Circular RNA, chimeric antigen receptor T cells and construction methods and uses

Abstract

The application belongs to the technical field of biology, and discloses a circular RNA, a chimeric antigen receptor T cell, a construction method and application. The circular RNA provided by the application comprises a UTR-1 fragment with a special nucleotide sequence and a UTR-2 fragment with a special nucleotide sequence. The UTR-1 fragment, the UTR-2 fragment and the introduced IRES sequence are cooperated to make the circular RNA have excellent translation efficiency in T cells. The circular RNA also has excellent anti-degradation ability in cells, so that the circular RNA can be continuously and efficiently expressed in T cells for a long time, which is beneficial to the realization of the sustained killing effect of T cells and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a circular RNA, a chimeric antigen receptor T cell, its construction method, and its application. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy is a revolutionary cancer immunotherapy that uses genetically engineered T cells to recognize and attack cancer cells. However, currently approved CAR-T cell products face several significant challenges. First, the preparation of traditional CAR-T cells involves complex genetic engineering processes, including the use of viral vectors, which not only increases production complexity and time but also leads to extremely high treatment costs. Second, gene transduction using viral vectors may introduce genotoxic risks such as insertional mutations, affecting treatment safety. Furthermore, the limited in vivo duration of existing CAR-T cells may affect long-term treatment efficacy. These factors collectively limit the widespread application of CAR-T therapy. Therefore, the development of novel T-cell engineering methods is crucial for the advancement of CAR-T therapy.

[0003] mRNA technology, which delivers in vitro synthesized mRNA into cells to guide the synthesis of specific proteins, is a non-viral method. In mRNA technology, the introduced mRNA does not integrate into the host genome, reducing the risk of genotoxicity; it can be rapidly synthesized through in vitro transcription, simplifying the production process and resulting in lower production costs compared to viral vectors; these advantages make mRNA a highly attractive method for CAR-T cell engineering.

[0004] However, linear mRNAs have a short half-life and short expression duration, which limits their sustained expression time in CAR-T cells and makes it difficult to achieve sustained killing effects of T cells, thus having significant limitations. Summary of the Invention

[0005] The primary objective of this invention is to address the problem of short mRNA expression duration in the application of existing mRNA technology to CAR-T cell engineering, and to provide a circular RNA.

[0006] A second objective of this invention is to provide a construct for constructing the aforementioned circular RNA.

[0007] A third objective of this invention is to provide a method for constructing the aforementioned circular RNA.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned circular RNA in the construction of chimeric antigen receptor cells.

[0009] The fifth objective of this invention is to provide a chimeric antigen receptor T cell.

[0010] Specifically, the circular RNA provided by this invention comprises the structure shown in formula (1):

[0011]

[0012] Wherein, the UTR-1 fragment is any segment of the nucleotide fragments with sequences as shown in SEQ ID NO:1 to 8; the UTR-2 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NO:11, 12 or 14.

[0013] Further, the UTR-1 fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:2 or 6, and the UTR-2 fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:11 or 14.

[0014] Furthermore, the coding fragment includes a nucleotide fragment with a sequence as shown in SEQ ID NO:28 or 29.

[0015] Furthermore, the IRES sequence is any segment of a nucleotide fragment as shown in SEQ ID NO:22-24.

[0016] Furthermore, the length of the Scar segment is 0 to 50 nt.

[0017] The construct provided by the present invention for constructing the above-mentioned circular RNA includes, along the 5' to 3' direction, a UTR-1 fragment, an IRES fragment, a coding fragment, and a UTR-2 fragment.

[0018] The method for constructing the circular RNA provided by the present invention includes: taking the above-mentioned construct and performing an in vitro transcription reaction and an in vitro circularization reaction to obtain the circular RNA.

[0019] This invention provides the application of the above-mentioned circular RNA in the construction of chimeric antigen receptor T cells.

[0020] The chimeric antigen receptor T cell provided by the present invention includes the above-mentioned circular RNA.

