JP5900895B2 - Novel carrier molecules and methods for delivering RNA into the cytoplasm by near infrared light - Google Patents

Description

  The present invention relates to a method for expressing a function by introducing RNA (functional RNA) capable of expressing a predetermined function into a cell and irradiating light having a predetermined wavelength, and a carrier used in the method It relates to molecules.

  RNAi is a phenomenon in which the expression of a specific gene is suppressed depending on the nucleotide sequence of shRNA or siRNA, and since it can be applied to the treatment of diseases based on the suppression of the expression of a gene causing a disease or a viral gene, it has recently received much attention. Has been. For this purpose, research and development of means for efficiently introducing RNA into the cytoplasm is underway.

  In Patent Document 1, RNA having a recognition sequence for an RNA-binding protein and an arbitrary sequence is bound to a fusion protein comprising an RNA-binding protein and a membrane-permeable carrier peptide, and this conjugate is mixed with an arbitrary cell. Thus, a method for introducing the RNA into cells is described.

  Non-Patent Documents 1 and 2 include a fusion protein containing a fluorescent dye (for example, Alexa Fluor 546, excitation wavelength 530-550 nm) that is excited with light having a wavelength in the visible region in addition to an RNA-binding protein and a membrane-permeable carrier peptide. Complexes with siRNA bound are described. The conjugate is then brought into contact with the cell, and the conjugate is taken up into the cell by endocytosis and localized in the endosome, and then irradiated with excitation light, whereby the fluorescent dye acts as a photosensitizer. , It is described that siRNA can be released from endosomes into the cytoplasm and achieve a predetermined RNAi effect (see FIG. 1). Non-Patent Documents 1 and 2 neither describe nor suggest that the above-described effects can be achieved even when a fluorescent dye excited by light having a wavelength in the near infrared region is used.

  On the other hand, in Patent Document 2, gold nanorods are used as a functional molecular carrier, a complex of gold nanorod aggregates and DNA is added to cultured cells, and irradiated with a pulsed laser in the near infrared region (wavelength 1064 nm). A method is described in which DNA is detached from gold nanorods and a gene from the DNA is efficiently expressed (see Examples). However, the gold nanorod has a photothermal conversion function, and is also a material that is used for photothermal treatment of the tissue around the target site where the gold nanorod is accumulated (to kill tumor cells and the like with the generated heat) ( For example, see Patent Document 3), there is a concern about cytotoxicity and safety in such a use that is not intended.

JP 2006-280261 A JP 2005-255582 A JP 2010-083803 A

T. Endoh, M. Sisido and T. Ohtsuki (2008) Cellular siRNA delivery mediated by a cell permeant RNA-binding protein and photoinduced RNA interference.Bioconjugate chemistry 19 (5), 1017-1024. T. Endoh, M. Sisido and T. Ohtsuki (2009) Spatial regulation of specific gene expression through photoactivation of RNAi. Journal of Controlled Release 137, 241-245.

  When RNA is introduced into cells by the methods described in Non-Patent Documents 1 and 2, visible light is used as excitation light, but visible light is inferior in permeability into the living body. Therefore, it can be applied to the introduction of RNA into cells on the surface of the living body (for example, the skin surface), but is inappropriate for the introduction of RNA into cells deep in the living body.

  The present invention provides a means for introducing RNA into cells using near-infrared light having good biological permeability (preferably having a wavelength of about 700 to 1000 nm) without causing cell damage or fever problems. Objective.

  The present inventors irradiate near-infrared light when a specific one of fluorescent dyes having near-infrared light as excitation light is used as a fluorescent dye in a complex described in Non-Patent Document 1 or the like. Has been found to have the ability to act as a photosensitizer and to release RNA from the endosome into the cytoplasm (see FIG. 1). Furthermore, it has been found that the efficiency of introducing RNA into the cytoplasm can be further increased by further adding a specific peptide to the fusion protein in the complex, and the present invention has been completed.

That is, the present invention includes the following matters.
[1] It functions with light having a wavelength in the near-infrared region connected to a carrier protein including a cell membrane permeable peptide (CPP) and an RNA-binding protein (RBP) and the N-terminal side or C-terminal side of the carrier protein. A carrier molecule having a structure comprising a photosensitizer (PST).

