Method for maintaining and / or enhancing effect of activator, method for producing object cell, and delivery carrier set and kit

Abstract

To provide a technique for obtaining a desired effect of an activator on a target cell.SOLUTION: According to one embodiment, a method for maintaining and / or enhancing the effect of an activator in a target cell includes bringing a first lipid particle encapsulating a first activator into contact with a target cell in a first state, generating a target cell in a second state from the target cell in the first state, bringing a second lipid particle encapsulating a second activator into contact with the target cell in the second state, generating a target cell in a third state from the target cell in the second state, and maintaining and / or enhancing the effect of the activator in the target cell. Each of the first and second lipid particles has a lipid composition that is designed to exhibit an affinity suitable for the corresponding target cell in the first and second states, respectively.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Embodiments of the present invention relate to a method for maintaining and / or enhancing an activator effect, a method for producing target cells, an introduction carrier set, and a kit.

Background Art

[0002] Introduction of genes into target cells is widely used from basic research to clinical applications, for example, for the treatment, prevention, and diagnosis of various diseases such as cancer, or for silencing specific genes and cell reprogramming. Such gene introduction can obtain the desired effect by the gene introduced into the target cell exhibiting specific activity in the target cell.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The problem to be solved by the present invention is to provide a technique for obtaining a desired effect of an activator on a target cell.

Means for Solving the Problems

[0004] The method according to the embodiment maintains and / or enhances the activator effect in the target cell. The method includes contacting a first lipid particle encapsulating a first activator with a target cell in a first state, generating a target cell in a second state from the target cell in the first state, contacting a second lipid particle encapsulating a second activator with the target cell in the second state, generating a target cell in a third state from the target cell in the second state, and maintaining and / or enhancing the activator effect in the target cell. The first and second lipid particles each have a lipid composition designed to exhibit appropriate affinity for the corresponding target cells in the first and second states, respectively. The lipid composition of the second lipid particle is different from the lipid composition of the first lipid particle.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0006] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially the same constituent parts are denoted by the same reference numerals, and the description thereof may be partially omitted. The drawings are schematic, and the relationship between the thickness and the planar dimensions of each part, the ratio of the thicknesses of each part, etc. may be different from the actual ones.

[0007] (First Embodiment) The first embodiment is a method for maintaining and / or enhancing the effect of an active agent in target cells. In this method, the introduction carrier set 1001 shown in FIG. 1 is used to activate the target cells. An example of the introduction carrier set will be described with reference to FIG. 1. The introduction carrier set 1001 includes a first introduction carrier 101a and a second introduction carrier 101b. The first introduction carrier 101a includes a lipid particle 11a and an active agent 12a. The lipid particle 11a is a hollow body formed of a lipid membrane, for example, it is a liposome. Inside the lipid particle 11a, that is, in the hollow part, the active agent 12a is encapsulated. The second introduction carrier 101b includes a lipid particle 11b and an active agent 12b. The lipid particle 11b is a hollow body formed of a lipid membrane, for example, it is a liposome. Inside the lipid particle 11b, that is, in the hollow part, the active agent 12b is encapsulated. In the example shown in FIG. 1, the active agent 12a and the active agent 12b are different types of active agents from each other.

[0008] The lipid particle 11a has an appropriate affinity for the target cells in the first state. Here, "appropriate affinity" means, for example, when referring to the target cells in the first state, it has a higher affinity compared to a similar introduction carrier with a general design under normal and / or general contact conditions with the target cells in the first state. Such appropriate affinity will be achieved by adjusting the lipid composition of the lipid particle, which will be described in detail later.

[0009] Here, "target cells" refers to the cells to which the active agent is to act. For example, the target cells in the first state can become the target cells or the target cells in the second state by the action of the active agent. For example, the target cells in the first state are the cells that should undergo changes by the introduction or action of the active agent. For example, they may be diseased cells to be treated by the introduction of the active agent, or they may be source or origin material cells for the target cells to be obtained. The target cells may be selected according to the purpose.

[0010] The "target cell" can be, for example, a cell in a state after an activator has contacted or acted on a target cell as a source cell, a cell in a state after an activator has acted, a cell in a state after reacting with an activator, or a cell in a state after an activator has been introduced. What kind of target cells to produce and / or acquire may be determined by the implementer as desired. In cases such as when repeating the method and the steps included in the method, in some cases, the target cell can become a further source cell to which an activator should be introduced or allowed to act subsequently. The "source cell" can be used interchangeably with the terms "initial source cell", "source cell in the first state", and "seed cell". For example, when using a plurality of activators and performing repeated introductions multiple times, the source cell is in the first state before introduction, and depending on the number of repetitions of the steps required to finally obtain the target cell, the source cell, i.e., the target cell, can continue to change in its properties or characteristics. Here, for the sake of convenience, the term "state" is used for its state, properties, or the presence or absence of contact with a specific activator, and it is also described as "source cell in the second state", "source cell in the third state"... "source cell in the nth state" (n is an integer of 3 or more) according to the number of introductions or actions of the activator. Whether to use the "target cell" as a "source cell" for subsequent steps after the "source cell" has made contact with a specific activator or as the "target cell" to be finally obtained can be determined according to the desire of the implementer or according to subsequent procedures.

[0011] As described above, the "target cell" can be the "cell in the desired state" or the cell to be produced. Examples of target cells can be any desired cells configured to have properties different from those of the initial source cell, the target cell, by introducing an activator. Such cells can be, for example, any cells formed by introducing genes. Examples of target cells are cells used in the laboratory level, basic research field, therapeutic field, regenerative medicine field, etc., such as treated cells, healthy cells, cells treated for disorders or defects, cells in which disorders or defects have been resolved, modified cells, or reprogrammed cells, and induced pluripotent stem cells (iPS cells) and other artificial pluripotent stem cells.

[0012] Here, the "activator" may be an active ingredient that contacts, introduces and / or acts on the target cells, and / or reacts with the target cells to change them into target cells having properties, physical properties and / or forms different from the original target cells, transfer them to the next state, or transfer them to the target cells, or bring them into the target state. Examples of the activator may be substances for recombining the genome of desired cells, etc., and generally may be substances referred to as genes. Specifically, for example, the activator may be a natural product, a compound, an extract, a nucleic acid, a peptide, a protein, etc. For example, the nucleic acid may be a nucleic acid fragment, a nucleic acid construct, DNA, RNA, etc. The activator applied to the material cells may be one type or a combination of two or more types. Alternatively, the activities of the first activator and / or the second activator may be protein synthesis activity, protein cleavage activity, activity affecting protein expression status, enzyme activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, genome editing activity, etc. For example, the first activator and the second activator may be nucleic acid substances encoding any gene having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity and genome editing activity. Also, for example, for uniform introduction, it is also preferable that the activator contained in one liposome be one type. However, the activator may consist of one type of component, or may consist of a plurality of components of different types. In the method, a plurality of types of activators may be used in one step, a plurality of types of activators may be used respectively in a plurality of steps, or one type of activator may be used in one step, and the same type of activator may be used respectively in a plurality of steps.

