Application of CUL4A as rheumatoid arthritis diagnostic marker

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of CUL4A as a diagnostic marker for rheumatoid arthritis. Experiments prove that the CUL4A level in rheumatoid arthritis involved parts such as serum and synovial tissues of rheumatoid arthritis patients is remarkably increased, CUL4A and rheumatoid arthritis are positively correlated, and the CUL4A gene or CUL4A protein can be used as a molecular marker for screening or diagnosis or prognosis evaluation of rheumatoid arthritis. Experiments prove that the targeted CUL4A can be used as an effective way for treating rheumatoid arthritis. The application has important meanings for researching the effect of CUL4A in rheumatoid arthritis, understanding the mechanism of RA disease and developing a new treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of CUL4A as a diagnostic marker for rheumatoid arthritis. Technical Background

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease with an increasing incidence. Its pathological features are synovial tissue hyperplasia, persistent synovial inflammation, pannus formation, erosion of adjacent articular cartilage, and progressive destruction of bone, ultimately leading to joint dysfunction or even loss.

[0003] In recent years, with the advancement of technology, our understanding of the pathogenesis of RA has continued to deepen. Fibroblast-like synoviocytes (FLS) have been revealed to play an important role in the pathogenesis of RA. FLS abnormally proliferate and activate in the synovium of RA patients' joints, not only participating in local inflammatory responses, but also damaging surrounding tissues by secreting multiple cytokines and matrix metalloproteinases, accelerating the progression of joint lesions.

[0004] Currently, the clinical treatment of rheumatoid arthritis mainly relies on non-steroidal anti-inflammatory drugs and immunosuppressants to control the inflammatory response and delay the progression of the disease. However, these traditional treatments generally have problems such as slow onset of effect and significant toxic side effects, which have become the main difficulties in clinical treatment. With the continuous deepening of research on the pathological mechanism of rheumatoid arthritis, early intervention treatment targeting specific molecular targets has become a new research direction. This type of targeted treatment has shown significant efficacy in patients diagnosed at the early stage, not only effectively controlling the progression of the disease, but also significantly improving the patient's prognosis and quality of life.

[0005] In recent years, autoantibodies such as rheumatoid factor (RF), C-reactive protein (CRP), and anti-cyclic citrullinated peptide (Anti-CCP) have been proposed as laboratory diagnostic markers for rheumatoid arthritis. However, serum tests for these autoantibodies are subject to a certain degree of false positives. Studies have reported that RF may also be present in patients with systemic lupus erythematosus (SLE) and hepatitis C (HCV) infection, leading to a high rate of misdiagnosis. These markers alone are not sufficient for a definitive diagnosis of rheumatoid arthritis.

[0006] Some studies have also proposed various new diagnostic markers for RA. For example, patent CN113881764A suggests that Jmjd1c gene expression is closely associated with rheumatoid arthritis and can be used as an RA diagnostic marker. Patent CN109486942A proposes RPN2 mRNA or RPN2 protein as RA diagnostic markers. However, these markers are still in the early stages of exploration. Given the complexity of RA etiology and to improve diagnostic accuracy, there is an urgent need to explore new targets and conduct further experimental research to assist in the diagnosis of rheumatoid arthritis. Summary of the Invention

[0007] One of the objectives of the present invention is to provide the use of CUL4A gene or CUL4A protein as a molecular marker for screening, diagnosis or prognosis evaluation of rheumatoid arthritis.

[0008] A second object of the present invention is to provide a use of the CUL4A gene or CUL4A protein in the preparation of a product for screening, diagnosis or prognosis assessment of rheumatoid arthritis.

[0009] Preferably, the product includes reagents and kits.

[0010] A third object of the present invention is to provide a product for screening, diagnosis or prognosis assessment of rheumatoid arthritis, characterized in that the product is a reagent for detecting the mRNA expression level of CUL4A and / or the expression level of CUL4A protein, or the product contains a reagent for detecting the mRNA expression level of CUL4A and / or the expression level of CUL4A protein.