[0021] Furthermore, the chimeric antigen receptor T cell synthesizes and expresses a chimeric antigen receptor sequence as shown in SEQ ID NO:26.

[0022] Beneficial effects:

[0023] The circular RNA provided by this invention includes a UTR-1 fragment with a specific nucleotide sequence and a UTR-2 fragment with a specific nucleotide sequence. Through the synergistic cooperation of the introduced UTR-1 fragment, UTR-2 fragment, and IRES sequence, the circular RNA exhibits excellent translation efficiency in T cells. Furthermore, the circular RNA also possesses excellent anti-degradation ability within the cell, enabling it to be continuously and efficiently expressed in T cells for a long period of time. This is beneficial for achieving a sustained killing effect of T cells and has promising application prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structure of circular RNAs 1 to 9 provided in Example 1 of the present invention.

[0025] Figure 2 This is a diagram showing the experimental results of screening UTR-1 fragments provided in Example 1 of this invention.

[0026] Figure 3 This is a schematic diagram of the structure of circular RNA 10-18 provided in Example 2 of the present invention.

[0027] Figure 4 This is one of the experimental results of screening UTR-2 fragments provided in Example 2 of this invention.

[0028] Figure 5 The second figure shows the experimental results of screening UTR-2 fragments provided in Example 2 of this invention.

[0029] Figure 6 This is a diagram showing the experimental results of screening the IRES fragment provided in Example 3 of this invention.

[0030] Figure 7 This is a schematic diagram of the structure of circular RNA 25-28 provided in Example 4 of the present invention.

[0031] Figure 8 This is one of the experimental results of screening the coding gene of the chimeric antigen receptor provided in Example 4 of this invention.

[0032] Figure 9 This is the second figure showing the experimental results of screening the coding gene of the chimeric antigen receptor provided in Example 4 of this invention.

[0033] Figure 10 This is an experimental result diagram of the ability of the circular RNA molecule provided in Example 8 of the present invention to be continuously and efficiently expressed in T cells over a long period of time. Detailed Implementation

[0034] Based on a profound understanding of the factors affecting the sustained expression of mRNA in cells over a long period of time, the inventors of this invention creatively used circular RNA as a carrier for chimeric antigen receptor encoding genes. In response to the problem of low translation efficiency of circular RNA in T cells, the inventors conducted extensive and in-depth research and a large number of experiments, thereby obtaining the circular RNA provided by this invention.

[0035] In this invention, the circular RNA specifically comprises the structure shown in formula (1):

[0036]

[0037] In this invention, the UTR-1 fragment is specifically any segment of the nucleotide fragments shown in SEQ ID NO:1 to 8. In some specific embodiments, the UTR-1 fragment is preferably a nucleotide fragment with a sequence shown in SEQ ID NO:2 or 6. In this case, the circular RNA incorporating the UTR-1 fragment exhibits excellent translation efficiency in T cells.

[0038] In this invention, the UTR-2 fragment is specifically a nucleotide fragment with the sequence shown in SEQ ID NO: 11, 12, or 14. In some specific embodiments, the UTR-2 fragment is preferably a nucleotide fragment with the sequence shown in SEQ ID NO: 11 or 14. In this case, the circular RNA with the UTR-2 fragment introduced exhibits excellent translation efficiency in T cells.

[0039] In this invention, the IRES fragment refers to the internal ribosome entry site sequence, which can recruit ribosomes to translate mRNA. It is a type of functional element commonly used in the construction of existing recombinant proteins. This invention does not specifically limit it. Specific examples include, but are not limited to, one or more of the following: CVB3 IRES fragment, EMCV IRES fragment, and FMDVIRES fragment.

[0040] In this invention, the inventors creatively designed the IRES fragment based on the constructed circular RNA and the target cells for expression, and obtained an IRES fragment with a sequence as shown in any of SEQ ID NO:22 to 24.