[0013] For example, when the active agent is introduced into the target cell, during or after the introduction, for example, at least one of the time points or locations such as when approaching the target cell, when contacting the target cell, when entering the interior of the target cell, when contacting the nucleus of the target cell, or during or after entering the nucleus, it can be a substance that acts on the target cell and / or reacts with the target cell. The "introduction" of the active agent into the cell, in a narrow sense, refers to bringing the active agent to a distance where it can act on and / or react with the cell. In a broad sense, in addition to that, it comprehensively includes the active agent contacting the cell, the active agent being brought into the cell, the active agent being brought into a state where it can act on the nucleus of the cell, the active agent being brought into a state of contacting the cell, the active agent being brought into the nucleus of the cell, etc. Also, its "activity" can be that the active agent acts on the cell, the active agent reacts with the cell, or both. For example, the introduction of the active agent into the cell can be carried out by bringing the active agent into a solution containing the cell. For example, the introduction of the active agent into the cell, for example, the contact with the cell, may be carried out by incubation at a certain temperature. The incubation may be carried out while being sent through a flow path, for example, may be carried out while being held at a specific position by a cell capture mechanism or device, or may be carried out in one container. For example, the incubation temperature can be about 35°C to about 38°C, etc., depending on the types of cells and active agents. When the activator is introduced, depending on the types and situations of the activator and / or the target cell, a specific state of the target cell becomes different from its initial state, and / or the target cell becomes the target cell.

[0014] As described above, the lipid particles can be lipid liposomes. It can be particles with a lipid membrane encapsulating an aqueous core, for example, particles with a lipid bilayer membrane. Any liposome known per se can be used as the liposome. For example, the lipid composition forming the liposome may contain, as its constituent components, the first lipid (FFT-10) of formula (I) and / or the second lipid (FFT-20) of formula (II). These lipids are biodegradable lipids. By adjusting the lipid composition of the lipid particles using these lipids, appropriate affinity can be achieved. [Chemical formula]

[0015] In addition to the above-described first lipid and second lipid, the lipid particles may contain further lipids. Among the compositions of the lipid molecular materials constituting the lipid particles, the fraction consisting of the first lipid and the second lipid is hereinafter referred to as the "first fraction". Also, the fraction consisting of lipid molecular materials other than the first lipid and the second lipid is hereinafter referred to as the "second fraction". The lipids contained in the second fraction are collectively hereinafter also referred to as the "third lipid".

[0016] The terms "first fraction" and "second fraction" represent the composition of the constituent components of the lipid particles and do not indicate the physical positions of the lipids contained therein. For example, the constituent components of the first fraction and the second fraction do not necessarily form one lump in the lipid particles, and the lipids contained in the first fraction and the lipids contained in the second fraction may be mixed and present. The blending ratio of the first fraction with respect to the entire lipid material constituting the lipid particles may be 5% or more, 10% or more, 15% or more, for example, 10% to 80%, or 15% to 60%, etc.

[0017] In other words, the total content of FFT-10 and / or FFT-20 may be 5% or more, 8% or more, 10% or more, 15% or more as the blending ratio of the lipid particles, for example, 10% to 60%, or 15% to 60%, 15% to 50%, etc. The maximum content of FFT-10 and FFT-20 in the lipid particles may be, for example, an amount such that the lipid particles can form liposomes. The blending ratio of the second lipid in the first fraction may be 0% or more to 100%, for example, 15% to 75, 20% to 60%, 24% to 50%, etc. Similarly, the blending ratio of the first lipid in the first fraction may be 0% or more to 100%, for example, 15% to 75%, 20% to 60%, 24% to 50%, etc. Here, the percentage is shown as mol / mol% unless otherwise specified.

[0018] Depending on the mixing ratio of the first lipid and the second lipid in the first fraction, the particle size and cell permeability of the lipid particles may change. For example, the larger the amount of the second lipid, the larger the particle size of the lipid particles can be. The average particle size of the lipid particles can be changed according to the application. For example, it may be adjusted to about 50 nm to about 300 nm. For example, it may be about 70 nm to about 100 nm.

[0019] The type of the third lipid contained in the second fraction of the lipid particles is not limited. For example, the second fraction contains a base lipid. As the base lipid, for example, a lipid that is a main component of a biological membrane can be used. The base lipid can be a phospholipid or a sphingolipid, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin or cerebroside, or a combination thereof, etc.

[0020] For example, as the base lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol, or a combination thereof, etc. is preferably used. As the above base lipid, it is particularly preferable to use a lipid such as a cationic lipid or a neutral lipid, and the acid dissociation constant of the lipid particles can be adjusted by its content. For example, it is also preferable to use DOTAP as the cationic lipid, and it is also preferable to use DOPE as the neutral lipid.

[0021] The blending ratio of cationic lipids such as FFT10, FFT20, and DOTAP to the whole lipid particles is preferably, for example, 65% or less in order to adjust the appropriate affinity for target cells. For example, it may be in the range of 5% - 65%, 10% - 65%, 20% - 65%, 30% - 65%, 40% - 65%. In addition, the adjustment of the lipid composition for obtaining an appropriate affinity can be achieved, for example, by changing the component ratio of the cationic lipid contained in the lipid particles or by providing a gradient according to the type and state of the target cells, so as to design a lipid composition that shows an appropriate affinity for each of a plurality of types or states. In order to provide an appropriate affinity for target cells in a specific state, the component ratio of the cationic lipid can be adjusted, and by changing the component ratio of the cationic lipid between the first lipid particles and the second lipid particles, it is possible to achieve an appropriate affinity for target cells in each state. For example, an appropriate affinity can be achieved by making the component ratio of the cationic lipid contained in the second lipid particles larger than the component ratio of the cationic lipid of the first lipid particles.

[0022] The second fraction preferably also contains a lipid that prevents aggregation of lipid particles. For example, the lipid that prevents aggregation may further contain a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristoyl glycerol (DMG-PEG), a polyamide oligomer derived from an omega-amino (oligoethylene glycol) alkanoic acid monomer (U.S. Patent No. 6,320,017), or monosialoganglioside, etc.

[0023] The second fraction may further contain lipids such as a relatively low-toxicity lipid for adjusting toxicity; a lipid having a functional group for binding a ligand to the lipid particle; a lipid for suppressing leakage of inclusions such as sterols, for example, cholesterol. In particular, it is preferable to include cholesterol.

[0024] The type and composition of the lipid used in the second fraction may be appropriately selected in consideration of the acid dissociation constant (pKa) of the target lipid particle, the particle size of the lipid particle, the type of the included active agent, or the stability in cells, etc.

[0025] For example, when the second fraction contains DOPE, DOTAP, cholesterol, and DMG-PEG, it is preferable because the delivery efficiency of the active agent is particularly excellent.

