WO2011049059A1 - Composition comprising rna-enclosing carrier - Google Patents

Abstract

Provided is a composition comprising one or more kinds of RNA-enclosing carriers, said carriers being selected from among a monocyte, a macrophage and a shedding microvesicle originating in these cells. The RNA-enclosing carrier(s) in the aforesaid composition can maintain the RNA in a stable state in vivo.

Description

RNA-encapsulating carrier composition

This application claims priority based on Japanese Patent Application No. 2009-243466 filed on Oct. 22, 2009, the entire contents of which are incorporated herein by reference. The disclosure herein relates to compositions suitable for in vivo delivery of RNA, such as microRNA.

RNA such as siRNA and microRNA is expected as a nucleic acid medicine. On the other hand, RNA is considered to have a problem in delivery to a target site. That is, in the living body, there are abundant nucleases that degrade RNA. Here, as a method for avoiding attacks from nucleases, it is possible to encapsulate and administer nucleic acids in liposomes or nanopolymers (Patent Document 1).

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However, the liposome is about 5 nm to 200 nm, and even if RNA is embedded, it is taken up by macrophages and decomposed. Liposomes induce an allergic reaction and may cause damage to the liver and kidneys. For these reasons, the actual dose of functional RNA is limited, and the current situation is that a sufficient effect cannot be expected.

Therefore, the disclosure of this specification is intended to provide a novel delivery system for RNA and to use it together.

The present inventors focused on monocytes, which are a type of white blood cell. That is, RNA of interest is introduced into monocytes collected from patients, and monocytes after introduction, macrophages obtained by differentiating monocytes, and microvesicles (Shedding microvesicles (SMV)) obtained from these culture supernatants are administered. It was found that the above problems can be solved. According to the disclosure of the present specification, the following means are provided.

According to the disclosure of the present specification, there is provided a composition comprising one or more carriers selected from monocytes, macrophages, and microvesicles of these cells, and a carrier containing RNA. The RNA may be exogenous RNA, and the exogenous RNA may contain microRNA. The foreign RNA may have antitumor activity, and such a composition can be used as an antitumor composition. Moreover, such a composition may be a composition for RNA delivery into the living body of animals including humans.

According to the disclosure of the present specification, a method for producing an RNA-encapsulating carrier is provided, which comprises the step of introducing RNA or a DNA construct that expresses the RNA into monocytes or macrophages. The manufacturing method further includes the following steps:
(A) A step of culturing monocytes introduced with the RNA to differentiate into macrophages may be provided. Further, the manufacturing method may include (b) a step of releasing microvesicles from the monocytes and / or the macrophages. The manufacturing method may be a method for manufacturing an antitumor composition.

It is a figure which shows the outline | summary of the delivery of RNA by the RNA inclusion carrier of an indication of this specification. FIG. 3 is a graph comparing the amount of introduced miR-143 in human monocyte leukemia cell THP-1. It is a figure which shows the measurement result of the quantity of miR-143 in the precipitate which concentrated the microvesicle by the ultracentrifugation from the culture supernatant when the cell in which miR-143 was introduce | transduced was serum-starved. It is a figure which shows the measurement result of mRNA of GAPDH gene and mRNA of ACTB gene in the precipitate which concentrated the microvesicle by the ultracentrifugation from the culture supernatant when the cell which introduce | transduced miR-143 was serum-starved. It is a figure which shows the electron micrograph of the microvesicle concentrated by the ultracentrifugation from the culture supernatant. It is a figure which shows the measurement result of the amount of miR-143 in the tumor site | part of a tumor bearing nude mouse. It is a figure which shows the relationship between the density | concentration of miRNA-143 introduce | transduced into K562 cell, and the density | concentration of miRNA-143 in a microvesicle. It is a figure which shows the immunoelectron micrograph of the intracellular (a) THP-1 extracellular (b) of the THP-1 cell which introduce | transduced miRNA-143BP labeled with Cy5. It is a figure which shows accumulation | storage to the tissue (kidney) of the miRNA-143BP in the tumor bearing mouse | mouth which administered the THP-1 cell which introduce | transduced miRNA-143BP. It is a figure which shows the change of the blood level of miRNA-143BP in the tumor bearing mouse | mouth which administered the THP-1 cell which introduce | transduced miRNA-143BP.