[0041] In this invention, compared with conventionally used IRES fragments such as the CVB3 IRES fragment, any segment of the nucleotide fragments with sequences as shown in SEQ ID NO:22-24 is used as the IRES fragment in the circular RNA, which can further improve the expression efficiency of the circular RNA in T cells, thereby better achieving the continuous and efficient expression of the circular RNA in T cells.

[0042] In this invention, the coding fragment refers to the coding gene in the circular RNA used to guide protein synthesis and expression. The specific sequence is determined by the protein to be expressed in T cells, and this invention does not impose any particular limitation on it.

[0043] In this invention, with the aim of synthesizing and expressing a chimeric antigen receptor with a sequence as shown in SEQ ID NO:26 in T cells, the inventors have creatively designed the coding gene to obtain a coding gene with a sequence as shown in SEQ ID NO:28 or 29.

[0044] In this invention, a nucleotide fragment with a sequence such as SEQ ID NO:28 or 29 is used as the coding gene that guides the synthesis and expression of the chimeric antigen receptor, which enables the efficient expression of the chimeric antigen receptor in T cells.

[0045] In this invention, the Scar fragment refers to the scar sequence contained in the construct used to construct circular RNA and which remains in the circular RNA after in vitro transcription and in vitro circularization. The presence or absence of the Scar fragment and its specific length and nucleotide sequence are determined by the construct and its splicing and circularization method. The presence of the Scar fragment does not have a significant impact on the translation efficiency of the circular RNA, and this invention does not impose any particular limitations on it.

[0046] In some specific embodiments, the length of the Scar fragment is preferably 0–50 nt, such as 0 nt, 2 nt, 5 nt, 8 nt, 10 nt, 15 nt, 25 nt, 30 nt, 50 nt, or any value between them. In this case, the Scar fragment retained in the circular RNA is relatively short, and its presence has little impact on the transfection of the circular RNA to T cells, while the circular RNA still has excellent transfection efficiency for T cells.

[0047] In some specific embodiments, the Scar fragment is preferably 0 nt in length; that is, the 5' end of the UTR-1 fragment is directly connected to the 3' end of the UTR-2 fragment to form a circular structure. In this case, there is no residual exogenous scar sequence in the circular RNA, and it still has excellent transfection efficiency for T cells.

[0048] With the aim of obtaining the above-mentioned circular RNA, the present invention also provides a construct for constructing the above-mentioned circular RNA.

[0049] In this invention, the construct comprises at least a UTR-1 fragment, an IRES fragment, a coding fragment, and a UTR-2 fragment sequentially along the 5' to 3' direction. The UTR-1 fragment, IRES fragment, coding fragment, and UTR-2 fragment in the construct are determined by the desired circular RNA, which will not be elaborated further here.

[0050] In this invention, the construct may further include functional elements for promoting in vitro transcription and in vitro circularization to obtain circular RNA. Specific examples of the functional elements include, but are not limited to, one or more of the following: T7 promoter, T4 promoter, type I intron, and type II intron.

[0051] With the aim of obtaining the above-mentioned circular RNA, the present invention also provides a method for constructing circular RNA, which specifically includes: taking the above-mentioned construct and performing an in vitro transcription reaction and an in vitro circularization reaction to obtain the circular RNA.

[0052] In this invention, the in vitro transcription reaction and in vitro circularization reaction are conventional techniques used in mRNA technology. Those skilled in the art can make adaptive choices as needed, limited to obtaining the above-mentioned circular RNA. This invention does not impose any particular limitations on them.

[0053] Based on the ability of the circular RNA to be expressed efficiently and continuously in T cells over a long period of time, the present invention also provides the application of the circular RNA in the construction of chimeric antigen receptor T cells.

[0054] Based on the application potential of the above-mentioned circular RNA in the construction of chimeric antigen receptor T cells, the present invention also provides a chimeric antigen receptor T cell comprising the above-mentioned circular RNA.

[0055] In this invention, when the circular RNA includes a coding fragment with a sequence as shown in SEQ ID NO:28 or 29, the chimeric antigen receptor T cell is able to synthesize and express a chimeric antigen receptor with a sequence as shown in SEQ ID NO:26.