[2] The carrier molecule according to [1], wherein the photosensitizer (PST) functions with light having a wavelength of 700 nm to 1000 nm.
[3] The photosensitizer (PST) is a compound represented by the following structural formula (DY750), a compound having the fluorescence spectrum shown in FIG. 3 (Alexa Fluor750), or a compound having the absorption spectrum shown in FIG. 4 (IRDye800CW). The carrier molecule according to [1] or [2].

  [4] The carrier molecule according to any one of [1] to [3], wherein the carrier protein further comprises a proteasome degradation signal sequence tag (PDS) and / or a His-rich tag (HR).

  [5] In the carrier protein, cell membrane permeable peptide (CPP), RNA binding protein (RBP), proteasome degradation signal sequence tag (PDS) and / or His-rich tag (HR) are arranged in this order from the N-terminal side. The carrier molecule according to any one of [1] to [4], wherein the photosensitizer (PST) is linked to the C-terminal side of the carrier protein.

[6] The CPP is a peptide consisting of 12 amino acids in a human immunodeficiency virus-derived Tat (trans-activator of transcription) protein having the amino acid sequence represented by SEQ ID NO: 1. The carrier molecule according to any one of the above.
YGRKKRRQRRRG SEQ ID NO: 1

[7] The carrier molecule according to any one of [1] to [6], wherein the RBP is an RNA binding domain of human-derived U1A (U1 small nuclear riboprotein A) having the amino acid sequence represented by SEQ ID NO: 2. .
AVPETRPNHTIYINNLNEKIKKDELKYKSLYAIFSQFGQILDILVVSLSLKMRGQAFVIFKEVSSATNANLRSQGFPFYDKPMRIQYAKTDSDIIAKMK SEQ ID NO: 2

[8] A carrier molecule / RNA complex having a structure comprising the carrier molecule according to any one of [1] to [7] and RNA bound to RBP in the RNA carrier molecule.
[9] The RNA complex according to [8], wherein the RNA is short hairpin RNA (shRNA), small interfering RNA (siRNA), or microRNA (miRNA).

[10] A gene therapy agent or diagnostic agent comprising the carrier molecule / RNA complex according to [8] or [9].
[11] The gene therapy agent or diagnostic agent according to [10], which targets cancer or viral diseases.

  [12] A step of contacting the carrier molecule / RNA complex according to [8] or [9] with a cell to localize it in an endosome in the cell in vitro, and in the carrier molecule / RNA complex A method of delivering RNA into the cytoplasm comprising the step of irradiating light having a wavelength in the near-infrared region in which the PST functions to diffuse the carrier molecule / RNA complex into the cytoplasm.

  Since most of the RNA carrier according to the present invention can be synthesized with a protein which is a biomolecule, it can be used as a safe RNA carrier that is biodegradable and non-cytotoxic. Furthermore, since RNA diffuses into the cytoplasm only when irradiated with near-infrared light, the RNA can be delivered into the cytoplasm by specifying the position and timing, and the expression of RNA functions such as RNAi can be caused.

FIG. 1 shows an outline of a method for delivering RNA into the cytoplasm by irradiation with light having a predetermined wavelength. A) RNA carrier protein (CPP-RBP) and RNA complex. B) Translocation of CPP-RBP / RNA complex into cytoplasm via endocytosis. CPP-RBP to which a fluorescent group is added is first introduced into cells via 1) CPP, and 2) taken up into endosomes via endocytosis. Next, 3) by irradiating light with the excitation wavelength of the fluorescent dye used, the endosomal membrane is disrupted, 4) the CPP-RBP / RNA complex diffuses into the cytoplasm, and 5) the function of RNA is expressed ( DNAi is induced when the introduced RNA is shRNA or the like). FIG. 2 represents the excitation spectrum (left) and fluorescence spectrum (right) of DY750. FIG. 3 shows the fluorescence spectrum (right) of Alexa Fluor 750 (for comparison, the fluorescence spectrum of Cy7 (left) is also shown). FIG. 4 represents the excitation spectrum of IRDye800CW (fluorescence spectrum is also shown). FIG. 5 is an image showing the presence / absence of transfer of the carrier / shRNA complex to the cytoplasm by irradiation with near-infrared light performed in Example 3). Alexa Fluor 750, DY750 and IRDye800CW show RNA in the cytoplasm, especially for DY750, and these fluorescent dyes can diffuse the carrier / shRNA complex into the cytoplasm upon irradiation with near infrared light. It was confirmed that it was possible. FIG. 6 is a graph showing the RNAi effect by near-infrared light irradiation using TatU1A-DY750 produced in Example 4). FIG. 7 is a graph showing the RNAi effect of near-infrared light irradiation using TatU1A-His6-DY750 produced in Example 6). FIG. 8 is a graph showing the RNAi effect in human tumor cells performed in Example 7). (A) Measurement results by flow cytometry. (B) Average fluorescence intensity calculated from the results of flow cytometry.