The disclosure of the present specification includes one or more carriers selected from monocytes, macrophages, and microvesicles derived from these cells, a composition containing a carrier encapsulating RNA, and a composition of such an RNA-encapsulating carrier. It relates to a manufacturing method. The composition disclosed in the present specification avoids degradation by macrophages or nucleases in the administered living body by containing such an RNA-encapsulating carrier. For this reason, RNA can be present in the living body at a higher concentration. Moreover, since these carriers are derived from living bodies, side effects that may occur in liposomes and the like are avoided. Furthermore, when the foreign substance recognition ability memorized by monocytes can be maintained in the individual from which these carriers are acquired, when they are administered again to this individual, the RNA contained in the carrier can be delivered to the foreign bodies. In particular, microvesicles are preferred carriers in that they are stable to nucleases and distributed by the bloodstream throughout the body.

In the present specification, monocytes are cells in a stage before differentiation into macrophages of macrophage cells, and mean cells that can exist mainly in blood in a living body. Also called mononuclear sphere. In the present specification, the term “monocytes” may include monocytes and pre-monocytes before differentiation into monocytes. Monocytes can have antigen presenting action, adhesion and phagocytosis. In the present specification, macrophages mean phagocytic cells differentiated from monocytes and have phagocytic ability. Macrophages can have cytotoxic effects such as antigen presenting action, adhesion, motility, misdifferentiated tumor cells and the like. In the present specification, monocytes and macrophages do not include dendritic cells.

FIG. 1 shows an outline of RNA delivery using the composition disclosed in the present specification. The RNA delivery disclosed in the present specification uses monocytes, macrophages and microvesicles derived from the membranes of these cells as RNA packaging carriers. These cells, particularly monocytes and macrophages, are themselves vehicles that deliver to the target site, etc., in the immune system in the living body, are carriers for packaging RNA, and microvesicles that contain RNA. It is presumed to have a function of releasing. In particular, the microvesicle is presumed to have a function of encapsulating RNA and delivering it to a target.

As shown in FIG. 1, the RNA-encapsulating carrier can take various forms. Examples include monocytes introduced with RNA and the like, macrophages obtained by inducing differentiation of the monocytes, macrophages introduced with RNA and the like, and microvesicles released from these monocytes and macrophages. The RNA-encapsulating carrier composition may contain only one type selected from these various types of RNA-encapsulating carriers, or may contain two or more types in combination.

In this specification, the microvesicle means a vesicle obtained in the culture supernatant of monocytes introduced with foreign RNA or macrophages obtained by differentiating the monocytes. These vesicles can be generated from biological membranes of monocytes and macrophages. Such vesicles can include shedsome microvesicles (SMV, secretory microvesicles) as well as exosomes. Here, SMV is a vesicle that sprouting directly from the cell membrane. SMV is thought to sprout from various cells such as monocytes and macrophages. These particles may have a substantially spherical membrane structure.

(RNA-containing carrier-containing composition)
In the following description, one or more particles (carriers) selected from monocytes, macrophages, and microvesicles derived from these cells, and particles (carriers) containing RNA that may be exogenous ) Is referred to as an RNA-containing carrier.

Monocytes and macrophages may be cultured cells in addition to cells collected from living organisms, or may be obtained by differentiation from iPS cells or stem cells. Monocytes can be collected by a known method from the blood of an individual organism, specifically, a mammal such as a human. Macrophages can be obtained as macrophages by differentiating collected monocytes by a known method, and can also be collected from tissues such as mammals. In order to differentiate monocytes into macrophages, for example, methods such as stimulation with TPA and certain cytokines can be mentioned. Microvesicles can be obtained from a supernatant fraction obtained by culturing monocytes and / or macrophages. Microvesicles can be confirmed by observing the tsg101 protein by Western blot, as well as by observing the culture supernatant with an electron microscope.

The origin of monocytes, macrophages, and microvesicles is not particularly limited. However, in view of administration to an individual organism, from the viewpoint of suppressing rejection, it is preferably an allogeneic cell rather than a heterologous cell. Autologous cells are preferred. For example, when the present composition is used for prevention / treatment, the cell acquisition source is preferably an individual subject to prevention / treatment (such as a patient).