[0056] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0057] Example 1

[0058] This embodiment illustrates the screening of UTR-1 fragments, specifically including:

[0059] (1) Preparation of T cell suspension: T cells (Oricells biotech) were seeded at a rate of 1×10⁶ cells / mL and added to RPMI 1640 medium (Gibco, catalog number 61870010). FBS (Gibco, catalog number 10099141) and interleukin-2 (PeproTech, catalog number 200-02) were added to the medium at final concentrations of 10% and 50 ng / mL, respectively. The cells were cultured at 37℃, 200 rpm, and 5% CO₂ for 48 h. The cells were then collected and subjected to Neo TM Cells were resuspended after transfection with buffer R (Invitrogen, catalog number MPK10096) and the cell density was adjusted to 2 × 10⁻⁶. 6 T cell suspension was obtained by dispersing cells / mL.

[0060] (2) Introduction of circular RNA: 1 μg of circular RNA 1-9 was mixed with 10 μL of T cell suspension and added to an electroporation cuvette. The T cells were electroporated at 1500 mV and 10 ms to introduce circular RNA 1-8 into the T cells.

[0061] Among them, circular RNAs 1–9 all possess… Figure 1 The structures shown differ in that circular RNA 1 does not contain a UTR-1 fragment, while circular RNAs 2–9 contain different UTR-1 fragments. The nucleotide sequences of each fragment are shown in Table 1.

[0062] Table 1.

[0063]

[0064]

[0065]

[0066] It should be noted that circular RNAs 1–9 were all obtained using the same type I intron system, through the same in vitro transcription and in vitro circularization induction conditions, and contained the same Scar fragment. The Scar fragment is only 88 nt in length, and its presence does not affect the translation efficiency of circular RNAs 1–9.

[0067] (3) Electrotransfected T cells were added to preheated RPIM 1640 medium and cultured at 37°C, 200 rpm, and 5% CO2 for 24 h. Flow cytometry was then used to detect and calculate the average fluorescence intensity of the T cells after the introduction of each circular RNA. The results are as follows: Figure 2 As shown.

[0068] Depend on Figure 2 The test results show that, compared with circular RNA 1 without the introduction of the URT-1 fragment, introducing any segment of the nucleotide fragments with sequences as shown in SEQ ID NO:1 to 8 as the UTR-1 fragment can significantly improve the fluorescence intensity of T cells transfected with circular RNA. Circular RNA has good translation efficiency in T cells, and UTR-1 fragment-2 and UTR-1 fragment-6 show even better translation efficiency improvement effects.

[0069] Example 2

[0070] This embodiment illustrates the screening of UTR-2 fragments, specifically including:

[0071] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cells / mL was prepared using the method provided in Example 1.

[0072] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 10–18 provided in Example 1 were mixed with 10 μL of T cell suspension and added to an electroporation cuvette. T cells were electroporated at 1500 mV for 10 ms to introduce circular RNA 3 and 10–18 into the T cells. Circular RNA 10–18 all possess… Figure 3 The structures shown differ in that circular RNAs 10–18 contain different UTR-2 fragments, the nucleotide sequences of which are shown in Table 2.

[0073] Table 2.

[0074]

[0075]

[0076] It should be noted that circular RNAs 3 and 10–18 were all obtained using the same type I intron system, induced by the same in vitro transcription and in vitro circularization induction conditions, and contain the same Scar fragment. The Scar fragment is only 88 nt in length, and its presence does not affect the translation efficiency of circular RNAs 3 and 10–18.

[0077] (3) Electrotransfected T cells were added to preheated RPIM 1640 medium and cultured at 37°C, 200 rpm, and 5% CO2 for 24 h. Flow cytometry was then used to detect and calculate the transfection efficiency of the circular RNA and the average fluorescence intensity of T cells after successful introduction of each circular RNA. The specific results are as follows: Figure 4 and5 As shown.