―Carrier protein―
The carrier protein in the present invention contains at least a cell membrane permeable peptide (CPP) and an RNA binding protein (RBP) as essential components, and preferably further comprises a proteasome degradation signal sequence tag (PDS) and / or a His-rich tag ( HR) refers to a peptide (fusion protein) containing a constituent element.

The elements of CPP, RBP, PDS, and His ₆ may be directly linked via a peptide bond or linked via a peptide peptide (usually consisting of about 1 to 20 amino acids) as a linker. May be. In addition, it is desirable that the entire carrier protein including the predetermined element and the linker is composed of only a peptide (amino acid). However, a carrier protein using a compound other than the peptide (amino acid) as the linker, for example, PEG (polyethylene glycol) or the like. It is also possible to configure.

  If necessary, a peptide other than the above-mentioned elements may be contained inside or at the end of the carrier protein. For example, a His tag or GST tag for purifying and recovering the carrier protein of the present invention produced by the expression vector by affinity chromatography may be linked to the vicinity of the N-terminus of the carrier protein (after the recovery of the carrier protein, the His When the tag is not cleaved, the His tag can be used as an HR for enhancing membrane permeability, and such HR may be linked to both the N-terminal side and the C-terminal side of the carrier protein. .) Further, in order to link PST to a carrier protein by a method as described later, cysteine may be located near the terminal.

・ Cell membrane permeable peptide (CPP)
CPP is generally known as a polypeptide having an amino acid sequence rich in basic amino acids such as arginine and having a function of binding to a cell membrane and incorporating itself into a cell. A protein transduction domain (Protein Transduction Domain) : PTD). Initially, CPP was named “cell membrane permeable peptide” because it was thought to permeate the cell membrane and spontaneously enter the cell, but now it is generally via the cell endocytosis pathway. It is thought to be taken up into cells. And it is often localized to the endosome as it is due to the nature of the cargo that is connected to the CPP and carried into the cell. In the present invention, CPP is defined as a term that refers to a peptide that has the ability to localize to the endosome along with other elements of the complex linked thereto.

  In the present invention, various CPPs can be used as long as they correspond to the target cells (usually mammalian cells), and the kind thereof is not particularly limited. The CPP may have a wild-type amino acid sequence, or may be substituted for the wild-type amino acid sequence as long as it has the ability to enter cells (preferably better than the wild-type). , May have a mutant amino acid sequence that has been deleted or added.

For example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 contained in a trans-activator of transcription protein (Tat protein) derived from human immunodeficiency virus type I (HIV-1) is suitable as the CPP in the present invention. It is.
YGRKKRRQRRRG SEQ ID NO: 1

Further, a polypeptide having the amino acid sequence represented by SEQ ID NO: 3 derived from flockhouse virus (FHV) and CTP512 (Cytoplasmic Transduction Peptide) having the amino acid sequence represented by SEQ ID NO: 4 are also cited as preferred CPPs in the present invention. It is done.
RRRRNRTRRNRRRVR SEQ ID NO: 3
YGRRRRRRRRR SEQ ID NO: 4
In addition, Rev peptide, feline herpesvirus Coat protein-derived peptide, polyarginine and the like are also exemplified as CPP.

RNA binding protein (RBP)
RBP is known as a protein that binds in an independent or independent manner to the base sequence or three-dimensional structure of RNA.

  In the present invention, various RBPs can be used, and the kind thereof is not particularly limited, but usually, those that recognize a specific base sequence or three-dimensional structure of RNA and specifically bind to specific RNA. Use it. The RBP may have a wild-type amino acid sequence, or may be substituted for the wild-type amino acid sequence as long as it has the ability to bind to RNA (preferably better than the wild-type). It may have a mutant amino acid sequence subjected to deletion or addition.