[0026] In addition to the active agent, further components may be encapsulated in the lipid particle as needed. The further components are, for example, a pH adjuster, an osmotic pressure adjuster, a gene activator, etc. The pH adjuster is, for example, an organic acid such as citric acid and its salts, etc. The osmotic pressure adjuster is sugar or an amino acid, etc. Here, the gene activator can be any substance that promotes or supports the activity of the active agent when the active agent is a gene.

[0027] Lipid particles encapsulating an active agent and, if necessary, other substances can be produced using, for example, known methods used when encapsulating small molecules in lipid particles, such as the Bangham method, the organic solvent extraction method, the surfactant removal method, or the freeze-thaw method. For example, a lipid mixture obtained by dissolving the material of the lipid particles in an organic solvent such as alcohol at a desired ratio and an aqueous buffer containing components to be encapsulated such as an activator are prepared, and the aqueous buffer is added to the lipid mixture. By stirring and suspending the resulting mixture, lipid particles encapsulating the activator and the like are formed. The lipid particles thus obtained are an example of liposomes.

[0028] By using such liposomes, it is possible to improve the introduction of the activator into cells, for example, the gene introduction rate. In addition, the cell introduction rate of the activator becomes more uniform, and quality control becomes easier.

[0029] The method activates target cells using the introduction carrier set 1001 as described above. The method can be, for example, a method of maintaining and / or enhancing the effect of the active agent in the target cells. Specifically, as shown in FIG. 2, contacting the first lipid particles encapsulating the first active agent with the target cells in the first state (S21), generating the target cells in the second state from the target cells in the first state (S22), contacting the second lipid particles encapsulating the second active agent with the target cells in the second state (S23), generating the target cells in the third state from the target cells in the second state (S24), and at least including maintaining and / or enhancing the effect of the active agent in the target cells. Here, the first lipid particles have a lipid composition designed to show an appropriate affinity for the target cells in the first state. The lipid composition of the second lipid particles is different from the lipid composition of the first lipid particles and has a lipid composition designed to show an appropriate affinity for the target cells in the second state. The second active agent may be of the same type as the first active agent or of a different type.

[0030] Bringing S21 and S23 into contact can be performed, for example, by adding an activator to a place where the target cells are contained, such as an aqueous solution. Generating the target cells in the second or third state from the target cells in the first or second state in S22 and S24 may be achieved by the contact of S21 and S23 and / or by leaving them standing after such contact, for example, by incubation. The fact that the state of the target cells has changed from the initial state to a different state, that is, from the first state to the second state and from the second state to the third state, includes changes observable or less than observable in the appearance of the target cells, such as properties of the overall morphology and partial morphology, color and color tone, for example, the abundance, distribution, dynamics, functional changes, functional changes, presence or absence of synthesis of specific intracellular substances, magnitude of synthetic ability, amount of synthesis, etc. of amino acids, peptides, proteins, RNAs, salts, functional components, non-functional components, etc.

[0031] For example, when the activator causes changes less than observable in the target cells, the method can maintain or enhance such effects. For such a purpose, for example, an introduction carrier set 2001 according to a further embodiment as shown in FIG. 3 may be used. FIG. 3 is a cross-sectional view showing a further example of the introduction carrier set. The introduction carrier set 2001 includes a first introduction carrier 101a and a second introduction carrier 201b. The first introduction carrier 101a in this case is as described above. The second introduction carrier 201b in this case includes lipid particles 11b and an activator 12a. The lipid particles 11b are hollow bodies formed of a lipid membrane, for example, they are liposomes. Inside the lipid particles 11b, that is, in the hollow part, the activator 12a is encapsulated. This activator 12a is of the same type as the activator 12a contained in the lipid particles 11a and shows the same effect. The lipid composition of the lipid particles 11b is designed to show an appropriate affinity for the target cells that have undergone changes less than observable by the first introduction carrier 101a.

[0032] According to the method for maintaining and / or enhancing the effect of an activator in target cells using the introduction carrier set 2001, first, the introduction carrier 101a is brought into contact with the target cells in the first state (S21), and the target cells in the second state are generated from the target cells in the first state (S22). Next, the introduction carrier 201b is brought into contact with the target cells in the second state (S23), and the target cells in the third state are generated from the target cells in the second state (S24). Thereby, for example, the expression of a change that is less than observable by the activator is maintained or enhanced in the target cells. For example, if an easy-to-understand example is given, according to such a method, the activator 12a is more effectively introduced into the target cells, so the expression of the effect of the activator 12a is more effectively maintained or enhanced. As a result, for example, a change that is less than observable can be observed.

[0033] Regarding the concept of the method of the first embodiment, how it functions in target cells will be described with reference to FIG. 4. In this example, the introduction carrier set 2001 shown in FIG. 3 is used. The target cell 20a is in the first state represented by a white outline in FIG. 4. First, when the first introduction carrier 101a is introduced into the target cell 20a, for example, the introduction carrier 101a contacts the target cell 20a, enters its cytoplasm, decomposes, and the activator 12a further proceeds into the nucleus 21a. As a result, due to the activator 12a, the target cell 20a transitions from the first state represented by a white outline to the second state indicated by dots. Next, the second introduction carrier 201b having an appropriate affinity for this second state is introduced into the target cell 20b. The introduction carrier 201b proceeds, for example, into the interior of the target cell 20b in the second state, decomposes, and the activator 12a further migrates into its nucleus 21b. Thereby, the target cell 20c in the third state indicated by diagonal lines is obtained from the second state represented by dots. In this way, for example, by using introduction carriers having different lipid compositions each having an affinity adapted to the state for the same type of activator 12a, it is possible to enhance the effect of the activator 12a in the target cell in sequence or to sustain the effect of the activator 12a. However, even when using introduction carriers each containing different types of activators 12a and 12b, it is possible to sustain and / or enhance the effects of the activators 12a and / or 12b.

[0034] Using FIG. 5, a further example of the method will be described. In this example, the introduction carrier set 1001 shown in FIG. 1 is used. First, when the first introduction carrier 101a is introduced into the target cell 20a, the introduction carrier 101a contacts the target cell 20a, enters its cytoplasm, decomposes, and the activator 12a further proceeds into the nucleus 21a. As a result, the activator 12a forms, for example, substance a, for example, peptide a. In other words, due to the action of the activator 12a, the target cell 20a that was in the first state of not producing peptide a becomes the target cell 20b that is in the second state of producing peptide a. Next, a second introduction carrier 101b having an appropriate affinity for this second state is introduced into the target cell 20b. The introduction carrier 101b proceeds into the interior of the target cell 20b in the second state, decomposes, and the activator 12b further migrates into its nucleus 21b. Thereby, for example, substance b, for example, peptide b is produced. That is, due to the action of the activator 12b, the target cell 20b in the second state of producing peptide a but not producing peptide b becomes the target cell 20c in the third state of producing peptide a and peptide b. By this method, in the target cell 20c in the third state, the activity of the first activator 12a is enhanced by the second activator 12b. Such enhancement will occur, for example, by an event occurring between substance a and substance b, for example, an interaction, or some action known per se from one to the other. Such an action may be, for example, the binding of peptide a and peptide b.