In this specification, RNA means a polymer in which ribonucleotides are linked by phosphodiester bonds. RNA may be composed entirely or partly of a known nucleotide analog. In addition, some modification may be made in order to avoid or suppress degradation by nuclease or the like. Examples of the RNA included in the carrier include mRNA, tRNA, rRNA, tRNA and non-coding RNA (ncRNA) other than rRNA, ribozyme, and antisense RNA. Examples of ncRNA include micro RNA, siRNA, and shRNA. Moreover, RNA which has a base sequence complementary to these RNA is also mentioned. Such RNA can usually be artificially obtained by a known nucleic acid synthesis method. Furthermore, the RNA may be a DNA construct configured such that such RNA can be expressed in cells.

This composition can be used for introducing such RNA into the body of animals including humans. In particular, it can be used for tissue delivery. For example, by using a microRNA that is related to or likely to develop various diseases as RNA, for example, by administering a composition containing such an RNA-encapsulating carrier to an animal or the like, the functional study of the microRNA Individuals can be obtained for research and testing such as the onset process. In addition, an RNA-encapsulating carrier composition that encapsulates an RNA having a complementary base sequence of a disease-related RNA can be used for the prevention / treatment of such diseases or its test research.

By using a microRNA related to or possibly preventing or treating a disease, such a composition containing an RNA-encapsulating carrier can be administered to an animal such as a human to be used for prevention / treatment or a test study thereof. . In this embodiment, miR-143, miR-145, let-7, etc. are preferably used as the microRNA.

In order to administer this composition to a living body, in addition to administration via a vascular route such as intravenous administration, it can be injected into a tissue percutaneously or with an incision. it can.

The RNA in the RNA-encapsulating carrier may be RNA that is inherently present in an organism individual that is a source of cells such as monocytes, or a heterogeneous or artificial RNA that cannot inherently exist in the individual organism. It may be. In addition, the RNA may be exogenous introduced from the outside regardless of whether it is intrinsic to the acquisition source.

(Manufacture of RNA inclusion carrier)
To obtain an RNA-encapsulating carrier, for example, a monocyte or macrophage obtained by collecting or differentiating progenitor cells is introduced with RNA or a DNA construct capable of generating RNA as a transcript by a known method. There are various methods for introducing nucleic acids such as RNA and DNA into such cells. For example, electroporation, calcium phosphate method, lipofection method and the like can be mentioned. In order to obtain macrophages encapsulating RNA as an RNA-encapsulating carrier, RNA or a DNA construct that expresses the RNA may be introduced into monocytes and then differentiated into macrophages.

The microvesicle encapsulating RNA as an RNA-encapsulating carrier can be obtained in the culture supernatant when culturing monocytes or macrophages into which RNA or DNA construct has been introduced. Microvesicles are released by culturing monocytes or macrophages. The release of microvesicles is promoted by serum-starved culture of monocytes and macrophages. Monesin (calcium ionate) treatment can also be employed.

The RNA-encapsulating carrier may contain RNA in one or more carriers selected from monocytes, macrophages and microvesicles, and any of these carriers may contain the target RNA. In addition, the RNA-containing carrier-containing composition only needs to contain at least one RNA-encapsulating carrier, and even if it contains another carrier, the other carrier does not contain RNA. Good.

Hereinafter, the disclosure of the present specification will be specifically described by way of examples. However, the following examples do not limit the disclosure of the present specification.

In this example, cells D (−) and D (+) before and after differentiation induction of human monocyte leukemia cell THP-1 were prepared. For differentiation induction, TPA in the range of 20 to 160 nM was added to the medium. MiR-143 was introduced into these cells using liposomes (liofectamine, Invitrogen), and after culturing for 18 hours, the amount of miR-143 found in the cell fraction of each culture was confirmed. Specifically, miR-143 was introduced into liposomes by embedding 40 nM, and culture was performed in RPMI 1640 medium containing 10% calf serum. Note that miR-143 was detected using real-time PR. The results are shown in FIG.

As shown in FIG. 2, microRNAs are introduced into cells by using liposomes for THP-1 D (−) monocyte cells, and THP− after differentiation compared to cells before differentiation. It was found that when miRNA was introduced into 1 導入 D (+), more miR-143 was introduced and retained in the cell fraction. This supported that monocytes become macrophages by differentiating monocytes, but macrophages can retain more RNA in the cells than monocytes.