For example, the RNA binding domain of human-derived U1A (U1 small nuclear riboprotein A) having the amino acid sequence represented by SEQ ID NO: 2 is suitable as the RBP in the present invention.
AVPETRPNHTIYINNLNEKIKKDELKYKSLYAIFSQFGQILDILVVSLSLKMRGQAFVIFKEVSSATNANLRSQGFPFYDKPMRIQYAKTDSDIIAKMK SEQ ID NO: 2

  Further, the sex-lethal protein is slightly inferior in RNA carrying ability to the human-derived U1A, but may be used as the RBP of the present invention. In addition, a known RBP such as an aminoacyl-tRNA synthetase, an elongation factor Tu, or a protein having an RRM motif in its structure can also be used.

・ Proteasome degradation signal sequence tag (PDS)
A proteasome degradation signal sequence is an amino acid sequence added to the end of a protein, and ubiquitin binds to the sequence and is known to be capable of degrading a protein having the sequence in the proteasome. Sometimes referred to as a degradation promoting sequence.

  In the present invention, various PDSs can be used, and the kind thereof is not particularly limited. The PDS may have a wild-type amino acid sequence, or may be substituted for the wild-type amino acid sequence as long as it has the ability to be degraded by the proteasome (preferably better than the wild-type). , May have a mutant amino acid sequence that has been deleted or added. For example, known PDSs such as CL1, CL2, CL6, CL9, CL10, CL11, CL12, CL15, CL16, SL17, and PEST can be used.

  In addition, when PDS is used, after the complex is introduced into the cytoplasm, the carrier protein is degraded through the proteasome degradation system, and the dissociation of the carrier protein and RNA is promoted. Is considered to be high.

・ His-rich tag (HR)
HR is a peptide that is typically composed of 6 histidines (His) and can further increase the efficiency of introduction of the complex into the cell. A peptide consisting of 6 histidines is also known as the His tag, which is used for affinity chromatography. The HR in the present invention may be a peptide consisting only of (six) histidines as in the case of such a His tag, or the His has a capability of improving intracellular invasion compared to the case where HR is not used. A peptide in which ₆ is modified, for example, a peptide in which the number of histidines is increased to about 7 to 10 or a peptide in which amino acids other than histidine are interwoven may be used. The number of amino acids constituting HR is usually about 6 to 20, and the ratio of histidine in all amino acids is usually in the range of 50% to 100%.

-Preparation method The carrier protein containing said element can be prepared by a well-known means. Typically, 1) DNA having a base sequence corresponding to a polypeptide containing a predetermined element of the present invention is prepared using PCR, and 2) the prepared DNA is introduced into a vector such as a plasmid. ) Using a site-directed mutagenesis method, a vector having a base sequence corresponding to a polypeptide having a cysteine at the C-terminus was prepared, and 4) the resulting vector was expressed. A method of recovering the target carrier protein can be mentioned.

―Carrier molecule―
The carrier molecule in the present invention refers to a molecule in which the above-described carrier protein is linked to a photosensitizer that functions with light having a wavelength in the near infrared region (near infrared light).

・ Photosensitizer (PST)
A photosensitizer that "functions with light in the near-infrared region (near-infrared light)" is a complex that has been incorporated into cells and localized to endosomes when irradiated with near-infrared light. A photosensitizer that can be diffused into the cytoplasm to exhibit a predetermined function. It is assumed that the mechanism is that the photosensitizer excited by irradiation with near-infrared light generates singlet oxygen, which destabilizes (destroys) the endosomal membrane.

  In the present invention, the “near infrared region” refers to 700 nm to 2500 nm. In the present invention, it functions with light having a wavelength in the near-infrared region, more preferably 700 to 1000 nm, particularly preferably 700 to 850 nm, in other words, an optical enhancement in which the maximum excitation wavelength is in these regions. A sensitizer is used. The reason why the wavelength in the near-infrared region is preferable is that the wavelength is high in tissue permeability, avoiding absorption by hemoglobin and water in the living body.