[0035] According to such an embodiment, it is possible to maintain and / or enhance the effect of a desired activator in a desired target cell. For example, even when it is difficult to obtain an effect by a normal general method or a conventional method, the effect of the activator can be maintained for a desired period, and / or, for example, when it is difficult to observe or detect, or when it is difficult to sufficiently obtain a desired effect, it can be made observable or detectable, or a sufficient effect can be obtained.

[0036] (Second Embodiment) The second embodiment is, briefly speaking, a method of repeating the steps of the method of the first embodiment. Specifically, the method may be the same as the method of the first embodiment, except that, in addition to the method of the first embodiment, it includes further contact between the activator and the target cells and generation of target cells in a further state. By introducing such an activator, the state of the target cells changes sequentially. It is a method of sequentially using an introduction carrier having lipid particles appropriate for the state and allowing the activator encapsulated therein to act.

[0037] This method will be described with reference to FIG. 6. First, two examples of the introduction carrier sets used therein are shown in FIGS. 7 and 8, respectively. The introduction carrier set 7001 shown in FIG. 7 includes the same type of activator 12a encapsulated in lipid particles 11a to 11x designed to have appropriate affinity for target cells in each state. x is equal to n described later, and n is an integer of 3 or more. The introduction carrier set 8001 shown in FIG. 8 includes different types of activators 12a to 12x encapsulated in lipid particles 11a to 11x designed to have appropriate affinity for target cells in each state.

[0038] This method activates target cells, for example, using the introduction carrier set 7001 or 8001. This method can be, for example, a method of maintaining and / or enhancing the activator effect in target cells. Specifically, as shown in FIG. 6, contacting the first lipid particles encapsulating the first activator with the target cells in the first state (S61), generating the target cells in the second state from the target cells in the first state (S62), contacting the second lipid particles encapsulating the second activator with the target cells in the second state (S63), generating the target cells in the third state from the target cells in the second state (S64), contacting the nth lipid particles encapsulating the nth activator with the target cells in the nth state (S65), generating the target cells in the n + 1th state from the target cells in the nth state, and maintaining and / or enhancing the activator effect in the target cells (S67) at least.

[0039] Here, n is an integer of 3 or more, and n generations are sequentially performed from the generation of the target cells in the second state to the generation of the target cells in the (n + 1)-th state. Herein, the first to n-th lipid particles each have a lipid composition designed to exhibit appropriate affinity for the corresponding target cells in the first to n-th states. The first to n-th active agents may be of the same type as each other, or some of the active agents may be of the same type as each other, or some of the active agents may be of different types from each other. Alternatively, the first to n-th active agents may all be of different types from each other. Also, the first to n-th lipid particles are designed with a lipid composition to have appropriate affinity for the corresponding target cells. For example, all of the first to n-th lipid particles may have different lipid compositions from each other, or some of the lipid particles among the first to n-th lipid particles may have the same composition as each other, or some of the lipid particles may have different compositions from each other.

[0040] Here, n is an integer of 3 or more. For example, n may be an integer from 3 to 30, or an integer from 3 to 10, or an integer from 3 to 5. As described above, x is equal to n. The numbers of n and x, the number of states of the target cells, and the type and state of the target cells, etc. may be selected according to the purpose of the implementer.

[0041] According to such an embodiment, it is possible to maintain and / or enhance the effect of a desired active agent in a desired target cell. For example, even in a case where it is difficult to obtain an effect by a normal general method or a conventional method, the effect of the active agent can be maintained over a desired period, and / or even in a case where it is difficult to observe or detect, or where a desired effect is sufficiently difficult to obtain, it can be made observable or detectable, or a sufficient effect can be obtained. By such a method, it is possible to obtain a target cell.

[0042] (Third Embodiment) The third embodiment is a method for producing predetermined target cells from target cells by using the above-described first and second embodiments. In the method according to the first embodiment, the target cells in the third state may be used as the target cells, and in the method according to the second embodiment, the target cells in the (n + 1)-th state may be used as the target cells. In other words, the method may be designed such that the target cells are the target cells in the third state or the target cells in the (n + 1)-th state in advance.

[0043] (Fourth Embodiment) The fourth to seventh embodiments described below are introduction carrier sets for use in any of the above methods. For example, the introduction carrier set for use in the first embodiment may be, for example, the introduction carrier set as shown in FIG. 1 or FIG. 3. The fourth embodiment described next is the introduction carrier set shown in FIG. 1.

[0044] An introduction carrier set according to the fourth embodiment will be described with reference to FIG. 1. The introduction carrier set 1001 includes a first introduction carrier 101a and a second introduction carrier 101b. The introduction carrier 101a includes lipid particles 11a (for example, liposomes 11a) and an active agent 12a encapsulated therein. The lipid particles 11a have an appropriate affinity for target cells in a first state. The introduction carrier 101b includes lipid particles 11b (for example, liposomes 11b) and an active agent 12b encapsulated therein. The lipid particles 11b have an appropriate affinity for target cells in a second state. The active agent 12a and the active agent 12b are of different types. Here, "being of different types" means substances of different types from each other, which may be substances having the same activity or effect, or substances of different types from each other and having different activities or effects, or even substances classified into the same category or substances classified into different categories. The selection of the first active agent and the second active agent, or the selection of their combination, may be selected according to the target cells and / or the target cells and the desired purpose. By using the introduction carrier set 1001, the active agent to be introduced can be appropriately introduced into the target cells according to the state of the target cells. Thereby, it becomes possible to maintain and / or enhance the effect of the active agent to be introduced into the target cells. Thereby, it is possible to obtain the target cells more efficiently.

[0045] (Fifth Embodiment) The fifth embodiment will be described with reference to FIG. 3. The introduction carrier set 2001 shown in FIG. 3 includes a first introduction carrier 101a and a second introduction carrier 201b. The introduction carrier 101a includes lipid particles 11a (for example, liposomes 11a) and an active agent 12a encapsulated therein. The lipid particles 11a have an appropriate affinity for target cells in the first state. The introduction carrier 201b includes lipid particles 11b (for example, liposomes 11b) and the active agent 12a encapsulated therein. The lipid particles 11b have an appropriate affinity for target cells in the second state. The introduction carrier set 2001 can maintain and / or enhance the effect of the active agent by repeatedly introducing the same type of active agent. Thereby, it is possible to efficiently obtain desired target cells or homogeneous target cells.