[0078] Depend on Figure 4 and 5 The test results show that, compared with circular RNA 3 without the introduction of the URT-2 fragment, circular RNAs 10, 11, and 13 obtained by introducing any segment of the nucleotide fragments shown in SEQ ID NO: 11, 12, and 14 as the UTR-2 fragment can significantly improve the fluorescence intensity of T cells after transfection with circular RNA without affecting the transfection efficiency of circular RNA. Circular RNA has good translation efficiency in T cells, and UTR-2 fragment-1 and UTR-1 fragment-4 show even better translation efficiency improvement effects.

[0079] Example 3

[0080] This embodiment illustrates the screening of IRES fragments, specifically including:

[0081] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cells / mL was prepared using the method provided in Example 1.

[0082] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 19-24 provided in Example 1 were mixed with 10 μL of T cell suspension and added to an electroporation cuvette. T cells were electroporated at 1500 mV for 10 ms to introduce circular RNA 3 and 19-24 into the T cells. Circular RNA 19-24 has the same structure as circular RNA 3, but differs in the IRES fragments contained within them. The nucleotide sequences of each IRES fragment are shown in Table 3.

[0083] Table 3.

[0084]

[0085]

[0086]

[0087] It should be noted that circular RNAs 3 and 19–24 were all obtained using the same type I intron system, induced by the same in vitro transcription and in vitro circularization induction conditions, and contain the same Scar fragment. The Scar fragment is only 88 nt in length, and its presence does not affect the translation efficiency of circular RNAs 3 and 19–24.

[0088] (3) Electrotransfected T cells were added to preheated RPIM 1640 medium and cultured at 37°C, 200 rpm, and 5% CO2 for 24 h. Flow cytometry was then used to detect the relative fluorescence intensity of the T cells after the introduction of each circular RNA, and the results were calculated using the following formula: Figure 6 As shown.

[0089] Relative fluorescence intensity = X / X0

[0090] Where X is the average fluorescence intensity of T cells introduced with any one of the circular RNAs 19 to 24, and X0 is the average fluorescence intensity of T cells introduced with circular RNA 3.

[0091] Depend on Figure 6 The test results show that, compared with circular RNA 3, the circular RNA 21-23 obtained by introducing any segment of the nucleotide fragments shown in SEQ ID NO:22-24 as the IRES fragment can significantly improve the fluorescence intensity of T cells after transfection with circular RNA and exhibit better translation efficiency in T cells.

[0092] Example 4

[0093] This embodiment illustrates the screening of the coding gene for a chimeric antigen receptor with an amino acid sequence as shown in SEQ ID NO:26, specifically including:

[0094] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cells / mL was prepared using the method provided in Example 1.

[0095] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 25-28 provided in Example 1 were mixed with 10 μL of T cell suspension and added to an electroporation cuvette. T cells were electroporated at 1500 mV for 10 ms to introduce circular RNA 3 and circular RNA 25-28 into the T cells. Circular RNA 25-28 all possess... Figure 7 The structures shown differ in that circular RNAs 25–28 contain different chimeric antigen receptor coding genes, and the nucleotide sequences of each fragment are shown in Table 4.

[0096] Table 4.

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] It should be noted that circular RNAs 3 and 25–28 were all obtained using the same type I intron system, induced by the same in vitro transcription and in vitro circularization induction conditions, and contain the same Scar fragment. The Scar fragment is only 88 nt in length, and its presence does not affect the translation efficiency of circular RNAs 3 and 25–28.

[0103] (3) Electrotransfected T cells were added to preheated RPIM 1640 medium and cultured at 37°C, 200 rpm, and 5% CO2 for 24 h. Flow cytometry was then used to detect and calculate the transfection efficiency of the chimeric antigen receptor coding genes and the average fluorescence intensity of the chimeric antigen receptors in T cells after successful transfection of each chimeric antigen receptor coding gene. The specific results are as follows: Figure 8 and 9 As shown.