  Specific examples of the PST in the present invention include DY750 (manufactured by DYOMICICS GmbH), Alexa Fluor 750 (registered trademark, manufactured by Invitrogen), and IRDye800CW (registered trademark, manufactured by LI-COR). DY750 is a compound having a structural formula shown in the following formula and an excitation spectrum (maximum excitation wavelength is 747 nm) shown in FIG. 2 (see DYOMICS GmbH website, http://www.dyomics.com/dy-750.html). ). Alexa Fluor 750, whose structural formula has not been clarified, has a fluorescence spectrum shown in FIG. 3 and has a maximum excitation wavelength of 749 nm (Invitrogen website, http://www.invitrogen.jp/catalogue/molecular). # probes / alexa / alexa # index.html and http://www.invitrogen.jp/catalogue/molecular#probes/alexa/alexafluor750/index.html). IRDye800CW is a compound having an excitation spectrum (maximum excitation wavelength is 774 nm) shown in FIG. 4 although the structural formula is not clarified (catalog available from the website of LI-COR, http: // biosupport. see licor.com/docs/800CW#Micro#08618.pdf).

-Preparation method Carrier molecules can be prepared by linking a carrier protein and PST by a known method. Typically, the only cysteine in the carrier protein is placed near the N-terminus or C-terminus, while PST reacts selectively with the cysteine thiol group (other amino acid residues in the carrier protein). And a method in which a maleimide group is introduced and these functional groups are reacted to bind PST to a carrier protein. Such a method is to remove unreacted PST with a gel filtration spin column after mixing with a carrier protein containing cysteine at one end using a commercially available “PST with a maleimide group”. Can be done.

―Carrier molecule / RNA complex―
The carrier molecule / RNA complex (sometimes simply referred to as “complex”) in the present invention refers to a complex in which RNA is bound to the above-described carrier molecule via RBP. However, RNA may form a complex with a carrier molecule by specific binding to RBP and electrostatic / non-specific binding to CPP having a large positive charge (for example, Tat).

・ RNA
The RNA to be bound to the RBP in the carrier protein includes an oligonucleotide having a base sequence recognized at the RNA recognition site of the RBP and an oligonucleotide having a base sequence that exhibits a predetermined function when introduced into the cytoplasm (hereinafter referred to as the RNA). These are referred to as “functional RNA”), and may be bound directly or via an oligonucleotide serving as a linker.

  Various RNAs can be used as the functional RNA, and the type thereof is not particularly limited. Representative functional RNAs in the present invention include shRNA (short hairpin RNA), siRNA (small interfering RNA), and miRNA (micro RNA) for suppressing the expression of a specific gene. Moreover, mRNA for synthesizing a specific protein in a cell and modified tRNA for introducing an unnatural amino acid into a specific site of the protein can also be used as the functional RNA in the present invention. In addition, antisense RNA, aptamer RNA, ribozyme and the like can also be functional RNA in the present invention.

Formation Method The carrier molecule / RNA complex can be easily formed by mixing a carrier molecule and RNA prepared in advance in a solution. The ratio of these mixed amounts (moles) can be adjusted as appropriate. For example, in the case of carriers (TatU1A, TatU1A-CL1, TatU1A-His6) used in the examples, RNA: carrier molecule = 1: 5 10 is preferable.

-Delivery method of carrier molecule / RNA complex into cytoplasm-
The carrier molecule / RNA complex according to the present invention can be delivered into the cytoplasm by the following procedure.

  First, a carrier molecule / RNA complex is brought into contact with a cell to localize to the endosome in the cell. Usually, when a solution containing a carrier molecule / RNA complex is added to a cell, the carrier molecule / RNA complex is naturally taken up by endocytosis after a certain period of time and is localized in the endosome in the cell. . The cells are usually animal cells (including human cells) and may be in vivo or in vitro (cultured cells). That is, the method for delivering RNA into the cytoplasm according to the present invention can be applied both in vivo and in vitro.

  Next, the carrier molecule / RNA complex is diffused in the cytoplasm by irradiation with light having a wavelength in the near infrared region where the PST in the carrier molecule / RNA complex functions. Without such predetermined near-infrared light, the carrier molecule / RNA complex remains mostly localized in the endosome, and the predetermined function of RNA is not substantially expressed.

  The means for irradiating the excitation light is not particularly limited. Using a xenon arc lamp or dye pump laser that emits near-infrared light of a predetermined wavelength as the light source, and using an appropriate filter as necessary, the near-infrared light is applied to cells at the desired site where RNA functions are to be expressed. Irradiation is sufficient.