[0046] (Sixth Embodiment) The sixth embodiment is used, for example, for the method according to the second embodiment shown in FIG. 6. The sixth embodiment will be described with reference to FIG. 7. The introduction carrier set 7001 includes first to xth introduction carriers 101a to 101x. The first introduction carrier 101a includes lipid particles 11a (for example, liposomes 11a) and an active agent 12a encapsulated therein. The lipid particles 11a have an appropriate affinity for target cells in the first state. The introduction carrier 101b includes lipid particles 11b (for example, liposomes 11b) and the active agent 12a encapsulated therein. The lipid particles 11b have an appropriate affinity for target cells in the second state. x corresponds to n of the corresponding method and is an integer of 3 or more. The first to xth introduction carriers 101a to 101x respectively correspond to the target cells in the first to nth states and each have an appropriate affinity for the target cells in each state. Here, the first to xth introduction carriers all encapsulate the same type of active agent 12a. Thereby, it becomes easier for the active agent and / or the effect of the active agent to reach any of the target cells in each state. By reaching in a stacked manner, it becomes possible to maintain and / or enhance the effect of the active agent 12a in the target cells. Also, it is possible to produce target cells well, appropriately, efficiently, or homogeneously.

[0047] (Embodiment 7) Embodiment 7 is used, for example, for the method according to Embodiment 2 shown in FIG. 6. Embodiment 7 will be described with reference to FIG. 8. The introduction carrier set 8001 includes the first to xth introduction carriers 101a to 101x. The first introduction carrier 101a includes lipid particles 11a (for example, liposomes 11a) and an active agent 12a encapsulated therein. The lipid particles 11a have an appropriate affinity for target cells in the first state. The introduction carrier 101b includes lipid particles 11b (for example, liposomes 11b) and the active agent 12a encapsulated therein. The lipid particles 11b have an appropriate affinity for target cells in the second state. x corresponds to n of the corresponding method and is an integer of 3 or more. The first to nth lipid particles 11a to 11x respectively correspond to the target cells in the first to nth states and each have an appropriate affinity for the target cells in their respective states. The first to xth introduction carriers may all encapsulate different types of active agents 12a to 12x, or some of the first to xth introduction carriers may encapsulate the same type of active agent. Thereby, the active agent and / or the effect of the active agent can easily reach any of the target cells in each state. By sequentially delivering appropriate active agents according to the situation of the target cells and according to the desired plan, it is possible to maintain and / or enhance the effect of the active agents 12a to 12x in the target cells. Also, it is possible to produce target cells well or appropriately or efficiently or homogeneously.

[0048] (Embodiment 8) The introduction carrier sets according to the above-described fourth to seventh embodiments may be provided in a state immediately usable for desired target cells, or may be provided as an introduction carrier production kit in the form of a material so that the user of the above-described introduction carrier set can adjust it at the time of use. In that case, the kit may include, for example, a first activator to be introduced into target cells in a first state, and a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state, a material for first lipid particles for encapsulating the first activator, a second activator to be introduced into target cells in a second state, which may be of the same type or a different type from the first activator, a lipid composition having a lipid composition different from that of the first lipid particles and designed to exhibit an appropriate affinity for the target cells in the second state, and a material for second lipid particles for encapsulating the second activator.

[0049] These materials for the first and second lipid particles, and the first and second activators may be provided in containers in an appropriate state so that each is stably provided as a substance. An introduction carrier production instruction manual for the user to appropriately perform production may further be included.

[0050] Methods for introducing a plurality of genes into source cells to produce target cells have been reported. However, in practice, when introducing a plurality of desired genes into source cells, it is difficult for all genes to be sufficiently introduced and to achieve sufficient activity. For example, it is common for the activity of any one gene to be lower than that of the other. However, such problems can also be solved by any of the above-described embodiments. Also, by any of the above-described embodiments, it is possible to produce target cells into which an activator has been uniformly introduced, and it is also possible to efficiently and / or homogeneously produce target cells.

[0051] [Example] Hereinafter, an example of producing and using the lipid nanoparticles of the embodiment will be described.

[0052] Experiment 1. Production of lipid nanoparticles encapsulating the GFP gene As the nucleic acid encapsulated in the lipid nanoparticles, messenger RNA of green fluorescent protein (GFP) gene (manufactured by OZ Bioscience) was used. The nucleic acid was suspended in 10 mM HEPES (pH 7.3) to obtain a nucleic acid solution.

[0053] FFT-10, FFT-20, DOPE, DOTAP, cholesterol and DMG-PEG were each dissolved in ethanol at a molar ratio of 0:25.8:4.9:9.8:55.8:3.7 to obtain a lipid solution. The lipid solution and the above nucleic acid solution were mixed using a microfluidic chip and a syringe pump. The mixed solution was diluted 10-fold with 10 mM HEPES (pH 7.3) and then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain the lipid nanoparticles of Examples 1 to 4.

[0054] Experiment 2. Lipid quantification of lipid nanoparticles by LC / MS In order to compare the charged lipid composition ratio when preparing lipid nanoparticles with the composition ratio of lipids contained in the prepared lipid nanoparticles and evaluate the error range of the lipid composition ratio, lipid quantification was performed by liquid chromatography-mass spectrometry (LC / MS). Each lipid nanoparticle prepared in Experiment 1 was diluted with methanol, and FFT-10, FFT-20, DOPE, DOTAP, cholesterol and DMG-PEG contained in each lipid nanoparticle were quantified under the conditions shown in Table 1 using an LC / MS apparatus (ACQUITY UPLC / QToF system, Waters).

[0055]

Table 1

[0056] The measurement was performed according to the manual attached to the LC / MS apparatus. The ratio of each lipid was converted to mol% from the lipid quantification results, and the lipid composition ratio of the lipid nanoparticles was calculated. Tables 2 to 4 show the charged lipid composition ratio of the lipid nanoparticles, the lipid composition ratio calculated from the LC / MS analysis results, and the error amount.

[0057]

Table 2

[0058]

Table 3

[0059]

Table 4

[0060] As a result of calculating the error amount with the theoretical value of the charged lipid composition ratio and the measured value of the lipid composition ratio by LC / MS analysis, it was found that the error range in the above lipid nanoparticle production method is 0.1 to 4 mol%.

[0061] Experiment 3. Preparation of Lipid Nanoparticles Encapsulating GFP Gene As the nucleic acid encapsulated in the lipid nanoparticles, messenger RNA of the GFP gene was used. The nucleic acid was suspended in 10 mM HEPES (pH 7.3) to obtain a nucleic acid solution.

[0062] FFT-10, FFT-20, DOPE, DOTAP, cholesterol and DMG-PEG were dissolved in ethanol at the charged lipid composition ratios shown in Table 5 to obtain a lipid solution. The lipid solution and the above nucleic acid solution were mixed using a microfluidic chip and a syringe pump. The mixed solution was diluted 10-fold with 10 mM HEPES (pH 7.3), and then concentrated with an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain the lipid nanoparticles of Examples 5 to 9.