Further, the cultured THP-1 D (+) is cultured under serum-starved culture conditions without serum for 24 hours, and the culture supernatant is collected and centrifuged at 3000 rpm for 30 minutes, and the supernatant is further collected at 50,000 to 100,000. Ultracentrifugation was performed at rpm for 3 hours to collect microvesicles adhering to the tube wall. From these vesicle suspensions, miR-143, GAPDH gene, which is a cytoplasmic marker protein gene, and ACTB gene, which is a cytoskeletal protein gene, were measured. Serum starvation culture was performed for 24 to 28 hours using serum-free RPMI 1640 medium. miR-143, miR-22, GAPDH gene and ACTB gene were each measured by real-time PCR. The results are shown in FIGS.

As shown in FIGS. 3 and 4, when miR-143 is introduced into THP-1ＰD (+) and cultured under starvation conditions, miR-143 in microvesicles secreted into the culture supernatant depends on the concentration of the introduced It was found that it was increasing. miR-143 was considered to be miR-143 in microvesicles because it increased as a relative amount to miR-22 inherent in THP-1 D (+) cells. In addition, according to the results of measuring the amount of mRNA of genes that become internal standards such as Betha-Actin and GAPDH for the same sample, all the samples including the control contained almost the same amount, except for miR-143. It was shown that RNA was also present. This supported that the sample after ultracentrifugation was mainly composed of active vesicles, not cell dead bodies or fragments thereof.

Also, an electron micrograph of the microvesicles obtained by ultracentrifugation of the culture supernatant is shown in FIG. As shown in FIG. 5, SMV-like microvesicles were observed.

In this example, monocytes into which miR-143 was introduced via liposomes were intravenously administered to nude mice (BALB / c system, Charles River Japan, Inc.) bearing human colon cancer, and miR- 143 amount was confirmed. That is, tumor-bearing mice were prepared by transplanting 2 to 3 million human colon cancer DLD-1 cells subcutaneously in nude mice. MiR-143 and control miR-R 20 nM each were introduced into human monocytic cell line THP-1 (100,000 cells / ml) using Lipofectamine. One hundred thousand to 100,000 introduced THP-1 cells per tumor-bearing nude mouse were washed and administered through the tail vein of the mouse. Administration was once a day and twice a day (2 days). Five tumor-bearing nude mice were treated for each miR-143 and control. Twenty-four hours after the final administration, the tissue was dissected and organs and tumors were removed. RNA was extracted from the excised site with Trizol, and the amount of miR-143 was measured by real-time PCR. The results are shown in FIG.

As shown in FIG. 6, when the amount of miR-143 detected in the control tumor site was 1, accumulation of about twice as much miR-143 was confirmed in the tumor site of miR-143-introduced mice. From this result, when monocytes introduced with RNA are intravenously administered, monocytes or macrophages or microvesicles differentiated from monocytes selectively reach tumor cells, and miR-143 is observed in the tumor tissue. It was thought

In this example, according to Example 1, D (+) was prepared by inducing differentiation of human chronic myeloid leukemia cell line K562 cells. For differentiation induction, TPA in the range of 20 to 160 nM was added to the medium. MiR-143 was introduced into these cells using liposomes (liofectamine, Invitrogen), and after culturing for 18 hours, the amount of miR-143 found in the cell fraction of each culture was confirmed. Specifically, miR-143 was introduced into liposomes by embedding 10 to 60 nM, and culture was performed in RPMI 1640 medium containing 10% calf serum.

Further, K562 · D (+) after culturing was cultured under serum-starved culture conditions without serum for 24 hours, and the culture supernatant was collected and centrifuged at 3000 rpm for 30 minutes, and the supernatant was further added at 50,000 to 100,000 rpm. Then, ultracentrifugation was performed for 3 hours to collect microvesicles adhering to the tube wall. For the vesicle suspension, miR-143 and miR-21 were measured by real-time PCR. Serum starvation culture was performed for 24 to 28 hours using serum-free RPMI 1640 medium. The results are shown in FIG.