[0011] Preferably, the object of detection is selected from at least one of the following: peripheral blood, peripheral serum, synovial cells, synovial tissue, bone tissue, rheumatoid arthritis bone tissue, CUL4A protein or its mRNA.

[0012] Preferably, when the test results show that the CUL4A mRNA expression level and / or CUL4A protein expression level is significantly higher than the preset threshold of the healthy control group, it indicates that the test subject is at risk of rheumatoid arthritis and needs to be further confirmed in combination with other clinical indicators.

[0013] A fourth object of the present invention is to provide a method for treating rheumatoid arthritis, which is to inhibit the expression or activity of the CUL4A gene in rheumatoid arthritis fibroblast-like synoviocytes or synovial tissue.

[0014] Preferably, an inhibitory element targeting the CUL4A gene is introduced into fibroblast-like synoviocytes or synovial tissue, wherein the inhibitory element comprises an RNA interference molecule targeting the CUL4A gene or the inhibitory element is a CRISPR gene editing system targeting the CUL4A gene.

[0015] Preferably, the RNA interference molecule targeting the CUL4A gene is siRNA or shRNA.

[0016] Preferably, the CRISPR gene editing system targeting the CUL4A gene comprises a gRNA that specifically recognizes the CUL4A gene, a Cas9 protein that binds to the gRNA, and a pharmaceutically acceptable delivery vector.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] CUL4A (Cullin 4A) is a scaffold protein for the E3 ubiquitin ligase, belonging to the Cullin protein family. It participates in numerous cellular processes, such as the cell cycle, DNA repair, and transcriptional regulation. CUL4A forms complexes with proteins such as DDB1 and RBX1, regulating the degradation of key factors (such as transcription factors and cell cycle proteins), thereby modulating the activation, proliferation, and differentiation of immune cells. Abnormal expression of CUL4A is associated with numerous diseases, particularly cancer, neurodegenerative diseases, and immune system disorders.

[0019] This application experimentally confirms that the CUL4A levels in RA-affected areas such as synovial tissue, synovial cells and serum of patients with rheumatoid arthritis are significantly higher than those in normal people. CUL4A is positively correlated with the occurrence and development of rheumatoid arthritis and can be used as a molecular marker for screening, diagnosis or prognosis evaluation of rheumatoid arthritis.

[0020] Knockdown of CUL4A in RA FLS has been shown to inhibit FLS activation and inflammatory responses; knockdown of CUL4A expression in K / BxN STA mice has also been shown to significantly reduce inflammation and alleviate symptoms. Therefore, targeting CUL4A may be an effective approach for treating rheumatoid arthritis. This application is of great significance for studying the role of CUL4A in rheumatoid arthritis, understanding the disease mechanisms, and developing new treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Figure 4 shows the expression of CUL4A in the synovial tissues of KRN serum-induced arthritis mice (KRN STA) and K / BxN serum-induced arthritis mice (K / BXN STA). Panel A shows the infiltration of inflammatory cells in the synovial tissues of KRN STA and K / BXN STA mice by HE staining. Scale bars: 100 μm (20×), 50 μm (63×). Panel B shows the expression of CUL4A in the synovial tissues of KRN STA and K / BXN STA mice by immunohistochemical staining. Scale bars: 100 μm (20×), 50 μm (63×).

[0022] Figure 2-Figure 4 The expression of CUL4A in synovial tissues of OA patients and RA patients. Figure 2 The left side shows HE staining to detect the infiltration of inflammatory cells in the synovial tissues of OA patients and RA patients, and the right side shows immunohistochemical staining to detect the expression of CUL4A in the synovial tissues of OA patients and RA patients. Scale bars: 100 μm (20×), 50 μm (63×). Figure 3 Immunofluorescence double staining (IF) was used to detect the expression of CUL4A and TNF-α in synovial tissues of OA patients and RA patients. Scale bars: 100 μm (20×), 50 μm (63×); Figure 4 Panel A shows Western blot analysis of CUL4A expression in synovial tissue from patients with OA and RA, and in FLS activated with TNF-α. Panel B shows qRT-PCR analysis of CUL4A mRNA expression in synovial tissue from patients with OA and RA, and in FLS activated with TNF-α. Panel C shows CUL4A expression in peripheral blood from healthy controls and patients with RA. Data represent a single replicate experiment with three samples per group and are expressed as mean ± SD. ** P<0.01.