  After the carrier molecule / RNA complex is delivered into the cytoplasm by such a procedure, a predetermined function of RNA is exhibited by the action of various biological substances in the cytoplasm. For example, when siRNA or shRNA is used as a functional RNA, the linker sequence portion and the single-stranded loop sequence portion are cleaved and decomposed by the action of the Dicer enzyme, and the double-stranded portion is recognized by a specific cell-specific structure. By this, the translation reaction from mRNA is inhibited and RNAi is achieved.

―Use―
The carrier molecule of the present invention can introduce any RNA capable of binding to the carrier molecule into the cytoplasm, and its use is not particularly limited. Typically, the carrier molecule / RNA complex of the present invention can be used, for example, as a gene therapy drug for cancer and viral diseases. In this case, RNA (functional RNA) having an appropriate base sequence capable of suppressing the expression of a specific gene causing cancer or viral disease by RNAi is selected.

1) Preparation of carrier protein
A transmembrane domain consisting of 11 amino acids of human immunodeficiency virus-derived Tat (t rans- a ctivator of t ranscription) protein as CPP, a fusion of the RNA binding domain of the human U1A (U1 small nuclear ribonucleoprotein A) as RBP, A carrier protein TatU1A having the amino acid sequence represented by SEQ ID NO: 5 was prepared (FIG. 1A). In the amino acid sequence of SEQ ID NO: 5, the double underline (positions 13-24) is Tat, and the underline (positions 36-132) is U1A.

In addition, carrier proteins TatU1A-CL1 and TatU1A-His ₆ having the amino acid sequences shown in SEQ ID NOs: 6 and 7, respectively, added with CL1 as PDS or His ₆ tag as HR at the C-terminus of this protein are also present. Prepared. In the amino acid sequence of SEQ ID NO: 6, the double underline (positions 13-24) is Tat, the underline (positions 36-132) is U1A, and the alternate long and short dash line (positions 135-150) is CL1. During the following amino acid sequence SEQ ID NO: 7, the double underlined (position 13-24) is Tat, underlined (position 36-132) is U1A, two-dot chain line (position 135-141) is a sequence of His _6.

Each carrier protein has a cysteine at the C-terminus for later addition of a thiol-reactive dye (photosensitizer). This C-terminal cysteine is the only cysteine in the carrier protein.

2) Addition of near-infrared fluorescent dye (as photosensitizer) to carrier protein
A fluorescent dye excited by near-infrared light was added to the carrier protein prepared in 1). Fluorescent dyes DY750 (DYOMICS GMMBH), HiLyte Fluor750 (Anaspec), IRDye800CW (LI-COR), Alexa Fluor 750 (Invitrogen), DyLight750 (Takara), DyLight800 (Takara) ) Was used. The fluorescent dye addition rate was determined from the protein concentration of the carrier molecule determined using the Protein Assay Kit and the fluorescent dye concentration determined by measuring the absorbance using a spectrophotometer. Unlabeled carrier protein was added and prepared so that the addition rate was about 10 to 30%.

3) Introduction of RNA into intracellular endosome by carrier protein with near-infrared fluorescent dye and diffusion to cytoplasm by near-infrared light U1A recognizes carrier protein (here, TatU1A) labeled with various fluorescent dyes Attempts were made to transfer cytoplasm of shRNA to which sequences were added. Carrier / shRNA complex by mixing shRNA and TatU1A-fluorescent dye conjugate (hereinafter referred to as carrier) to a final concentration of 200 nM and 2 mM, respectively, and incubating at 37 ° C for 10 minutes under light-shielded conditions The body was prepared. Here, FAM-labeled shRNA was used to visualize the intracellular localization of the complex. The above-mentioned carrier / shRNA mixed solution was added to mammalian cells (CHO cells) cultured until they became about 70% confluent, and cultured in a CO ₂ incubator at 37 ° C. for 2 hours. Light was irradiated at the excitation wavelength of each fluorescent dye, and intracellular localization of the carrier / shRNA complex was evaluated by observation with a fluorescence microscope. Only when Alexa Fluor 750, DY750, and IRDye800CW were used as fluorescent dyes (photosensitizers), shRNA that was scattered in cells (localized in endosomes) diffused into the cytoplasm by irradiation with near infrared light (Figure 5). That is, it was shown that a carrier / shRNA complex can be introduced into the cytoplasm specifically with near-infrared light irradiation. At this time, when using HiLyte Fluor750, Alexa Fluor 750, and DyLight750, light of 750 ± 20 nm (epitex Infrared illuminator L750-66-60), when using IRDye800CW and DyLight800, light of 780 ± 20 nm ( Epitex Infrared illuminator L780-66-60) was used as excitation light.