[0104] Depend on Figure 8 and 9 The test results show that circular RNAs 26 and 27 obtained by introducing nucleotide fragments with sequences such as SEQ ID NO:28 or 29 as the coding gene of chimeric antigen receptors can efficiently synthesize and express chimeric antigen receptors in T cells with excellent transfection efficiency.

[0105] Examples 5-7

[0106] Examples 5-7 illustrate the preparation of circular RNA molecules for chimeric antigen receptor T cells. The circular RNA molecule includes, along the 5' to 3' direction, a Scar fragment (sequence shown as SEQ ID NO:31), a UTR-1 fragment, an IRES fragment, a GFP-encoding gene (sequence shown as SEQ ID NO:9), a chimeric antigen antibody-encoding gene, and a UTR-2 fragment connected in sequence. Specific information about each fragment in each example is shown in Table 5.

[0107] Table 5.

[0108]

[0109] Example 8

[0110] This embodiment illustrates the ability of the circular RNA molecules provided in Examples 5-7 to be expressed efficiently and continuously in T cells over a long period of time. The specific tests include:

[0111] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cells / mL was prepared using the method provided in Example 1.

[0112] (2) Introduction of circular RNA: Take 1 μg of the circular RNA molecules provided in Examples 5 to 7 and mix them with 10 μL of T cell suspension. Add the mixture to an electroporation cup and electroporate the T cells at 1500 mV and 10 ms to introduce the circular RNA molecules into the T cells.

[0113] (3) Electrotransfected T cells were added to preheated RPIM 1640 medium and cultured at 37°C, 200 rpm, and 5% CO2 for 24 h. Samples of the culture medium were collected at 6, 16, 24, 32, 48, and 60 h after the recovery culture was completed and analyzed by flow cytometry. The average fluorescence intensity of the chimeric antigen receptor on T cells was calculated, with linear mRNA used as a control. The results are detailed below. Figure 10 As shown.

[0114] The linear mRNA is modified with pseudouridine triphosphate and its structure specifically includes UTR-1 fragment-2 (SEQ ID NO:2), IRES fragment-3 (SEQ ID NO:22), GFP encoding gene (sequence shown in SEQ ID NO:9), chimeric antigen antibody encoding gene-1 (SEQ ID NO:27), and UTR-2 fragment-1 (SEQ ID NO:27) connected sequentially along the 5' to 3' direction.

[0115] Depend on Figure 10 The test results show that, compared with the linear mRNA modified with pseudouridine triphosphate, the circular RNA molecules provided in Examples 5-7 of this invention exhibit good expression efficiency in T cells, and their half-life in T cells is all above 40 hours. That is, the circular RNA molecules provided in Examples 5-7 can be continuously and efficiently expressed in T cells for a long time, which is beneficial for achieving sustained T cell killing effects and has good application prospects.

[0116] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A circular RNA, characterized in that, The circular RNA comprises a structure as shown in formula (1): Wherein, the UTR-1 fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:2, the UTR-2 fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:11, the IRES sequence is a nucleotide fragment with the sequence shown in SEQ ID NO:22, the coding fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:27, and the Scar fragment is a nucleotide fragment with the sequence shown in SEQ ID NO:

31.

2. A construct for building the circular RNA of claim 1, characterized in that, The construct includes, along the 5' to 3' direction, a UTR-1 segment, an IRES segment, a coding segment, and a UTR-2 segment.

3. A method for constructing circular RNA, characterized in that, The construction method includes: taking the construct described in claim 2 and performing an in vitro transcription reaction and an in vitro circularization reaction to obtain the circular RNA.

4. The use of the circular RNA according to claim 1 in the construction of chimeric antigen receptor T cells.

5. A chimeric antigen receptor T cell, characterized in that, The chimeric antigen receptor T cell includes the circular RNA as described in claim 1.

6. The chimeric antigen receptor T cell according to claim 5, characterized in that, The chimeric antigen receptor T cell synthesizes and expresses a chimeric antigen receptor sequence as shown in SEQ ID NO:26.

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