[0023] Figure 5-Figure 6 The purpose of this study was to analyze the correlation between CUL4A mRNA expression in peripheral blood mononuclear cells (PBMC) of RA patients with newly diagnosed RA and RA disease activity and inflammatory factors. Figure 5 To investigate the correlation between CUL4A mRNA expression in PBMCs of newly diagnosed RA patients and RA disease activity; Figure 6 The correlation between CUL4A mRNA expression in PBMCs of RA patients and inflammatory factors and chemokines.

[0024] Figure 7-Figure 8 The experimental results show that knocking down CUL4A can inhibit the activation and inflammatory response of FLS. Figure 7 Middle A is a scratch assay to detect the migration ability of FLS after TNF-α activation by knocking down CUL4A, scale bar: 1000 μm; Figure 7Middle B is Transwell assay to detect the invasive ability of FLS after TNF-α activation by knocking down CUL4A, scale bar: 1000 μm; Figure 7 Middle C is Western blot analysis of the protein expression of matrix metalloproteinases in FLS after TNF-α activation by knocking down CUL4A; Figure 7 Middle D is qRT-PCR detection of the expression of matrix metalloproteinase mRNA in FLS after TNF-α activation by knocking down CUL4A; Figure 8 Middle A is Western blot analysis of the protein expression of inflammatory factors in FLS after TNF-α activation by knocking down CUL4A; Figure 8 Middle B is qRT-PCR detection of the expression of inflammatory factors and chemokine mRNA in FLS after TNF-α activation by knocking down CUL4A; Figure 8 Middle C shows the expression of inflammatory factors in FLS after TNF-α activation after CUL4A knockdown by ELISA. Data represent one replicate experiment with three samples per group and are expressed as mean ± SD. ** P<0.01, ## P<0.01.

[0025] Figures 9-11 The results of the in vivo study on the inflammatory response of Cul4a-shRNA to K / BxN serum-transferred arthritis mice. Figure 9 Figure A in the middle is a schematic diagram of mouse modeling; Figure 9 Middle B is a visual diagram of the swelling degree of the mouse ankle joint, X-ray, and representative micro-CT images, scale bar: 1.0 mm. Figure 10 Figure A in the middle shows the infiltration of inflammatory cells in the synovial tissue of mice in the Nc-shRNA group and the Cul4a-shRNA group detected by HE staining. Scale bars: 200 μm (10×), 50 μm (63×); Figure 10 Middle B shows the expression of Cul4a, Il-1β, and Il-6 in the synovial tissues of mice in the Nc-shRNA group and the Cul4a-shRNA group detected by immunohistochemistry. Scale bars: 100 μm (20×), 50 μm (63×); Figure 11 A in the middle is the joint score and swelling test; Figure 11 Middle B shows the effect of ELISA on the expression of inflammatory factors in the serum of K / BxN STA mice after knockdown of CUL4A; compared with the K / BxN STA Sh-Nc group: ** P<0.01. DETAILED DESCRIPTION

[0026] The technical solution of this application is described in more detail below with reference to experiments and drawings.

[0027] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0028] Introduction and full comparison:

[0029] OA: Osteoarthritis.

[0030] RA: Rheumatoid Arthritis.

[0031] CUL4A: Cullin 4A, Cullin protein family member 4A.

[0032] HE staining: Hematoxylin-Eosin staining.

[0033] IHC: Immunohistochemistry.

[0034] IF: Immunofluorescence.

[0035] IL-1β: Nterleukin-1β, interleukin 1β.

[0036] IL-6: Interleukin 6, interleukin 6.

[0037] IL-8: Interleukin 8, interleukin 8.

[0038] CCL8: Chemokine ligand 8.

[0039] CXCL3: CXC Motif Chemokine Ligand 3, CXC motif chemokine ligand 3.