4) Introduction of RNA into the cytoplasm of mammalian cells by near-infrared light and confirmation of functional expression of the RNA (RNAi)
Using TatU1A (TatU1A-DY750) to which a fluorescent dye DY750 having absorption at around 750 nm was added, RNAi induction by near infrared light irradiation was examined. TatU1A-DY750 / shRNA complex was prepared by mixing shRNA and TatU1A-DY750 at final concentrations of 200 nM and 2 mM, respectively, and incubating at 37 ° C. for 10 minutes under light-shielded conditions. Here, in order to target EGFP mRNA, shRNA with anti-EGFP sequence was used. CHO cells stably expressing EGFP (EGFP-CHO cells) were cultured until they became about 70% confluent, the above TatU1A-DY750 / shRNA mixed solution was added, and further cultured in a CO ₂ incubator at 37 ° C. for 2 hours. After irradiating light at the excitation wavelength of DY750 and culturing in a CO ₂ incubator at 37 ° C. for 20 hours, the cells were collected, and the RNAi effect was examined by flow cytometry. As a result, the EGFP average fluorescence intensity of the cell group irradiated with light was remarkably reduced as compared with the cell group not irradiated with light (FIG. 6). That is, an RNAi effect on EGFP was observed in cells irradiated with light.

  From the above results, shRNA can be introduced into cells with TatU1A-DY750, and shRNA can be transferred to the cytoplasm only when irradiated with light, that is, when irradiated with near infrared light. RNAi (expression of shRNA function) Could be induced.

5) Selection of carrier protein with high RNA carrying capacity For TatU1A prepared in 1) above, CPP part is FHV (Flockhouse virus-derived arginine peptide; RRRRNRTRRNRRRVR) or CTP512 (cytoplasmic transduction peptide; YGRARRRRRRR). In consideration of easiness, the amino acid sequence shown in SEQ ID NO: 4 in which R at position 4 was deleted was used. However, the ability of FHV-U1A and CTP-U1A to transport RNA into the cytoplasm was not significantly different from TatU1A. Next, Tat was used as CPP, and the RBP part was made into Sxl protein or bacteriophage λ-N peptide. Although this TatSxl had RNA carrying ability, it was inferior to TatU1A. TatλN did not show any RNA carrying ability.

Next, when the RNA carrying ability of TatU1A-CL1, TatU1A-His ₆ and TatU1A prepared in 1) was examined, TatU1A-CL1 and TatU1A-His ₆ showed higher RNA carrying ability than TatU1A. That is, as the carrier protein portion, TatU1A-CL1 and TatU1A-His ₆ had the highest ability as an RNA carrier among those examined above.

6) Introduction of RNA into the cytoplasm of mammalian cells using near-infrared light and confirmation of its functional expression (RNAi)
As in 4), RNAi induction by near-infrared light irradiation was investigated using TatU1A-His ₆ (TatU1A-His ₆ -DY750) to which a fluorescent dye DY750 having absorption near 750 nm was added. Prepare TatU1A-His ₆ -DY750 / shRNA complex by mixing shRNA and TatU1A-His ₆ -DY750 to a final concentration of 200 nM and 2 mM, respectively, and incubating for 10 minutes at 37 ° C in the dark. did. Here, in order to target EGFP mRNA, shRNA with anti-EGFP sequence was used. Culturing CHO cells that stably express EGFP (EGFP-CHO cells) until they are about 70% confluent, add the above TatU1A-His _6- DY750 / shRNA mixture, and further culture in a CO ₂ incubator at 37 ° C for 2 hours did. After irradiating light at the excitation wavelength of DY750 and culturing in a CO ₂ incubator at 37 ° C. for 20 hours, the cells were collected, and the RNAi effect was examined by flow cytometry. As a result, the EGFP average fluorescence intensity of the cell group irradiated with light was remarkably reduced as compared with that obtained without light irradiation (FIG. 7). That is, an RNAi effect on EGFP was observed in cells irradiated with light. From the above results, TatU1A-His ₆ -DY750 introduced shRNA into cells, and was able to transfer shRNA to the cytoplasm in a light irradiation-specific manner, and to induce RNAi (expression of shRNA function).