As shown in FIG. 7, when miR-143 was introduced into K562 · D (+) and cultured under starvation conditions, miR-143 in microvesicles secreted into the culture supernatant increased depending on the introduction concentration. I found out. miR-143 was considered to be miR-143 in microvesicles because it increased as a relative amount to miR-21 inherent in K562５６D (+) cells.

In this example, chemically modified miR-143BP and miR-143BP / Cy5 labeled with the fluorescent substance Cy5 were introduced into THP-1 cells induced to differentiate in the same manner as in Example 1. Specifically, miR-143BP / Cy5 was introduced into liposomes (liofectamine, Invitrogen) by embedding 40 nM, and cultured for 18 hours. Culture was performed in RPMI 1640 medium containing 10% calf serum. Further, the cultured THP-1 cells were cultured under serum-starved culture conditions without serum for 24 hours, and the cells were reacted with anti-Cy5 immunogold and then observed with an electron microscope. Serum starvation culture was performed for 24 to 28 hours using serum-free RPMI 1640 medium. The results are shown in FIG.

As shown in FIG. 8 (a), microvesicles (secretory membrane vesicles) enclosing Cy5 could be detected in the intracellular multivesicular bodies (see arrows in the figure). Moreover, as shown in FIG.8 (b), miR-143BP / Cy5 was able to be observed in the secreted microvesicle extracellularly (refer the arrow in a figure).

In this example, chemically modified miR-143BP was introduced into THP-1 cells induced to differentiate in the same manner as in Example 1. Specifically, miR-143BP was introduced into liposomes (liofectamine, Invitrogen) by embedding 40 nM, and cultured for 18 hours. Culture was performed in RPMI 1640 medium containing 10% calf serum. Then, after culturing in a serum-free medium for 24 hours, THP-1 cells were intravenously administered to tumor-bearing mice at 1 to 5 × 10 ⁵ / mouse. The tumor-bearing mice were prepared by transplanting human colon cancer DLD-1 cells under the skin of nude mice at 2 to 3 million cells / mouse. Administration was once a day and twice a day (2 days). Twenty-four hours after the final administration, the patient was dissected and the kidney and tumor area were removed. RNA was extracted from the excised site with Trizol, and the amount of miR-143 was measured by real-time PCR. In addition, blood was collected from the mice 4 hours, 8 hours and 24 hours after the final administration, and miR-143 and miR-21 in the serum were measured by real-time PCR. The results are shown in FIGS.

As shown in FIG. 9, when the amount of miR-143BP detected in the kidney of the control mouse is 1, the amount of about twice the amount in the kidney of the mouse systemically administered with miR-143 / BP-introduced TPH-1 cells. Accumulation of miR-143 was confirmed. From this result, by administering monocytes introduced with RNA intravenously, macrophages or microvesicles differentiated from THP-1 cells or THP-1 cells selectively reach the kidney, and miR-143 levels in the kidney It was thought that an increase in was observed. In addition, it was thought that it may have accumulated in the glomeruli. Moreover, as a result of examining the distribution to the tumor part, selective accumulation was recognized also in the tumor part.

As shown in FIG. 10, it was found that the blood concentration of miR-143BP in the serum gradually decreased with the passage of time from the final administration. That is, it was found that the blood concentration was effectively increased by administration.

Claims (8)

1. A composition comprising one or more carriers selected from monocytes, macrophages, and microvesicles derived from these cells, the carrier encapsulating RNA.
2. The composition according to claim 1, wherein the RNA is exogenous RNA.
3. The composition according to claim 2, wherein the exogenous RNA includes micro RNA.
4. The composition according to claim 2 or 3, wherein the exogenous RNA has antitumor activity.
5. The composition according to any one of claims 1 to 4, which is used for RNA delivery into the living body of animals including humans.
6. A method for producing a carrier containing RNA, comprising:
   Introducing RNA or a DNA construct expressing the RNA into monocytes or macrophages;
   A manufacturing method comprising:
7. In addition, the following steps:
   (A) The production method according to claim 6, comprising a step of culturing the monocytes introduced with RNA to differentiate into macrophages.
8. The manufacturing method according to claim 6 or 7, further comprising (b) a step of releasing microvesicles from the monocytes and / or the macrophages.

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