[0040] CXCL5: CXC motif chemokine ligand 5, CXC motif chemokine ligand 5.

[0041] MMP2: matrix Metallopeptidase 2, matrix metalloproteinase-2.

[0042] MMP3: matrix Metallopeptidase 3, matrix metalloproteinase-3

[0043] MMP9: matrix metallopeptidase 9, matrix metalloproteinase-9.

[0044] TNF-α: tumor necrosis factor-α, tumor necrosis factor.

[0045] The KRN STA and K / BxN STA mouse models used in the examples of this application are serum-induced arthritis mouse models and can be used as RA animal models. With the consent of Professors Christophe Benoist and Diane Mathis of Harvard Medical School, KRN mice (transgenic mice carrying T cell receptors) were introduced for the construction of K / BxN mice. NOD mice (non-obese diabetic mice) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. KRN mice are selected and hybridized with NOD mice to obtain K / BxN mice. K / BxN mice can spontaneously develop arthritis 100% in 4-5 weeks. The serum of K / BxN mice was collected and stored at -80°C for the construction of K / BxN STA mice.

[0046] 1. Expression of CUL4A in synovial tissues of KRN STA and K / BxN STA mice

[0047] Establishment of the KRN-STA and K / BxN-STA mouse models: KRN mice were imported with the permission of Professors Christophe Benoist and Diane Mathis of Harvard Medical School. NOD mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and K / BxN mice were bred from the two. The KRN-STA and K / BxN-STA mouse models were induced by intraperitoneal injection of sera from KRN and K / BxN mice into naive C57 mice. The ankle joints were extracted and paraffin-embedded using conventional methods. Immunohistochemistry was used to evaluate the expression of CUL4A in the synovial tissue of the KRN-STA and K / BxN-STA mice.

[0048] HE staining results showed that K / BxN STA group mice showed obvious inflammatory cell infiltration; immunohistochemistry results showed that the positive staining degree of synovial tissue in K / BxN STA mice was significantly higher than that in KRN STA group mice, indicating that the expression of CUL4A in the synovial tissue of K / BxN STA mice was significantly higher than that of KRN STA mice. Figure 1 .

[0049] 2. Study on the difference of CUL4A expression in synovial tissues of OA patients and RA patients

[0050] Synovial tissues from patients with typical clinical presentations of OA and RA were collected from the Department of Rheumatology at the First Affiliated Hospital of Anhui Medical University. RNA was extracted using conventional methods, and qRT-PCR was used to evaluate CUL4A mRNA expression in synovial tissues from patients with OA and RA. Protein was extracted using conventional methods, paraffin-embedded, and Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence were used to evaluate CUL4A expression in synovial tissues from patients with OA and RA. RA FLS were isolated and cultured, induced with 10 ng / mL TNF-α, and then harvested for protein extraction. CUL4A expression was measured 48 hours after induction.

[0051] HE staining results showed that the infiltration of inflammatory cells in the synovial tissue of RA patients was significantly higher than that in the synovial tissue of RA patients. Immunohistochemistry results showed that the positive staining degree of synovial tissue of RA patients was significantly higher than that of OA patients, indicating that the expression of CUL4A in the synovial tissue of RA patients was significantly higher than that of OA patients. Figure 2 .

[0052] Immunofluorescence results showed that CUL4A was highly expressed in the synovial tissue of RA patients, and partially overlapped with the expression of TNF-α, showing the same trend. It was further found that TNF-α could promote the expression of CUL4A in synovial cells. Figure 3 .

[0053] Western blot results showed that the expression of CUL4A in the synovium of RA patients was significantly higher than that in OA patients, and the expression was significantly increased after TNF-α-induced activation. Figure 4 Figure A. Figure B shows the expression of CUL4A mRNA in the synovial tissues of OA patients and RA patients and in FLS after activation with TNF-α detected by qRT-PCR. Figure C shows the expression of CUL4A in the peripheral blood of normal subjects and RA patients. The qRT-PCR results showed that the expression of CUL4A mRNA in the synovial tissues of RA patients was significantly higher than that in OA patients, and the expression was significantly increased after TNF-α-induced stimulation and activation.