[0063]

Table 5

[0064] Experiment 4. Measurement of Gene Expression Intensity Human peripheral blood mononuclear cells (PBMC, Precision For Medicine) cultured in StemSpan SFEMII medium (Stemcell technologies) containing 6 types of cytokines (IL-6, SCF, TPO, Flt-3L, IL-3, G-CSF; all from Wako) were centrifuged and collected. After that, they were seeded into a 96-well culture plate at a density of 4×104 cells / 100 μL / well of StemFit AK03N medium (supplemented with Rock Inhibitor Y27632). Each lipid nanoparticle prepared in Experiment 3 was added at a concentration of 1.3 μL / well. The culture plate was placed in an incubator, and the cells were cultured at 37°C in a 5% CO2 atmosphere.

[0065] From the day after the addition of the lipid nanoparticles to day 6, the fluorescence intensity of the GFP protein expressed from the GFP gene was photographed with a fluorescence microscope (KEYENCE BZ-X810), and the average luminance in each well was measured using image processing software (ImageJ). The measurement was performed according to the manual attached to the fluorescence microscope. Figure 9 shows the measurement results of the relative fluorescence intensity of the GFP protein. Among the 5 types of lipid nanoparticles with different charged lipid composition ratios (Examples 5 - 9), only the lipid nanoparticle with a composition ratio of FFT-10:FFT-20:DOPE:DOTAP:cholesterol:DMG-PEG = 20:20:0:10:48:2 (mol%) (Example 5) showed a relative fluorescence intensity of approximately 2.5 exceeding the reference value of 1.5 on the first day of measurement (D1) and maintained a relative fluorescence intensity of 2.5 or more until day 6 of measurement (D6). None of the other lipid nanoparticles (Examples 6 - 9) showed a fluorescence intensity exceeding the measurement results of Example 5. From the above results, it was shown that the lipid nanoparticles composed of the lipid composition ratio of Example 5 can deliver nucleic acids to PBMC cultured under the above conditions with the highest efficiency.

[0066] Experiment 5. Comparison of lipid compositions for DNA recombination efficiency in breast cancer cells Using the decrease in luminescence as an indicator, a system was constructed to detect the DNA recombination performance of cells by the simultaneous action of two genes. Using this system, the DNA recombination efficiency in breast cancer cells was compared using different sets of transfection carriers with different lipid compositions.

[0067] As the first lipid particles for the transfection carrier set, liposomes encapsulating the DNA recombinase gene (Cre gene (NCBI Reference Sequence: NC_005856.1) synthesized and inserted into pcDNA4 / V5-HisB (manufactured by Thermo Fisher Scientific)) were prepared. As the second lipid particles, liposomes encapsulating the luminescence gene (loxP sequences 5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ (SEQ ID NO: 1) synthesized on the 3′ and 5′ sides of the NLuc gene and inserted into pcDNA4 / V5-HisB) were prepared. Specifically, FFT-10, FFT-20, DOPE, DOTAP, cholesterol, and DMG-PEG were dissolved in ethanol at the charged lipid composition ratios shown in Table 6 to obtain a lipid solution. The lipid solution and the above nucleic acid solution were mixed using a microfluidic chip and a syringe pump. The mixed solution was diluted 10-fold with 10 mM HEPES (pH 7.3) and then concentrated using an ultrafiltration filter (Amicon Ultra 0.5 Ultracel-50, manufactured by Merck) to obtain lipid nanoparticles encapsulating either the DNA recombinase gene or the luminescence gene in any of Liposomes of Example 10-1, Example 10-2, and Example 10-3. Breast cancer cells were used as the target cells. The introduction of liposomes into the cells was performed by first adding the first lipid particles to breast cancer cells cultured in a culture solution (MEM medium containing 10% fetal bovine serum) on a culture dish and then adding the second lipid particles 24 hours later. 24 hours after that, the luminescence of the cancer cells of the target cells was measured using a luminescence measuring device (Infinite F200 PRO plate reader (manufactured by Tecan)).

[0068]

Table 6

[0069] The results are shown in FIGS. 10 and 11. The graph in FIG. 10 has the light emission amount on the vertical axis and plots the measured light emission amounts. The conditions for each graph are as follows. The leftmost column shows the results obtained by sequentially introducing a first lipid particle (liposome) containing a DNA recombinase gene and having a lipid composition of Example 10-1 and a second lipid particle (liposome) containing a luminescence gene and having a lipid composition of Example 10-1 into breast cancer cells as target cells. The second column from the left shows the results obtained by sequentially introducing a first lipid particle (liposome) containing a DNA recombinase gene and having a lipid composition of Example 10-2 and a second lipid particle (liposome) containing a luminescence gene and having a lipid composition of Example 10-2 into breast cancer cells as target cells. The above two are comparative examples, and are examples where the lipid compositions of the liposomes sequentially introduced into the target cells are equal.

[0070] The third and fourth data from the left are examples. The lipid composition of the first lipid particle first introduced into the target cells is different from the lipid composition of the second lipid particle. In both cases, each composition is adjusted to suit the state of the target cells. Specifically, the third column from the left shows the results obtained by sequentially introducing a first lipid particle (liposome) containing a DNA recombinase gene and having a lipid composition of Example 10-2 and a second lipid particle (liposome) containing a luminescence gene and having a lipid composition of Example 10-1 into breast cancer cells as target cells. The fourth column from the left shows the results obtained by sequentially introducing a first lipid particle (liposome) containing a DNA recombinase gene and having a lipid composition of Example 10-3 and a second lipid particle (liposome) containing a luminescence gene and having a lipid composition of Example 10-2 into breast cancer cells as target cells.

[0071] In the conducted experiment, when the first lipid particles (liposomes) and the second lipid particles (liposomes) enter inside the target cells and exhibit an effect, the luminescence amount of the cells decreases. When the introduction of these lipid particles is insufficient and / or the activity of the activator is low, the luminescence amount of the cells shows a larger value. As shown in FIG. 10, in a genomic recombination activity measurement assay system for breast cancer in which the luminescence amount is suppressed by the cooperative action of two genes, compared with the case where the lipid compositions of the first lipid particles and the second lipid particles are the same, by making the lipid compositions of the first lipid particles and the second lipid particles different from each other, the obtained luminescence amount was suppressed by 6.9 times. From the graph of FIG. 10, it became clear that by changing the lipid compositions of the first lipid particles and the second lipid particles, the activity of the activator increases.

[0072] The results in FIG. 10 are the results of measuring the gene introduction amount when lipid particles having the lipid compositions of Example 10-1, Example 10-2, and Example 10-3 were added to cultured breast cancer cells alone. The measurement of the gene introduction amount was performed by measuring the luminescence amount of each cell with a plate reader using the Nano-Glo Luciferase Assay System (manufactured by Promega).

