such as functional fragments of antibodies, RNAs such as amino acids, DNA, and siRNA. Examples of the functional fragment of the antibody include active fragments obtained by enzymatic treatment of various Igs. Examples of the active fragment include Fab, Fab ′, F (ab ′) ₂ , Fv, disulfide bond Fv, single chain Fv, and polymers thereof.

  The formation method of the complex containing the substance to be delivered and the tag nucleic acid for delivery is not particularly limited, and can be appropriately determined according to the type of the substance to be delivered, for example, and can be performed by a known method.

  The composition of the present invention may contain various additives as appropriate depending on, for example, the administration form into the body, in addition to the complex. Moreover, the form is not particularly limited, and can be appropriately determined according to the administration form into the body.

<Treatment and prevention methods>
The treatment method and prevention method of the present invention include a step of administering the composition of the present invention into the body. In the composition, the substance to be delivered can be appropriately determined according to the purpose of treatment and prevention, and examples thereof include the above-mentioned therapeutic substance and prophylactic substance.

  The method for administering the composition is not particularly limited, and examples thereof include oral administration and parenteral administration. Examples of the parenteral administration include intravenous administration, arterial administration, administration to lymphatic vessels, intramuscular administration, subcutaneous administration, rectal administration, transdermal administration, intraperitoneal administration, and local administration. The administration target of the composition is not particularly limited, and examples thereof include humans and mammals other than humans such as mice and rats.

  According to the present invention, a nucleic acid that specifically accumulates in a desired organ can be obtained. Such a nucleic acid, for example, exhibits the property of being specifically accumulated in the same type of organ as the organ from which the nucleic acid was collected when administered into the body. Therefore, by using the obtained nucleic acid as a tag nucleic acid for delivering the delivery target substance, the delivery target substance is specifically delivered to the same type of organ as the organ from which the nucleic acid was collected. It becomes possible to do. For this reason, according to the present invention, efficient drug delivery is possible, so it can be said that this technique is extremely useful particularly in the medical field.

Claims (27)

An administration step of administering the candidate nucleic acid to the animal; and
A method for producing a nucleic acid, comprising: collecting a candidate nucleic acid specifically accumulated in the organ from the organ of the animal as a target nucleic acid. The production method according to claim 1, wherein the nucleic acid collected in the collecting step is a delivery tag nucleic acid capable of organ-specific delivery of a delivery target substance. The production method according to claim 1 or 2, wherein the candidate nucleic acid is introduced into at least one of a blood vessel and a lymphatic vessel of the animal in the administration step. The production method according to any one of claims 1 to 3, wherein the candidate nucleic acid is an aptamer. The manufacturing method according to any one of claims 1 to 4, wherein a plurality of types of candidate nucleic acids are introduced in the administration step. The production method according to any one of claims 1 to 5, wherein in the collecting step, candidate nucleic acids specifically accumulated in the organs are collected as target nucleic acids from a plurality of organs. The production method according to any one of claims 1 to 6, wherein in the collecting step, an organ is extracted from the animal, and candidate nucleic acids accumulated in the organ are collected as target nucleic acids. Furthermore, the manufacturing method as described in any one of Claim 1 to 7 including the amplification process which amplifies the target nucleic acid obtained at the said collection process. Furthermore, the manufacturing method as described in any one of Claim 1 to 8 which performs the said administration process and collection process about the amplified nucleic acid obtained at the said amplification process. The production method according to any one of claims 1 to 9, wherein the animal is at least one animal selected from the group consisting of non-human mammals and birds. The production according to claim 10, wherein the non-human mammal is at least one animal selected from the group consisting of mouse, rat, rabbit, guinea pig, hamster, cat, dog, pig, goat, horse, cow and baboon. Method. The production method according to any one of claims 1 to 11, wherein the animal is an immune response-suppressed animal. The production method according to claim 12, wherein the animal is a nude mouse. The animal is an animal transplanted with cells;
The production method according to any one of claims 1 to 13, wherein in the collecting step, the target nucleic acid is collected from an organ transplanted with the cells. The production method according to claim 14, wherein the cells transplanted into the animal are at least one selected from the group consisting of normal cells, mutant cells, and differentiated cells. The production method according to claim 15, wherein the differentiated cells are differentiated cells differentiated from iPS cells. The production method according to any one of claims 14 to 16, wherein the cells transplanted into the animal are human cells. A nucleic acid obtained by the production method according to any one of claims 1 to 17. The nucleic acid according to claim 18, wherein the nucleic acid is an aptamer. 20. A delivery tag nucleic acid capable of organ-specific delivery of a substance to be delivered, comprising the nucleic acid according to claim 18 or 19. 21. A composition capable of organ-specific delivery of the delivery target substance, comprising a complex comprising a delivery tag nucleic acid capable of organ-specific delivery of the delivery target substance according to claim 20 and the delivery target substance. The composition according to claim 21, wherein the delivery target substance and the delivery tag nucleic acid are bound to each other in the complex. The complex further includes a carrier that assists in the delivery of the substance to be delivered;
The composition according to claim 21 or 22, wherein the carrier is at least one selected from the group consisting of nanoparticles, liposomes, micelles, reverse micelles, polycations, cell membrane permeable peptides, magnetic particles, and calcium phosphate. 24. The composition of claim 23, wherein the nanoparticles are at least one nanocarbon of carbon nanohorns and carbon nanotubes. The composition according to claim 23 or 24, wherein the carrier carries the substance to be delivered and the tag nucleic acid for delivery. The composition according to any one of claims 21 to 25, wherein the substance to be delivered is a therapeutic drug or a preventive drug for a disease. 27. The composition according to claim 26, wherein the substance to be delivered is at least one of an anticancer substance and an immunosuppressive substance.