7) Infrared RNAi induction in human tumor cells
As in 4), RNAi induction by near-infrared light irradiation was investigated using TatU1A-CL1 (TatU1A-CL1-DY750) to which a fluorescent dye DY750 having absorption near 750 nm was added. TatU1A-CL1-DY750 / shRNA complex was prepared by mixing shRNA and TatU1A-CL1-DY750 at final concentrations of 200 nM and 2 mM, respectively, and incubating at 37 ° C. for 10 minutes under light-shielded conditions. Here, malignant mesothelioma cell 211H, which is a human tumor cell, was used. 211H cells stably expressing EGFP were cultured until they became about 70% confluent, the above TatU1A-CL1-DY750 / shRNA mixed solution was added, and further cultured at 37 ° C. for 2 hours in a CO ₂ incubator. After irradiating light at the excitation wavelength of DY750 and culturing in a CO ₂ incubator at 37 ° C. for 20 hours, the cells were collected, and the RNAi effect was examined by flow cytometry. As a result, the EGFP average fluorescence intensity of the cell group irradiated with light was remarkably reduced as compared with those not irradiated with light (FIG. 8). That is, an RNAi effect on EGFP was observed in cells irradiated with light.

Sequence number 4: CTP512
Sequence number 5: TatU1A
Sequence number 6: TatU1A-CL1
Sequence number 7: TatU1A-His6

Claims (10)

A carrier protein containing a cell membrane permeable peptide (CPP) and an RNA binding protein (RBP), and a photosensitization functioning with light having a wavelength of 700 nm to 1000 nm linked to the N-terminal side or C-terminal side of the carrier protein. A carrier molecule having a structure consisting of an agent (PST),
The photosensitizer (PST) is a compound represented by the following structural formula (DY750 : trademark ), a compound having a fluorescence spectrum shown in FIG. 3 (Alexa Fluor750 : registered trademark ), or a compound having an absorption spectrum shown in FIG. Carrier molecule characterized by being (IRDye800CW : registered trademark ).
  The carrier molecule according to claim 1, wherein the carrier protein further comprises a proteasome degradation signal sequence tag (PDS) and / or a His-rich tag (HR). In the carrier protein, a cell membrane permeable peptide (CPP), an RNA binding protein (RBP), a proteasome degradation signal sequence tag (PDS) and / or a His-rich tag (HR) are linked in this order from the N-terminal side. and it has, and the photosensitizer to the C-terminal side of the carrier protein (PST) is connected, the carrier molecule of claim 1 or 2. The CPP has the amino acid sequence shown in SEQ ID NO: 1 is a peptide consisting of 12 amino acids of human immunodeficiency virus-derived Tat (trans-activator of transcription) protein in, according to any of claims 1 to 3 Carrier molecule.
YGRKKRRQRRRG SEQ ID NO: 1 The carrier molecule according to any one of claims 1 to 4 , wherein the RBP is an RNA binding domain of human-derived U1A (U1 small nuclear riboprotein A) having the amino acid sequence represented by SEQ ID NO: 4.
AVPETRPNHTIYINNLNEKIKKDELKYKSLYAIFSQFGQILDILVVSLSLKMRGQAFVIFKEVSSATNANLRSQGFPFYDKPMRIQYAKTDSDIIAKMK SEQ ID NO: 4 A carrier molecule / RNA complex having a structure comprising the carrier molecule according to any one of claims 1 to 5 and RNA bound to RBP in the RNA carrier molecule. The carrier molecule / RNA complex according to claim 6 , wherein the RNA is short hairpin RNA (shRNA), small interfering RNA (siRNA) or microRNA (miRNA). A gene therapy drug comprising the carrier molecule / RNA complex according to claim 6 or 7 . The gene therapy drug according to claim 8 , which is intended for cancer or viral diseases. A step of bringing the carrier molecule / RNA complex according to claim 6 or 7 into contact with a cell to localize it in an endosome in the cell in vitro, and PST in the carrier molecule / RNA complex functions A method for delivering RNA into the cytoplasm comprising the step of irradiating light having a wavelength in the near infrared region to diffuse the carrier molecule / RNA complex into the cytoplasm.