[0073] Comparing the lipid compositions of Example 10-1, Example 10-2, and Example 10-3, the introduction rate of the initial breast cancer cells was high in the order of Example 10-2, Example 10-3, and Example 10-1. However, as is clear from FIG. 10, even if Example 10-2 with the highest introduction rate is used as the lipid composition of the first lipid particles and the second lipid particles, the activator is not introduced well. From the above results, it became clear that by sequentially introducing a plurality of lipid particles with different lipid compositions into target cells, it is possible to enhance the effect of the activator contained in the lipid particles, that is, liposomes.

[0074] Examples of further embodiments are described below. [1] Contacting the first lipid particles encapsulating the first activator with the target cells in the first state, Generating target cells in a second state from the target cells in the first state, Contacting second lipid particles encapsulating a second activator with the target cells in the second state, Generating target cells in a third state from the target cells in the second state, and Maintaining and / or enhancing the activator effect in the target cells at least including, The first lipid particles have a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state, The lipid composition of the second lipid particles is different from the lipid composition of the first lipid particles and has a lipid composition designed to exhibit an appropriate affinity for the target cells in the second state, The second activator is of the same type as or different from the first activator, A method for maintaining and / or enhancing the activator effect in target cells. [2] The method according to [1], wherein the second state is a state in which the effect of the first activator is exerted in the target cells in the first state, and the third state is a state in which the effect of the second activator is exerted in the target cells in the second state. [3] The method according to [1] or [2], wherein the first and / or second lipid particles are lipid particles composed of a lipid component containing biodegradable lipid FFT-10 and / or FFT-20. [4] The method according to any one of [1] to [3], characterized in that the proportion of the cationic lipid contained in the first and / or second lipid particles is 65% or less. [5] The method according to any one of [1] to [4], characterized in that the compositional ratio of the cationic lipid contained in the second lipid particles is larger than the compositional ratio of the cationic lipid of the first lipid particles. [6] The method according to any one of [1] to [5], wherein the first activator and / or the second activator is a substance having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, activity affecting protein expression state, enzyme activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity. [7] Contacting the nth lipid particle encapsulating the nth activator with the target cell in the nth state, and generating the target cell in the n + 1th state from the target cell in the nth state further comprising where n is an integer of 3 or more, and n generations are sequentially performed from the generation of the target cell in the second state to the generation of the target cell in the n + 1th state, the first to nth lipid particles each have a lipid composition designed to exhibit an appropriate affinity for the target cell in the corresponding state The method according to any one of [1] to [6]. [8] Contacting the first lipid particle encapsulating the first activator with the target cell in the first state, generating the target cell in the second state from the target cell in the first state, contacting the second lipid particle encapsulating the second activator with the target cell in the second state, and generating the target cell in the third state from the target cell in the second state at least comprising the target cell in the third state is a predetermined target cell the first lipid particle has a lipid composition designed to exhibit an appropriate affinity for the target cell in the first state, the lipid composition of the second lipid particle is different from the lipid composition of the first lipid particle and has a lipid composition designed to exhibit an appropriate affinity for the target cell in the second state, the second activator is of the same type as or different from the first activator, A method for producing a target cell from a target cell. [9] The method according to [8], wherein the second state is a state in which the effect of the first activator is exerted on the target cells in the first state, and the third state is a state in which the effect of the second activator is exerted on the target cells in the second state.

[10] The method according to [8] or [9], wherein the first and / or second lipid particles are lipid particles composed of a lipid component containing biodegradable lipids FFT-10 and / or FFT-20.

[11] The method according to any one of [8] to

[10] , wherein the proportion of the cationic lipid contained in the first and / or second lipid particles is 65% or less.

[12] The method according to any one of [8] to

[11] , wherein the compositional ratio of the cationic lipid contained in the second lipid particle is larger than the compositional ratio of the cationic lipid of the first lipid particle.

[13] The method according to any one of [8] to

[12] , wherein the first activator and / or the second activator is a substance having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity.

[14] Contacting the nth lipid particle encapsulating the nth activator with the target cells in the nth state, and generating the target cells in the n + 1th state from the target cells in the nth state further comprising wherein n is an integer of 3 or more, and n generations from the generation of the target cells in the second state to the generation of the target cells in the n + 1th state are sequentially performed, the first to nth lipid particles each have a lipid composition designed to exhibit appropriate affinity for the target cells in the corresponding state [8] to

[13] , the method according to any one of the items.

[15] An introduction carrier set for use in the method according to any one of [1] to

[14] , wherein the first activator to be introduced into the target cells in the first state, A first lipid particle encapsulating the first active agent and having a lipid composition designed to exhibit appropriate affinity for the target cells in the first state, A second active agent to be introduced into the target cells in the second state, which may be of the same type or different type from the first active agent, A second lipid particle encapsulating the second active agent, having a lipid composition different from that of the first lipid particle, and designed to exhibit appropriate affinity for the target cells in the second state A carrier set comprising the above. An introduction carrier set for use in the method according to

[16] , [7] or

[14] , A first to nth active agents to be introduced into the corresponding target cells in the first to nth states, which may be of the same type or different types from each other, First to nth lipid particles corresponding to the first to nth active agents respectively and encapsulating any one of the first to nth active agents Comprising the above, The first to nth lipid particles each have a lipid composition designed to exhibit appropriate affinity for the corresponding target cells in the state.

[17] A kit for preparing an introduction carrier for use in the method according to claim 1, A first active agent to be introduced into the target cells in the first state, A material for the first lipid particle having a lipid composition designed to exhibit appropriate affinity for the target cells in the first state and encapsulating the first active agent, A second active agent to be introduced into the target cells in the second state, which may be of the same type or different type from the first active agent, A material for the second lipid particle having a lipid composition different from that of the first lipid particle, designed to exhibit appropriate affinity for the target cells in the second state, and encapsulating the second active agent A kit comprising the above.

[18] The kit according to

[17] , wherein the target cell is selected from the group consisting of cells isolated from a human, cells capable of proliferation when isolated, isolated cells derived from a human, cells having a nucleus, fibroblasts, cancer cells, and breast cancer cells, and the active agent, the first and second active agents, are nucleic acid substances encoding any gene having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity.

[19] A kit for preparing an introduction carrier for use in the method according to [7] or

[14] , which should be introduced into the target cells corresponding to the first to nth states, respectively, and the first to nth active agents of the same or different types from each other, each having a lipid composition corresponding to the first to nth active agents and designed to exhibit an appropriate affinity for the target cells in the corresponding state, and the first to nth lipid particle materials for encapsulating any of the corresponding first to nth active agents and comprising, where n is an integer of 3 or more.

[20] The kit according to any one of

[17] to

[19] , wherein the target cell is selected from the group consisting of cells isolated from a human, cells capable of proliferation when isolated, isolated cells derived from a human, cells having a nucleus, fibroblasts, cancer cells, and breast cancer cells, and the active agent, the first and second active agents, are nucleic acid substances encoding any gene having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity.

[0075] Although some embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are included in the invention described in the claims and the equivalent scope thereof.

[0076] [Sequence Listing] 5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ (SEQ ID NO: 1)

Explanation of Symbols

[0077] 11a, 11b, 11x, 11a to 11x... lipid particles, 12a, 12b, 12x, 12a to 12x... active agents, 20a, 20b, 20c... target cells, 21a, 21b, 21c... nuclei of target cells, 101a... first introduction carrier, 101b, 201a... second introduction carrier, 101x... nth introduction carrier, 1001, 2001, 7001, 8001... introduction carrier sets, a... substance (active substance, e.g., peptide), b... substance (active substance, e.g., peptide)

Claims

1. Contacting first lipid particles encapsulating a first activator with target cells in a first state, generating target cells in a second state from the target cells in the first state, contacting second lipid particles encapsulating a second activator with the target cells in the second state, generating target cells in a third state from the target cells in the second state, and maintaining and / or enhancing the activator effect in the target cells at least including, wherein the first lipid particles have a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state, the lipid composition of the second lipid particles is different from the lipid composition of the first lipid particles and has a lipid composition designed to exhibit an appropriate affinity for the target cells in the second state, the second activator is of the same type as or different from the first activator, A method for maintaining and / or enhancing the activator effect in target cells.

2. The second state is a state in which the effect of the first activator is exerted in the target cells in the first state, and the third state is a state in which the effect of the second activator is exerted in the target cells in the second state. The method according to claim 1.

3. The method according to claim 1, wherein the first and / or second lipid particles are lipid particles composed of a lipid component containing biodegradable lipids FFT-10 and / or FFT-20.

4. The method according to claim 1, characterized in that the proportion of the cationic lipid contained in the first and / or second lipid particles is 65% or less.

5. The method according to claim 1, characterized in that the constituent ratio of the cationic lipid contained in the second lipid particles is larger than the constituent ratio of the cationic lipid of the first lipid particles.

6. The method according to claim 1, wherein the first activator and / or the second activator is a substance having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, activity affecting protein expression status, enzyme activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity.

7. Contacting nth lipid particles encapsulating an nth activator with target cells in an nth state, and generating target cells in an n+1th state from the target cells in the nth state further including, Here, n is an integer of 3 or more, and n generations are sequentially performed from the generation of the target cells in the second state to the generation of the target cells in the (n + 1)-th state. The first to n-th lipid particles each have a lipid composition designed to exhibit an appropriate affinity for the target cells in the corresponding state. The method according to claim 1.

8. Contacting first lipid particles encapsulating a first activator with target cells in a first state. Generating target cells in a second state from the target cells in the first state. Contacting second lipid particles encapsulating a second activator with the target cells in the second state, and Generating target cells in a third state from the target cells in the second state. at least including The target cells in the third state are predetermined target cells. The first lipid particles have a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state. The lipid composition of the second lipid particles is different from the lipid composition of the first lipid particles and has a lipid composition designed to exhibit an appropriate affinity for the target cells in the second state. The second activator is of the same type as or different from the first activator. A method for producing target cells from target cells.

9. The second state is a state in which the effect of the first activator is exerted in the target cells in the first state, and the third state is a state in which the effect of the second activator is exerted in the target cells in the second state. The method according to claim 8.

10. The method according to claim 8, wherein the first and / or second lipid particles are lipid particles composed of a lipid component containing biodegradable lipids FFT-10 and / or FFT-20.

11. The method according to claim 8, characterized in that the proportion of the cationic lipid contained in the first and / or second lipid particles is 65% or less.

12. The method according to claim 8, characterized in that the compositional ratio of the cationic lipid contained in the second lipid particles is larger than the compositional ratio of the cationic lipid of the first lipid particles.

13. The method according to claim 8, wherein the first activator and / or the second activator is a substance having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity.

14. Contacting nth lipid particles encapsulating an nth agent with target cells in an nth state, and generating target cells in an (n + 1)th state from the target cells in the nth state further comprising where n is an integer of 3 or more, and n generations from the generation of the target cells in the second state to the generation of the target cells in the (n + 1)th state are sequentially performed the first to nth lipid particles each have a lipid composition designed to exhibit an appropriate affinity for the target cells in the corresponding state The method according to claim 8

15. An introduction carrier set for use in the method according to claim 1, comprising a first agent to be introduced into the target cells in the first state a first lipid particle encapsulating the first agent and having a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state a second agent to be introduced into the target cells in the second state, which is of the same type or a different type from the first agent a second lipid particle encapsulating the second agent, having a lipid composition different from that of the first lipid particle, and designed to exhibit an appropriate affinity for the target cells in the second state A carrier set comprising

16. An introduction carrier set for use in the method according to claim 14, comprising first to nth agents to be introduced into the corresponding target cells in the first to nth states, respectively, which are of the same type or different types from each other first to nth lipid particles corresponding to the first to nth agents respectively and encapsulating any one of the first to nth agents comprising the first to nth lipid particles each have a lipid composition designed to exhibit an appropriate affinity for the target cells in the corresponding state

17. An introduction carrier production kit for use in the method according to claim 1, comprising a first agent to be introduced into the target cells in the first state a material for the first lipid particle having a lipid composition designed to exhibit an appropriate affinity for the target cells in the first state and encapsulating the first agent a second agent to be introduced into the target cells in the second state, which is of the same type or a different type from the first agent The first lipid particle has a lipid composition different from that of the second lipid particle, has a lipid composition designed to exhibit appropriate affinity for the target cells in the second state, and is a material for the second lipid particle for encapsulating the second activator. A kit comprising the same. **Claim 18** The target cells are selected from the group consisting of cells isolated from humans, cells capable of growing when isolated, isolated cells derived from humans, cells having a nucleus, fibroblasts, cancer cells, and breast cancer cells, and the first and second activators are nucleic acid substances encoding any gene having an activity selected from the group consisting of protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity. The kit according to claim 17. **Claim 19** An introduction carrier production kit for use in the method according to claim 14, First to nth activators, which should be introduced into the corresponding target cells in the first to nth states, respectively, and which may be of the same or different types from each other, Corresponding to the first to nth activators, each having a lipid composition designed to exhibit appropriate affinity for the target cells in the corresponding state, and being a material for the first to nth lipid particles for encapsulating any one of the corresponding first to nth activators. A kit comprising the same, where n is an integer of 3 or more. **Claim 20** The target cells are selected from the group consisting of cells isolated from humans, cells capable of growing when isolated, isolated cells derived from humans, cells having a nucleus, fibroblasts, cancer cells, and breast cancer cells, and the activator is a nucleic acid substance encoding any gene having an activity selected from the group consisting of the protein synthesis activity, protein cleavage activity, anti-cancer activity, reprogramming activity, nucleic acid cleavage activity, gene recombination activity, and genome editing activity of the first and second activators. The kit according to claim 19.

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