[0054] 3. Correlation between CUL4A mRNA expression levels in peripheral blood of newly diagnosed RA patients and clinical disease activity and synovial inflammation indicators.

[0055] Methods Peripheral blood samples were collected from patients with RA who were newly diagnosed in the Department of Rheumatology and Immunology at the First Affiliated Hospital of Anhui Medical University. Peripheral blood mononuclear cells (PBMCs) were extracted and the expression of CUL4A mRNA was analyzed by qRT-PCR to investigate its correlation with RA clinical disease activity (DAS28 score, ESR, CRP, RF, and CCP antibodies) and synovial inflammatory factors (TNF-α, IL-6, IL-1β, CCL3, and CCL8).

[0056] qRT-PCR results showed that the expression of CUL4A mRNA was positively correlated with DAS28, RF and PLT, and positively correlated with ESR and Anti-CCP, with statistical significance (see Table 1). Figure 5 Correlation analysis with RA synovial inflammatory factors showed that CUL4A mRNA expression was positively correlated with CCL8, IL-1β, IL-6, TNF-α, and CCL4, and there was a statistical difference. Figure 6 .

[0057] 4. In vitro study on the regulatory effect of targeting CUL4A on the activation response in FLS induced by TNF-α.

[0058] Synovial tissue from RA patients was collected, and RA FLS primary cells were extracted for culture. CUL4A-siRNA small interfering gene (F: GAGGGAGACAGAGUAAUCATT; R: UGAUUACUCUGUCUCCCUCTT) was constructed to knock down the expression of CUL4A in FLS. After 48 hours of induction activation with 10 ng / mL TNF-α (simulating the inflammatory microenvironment in RA patients), the migration of FLS after CUL4A knockdown was evaluated by scratch assay; the invasion of FLS after CUL4A knockdown was evaluated by transwell assay; total cell protein and RNA were extracted, and the expression of matrix metalloproteinases (MMP2, MMP3 and MMP9) in FLS after CUL4A knockdown was evaluated by Western blot and qRT-PCR experiments.

[0059] The results of the scratch assay showed that knockdown of CUL4A could significantly inhibit the migration ability of FLS. Figure 7 Middle A: Transwell assay results showed that knockdown of CUL4A could significantly inhibit the invasion ability of FLS. Figure 7 Middle B; Western blot results showed that knockdown of CUL4A could significantly inhibit the expression of MMP3 and MMP9 in FLS, see Figure 7 Middle C; qRT-PCR results showed that knockdown of CUL4A could significantly inhibit the expression of MMP2, MMP3 and MMP9 in FLS. Figure 7 Middle D.

[0060] 5. In vitro study on the regulatory effect of targeting CUL4A on inflammatory response in FLS induced by TNF-α.

[0061] Synovial tissues of RA patients were collected, and primary RA FLS cells were extracted for culture. CUL4A-siRNA small interfering knockdown of CUL4A expression in FLS was constructed. 10 ng / mL TNF-α was used to induce activation (simulating the inflammatory microenvironment in RA patients) for 48 hours. The cell supernatant was collected, and total cell protein and RNA were extracted. Western blot, qRT-PCR, and ELISA experiments were used to evaluate the expression of inflammatory factors (IL-Iβ, IL-6) in FLS.

[0062] Western blot results showed that knockdown of CUL4A could significantly inhibit the secretion of CUL4A, IL-1β and IL-6 in FLS. Figure 8 Middle A; qRT-PCR results showed that knockdown of CUL4A could significantly inhibit the secretion of CUL4A, inflammatory factors (IL-1β, IL-6 and IL-8) and chemokines (CCL8, CXCL3 and CXCL8) in FLS, see Figure 8 Middle B; ELISA results showed that knockdown of CUL4A could significantly inhibit the secretion of inflammatory factors (IL-Iβ and IL-6) of FLS in the cell supernatant, see Figure 8 Middle C.

[0063] 6. In vivo study on the regulatory effect of targeting CUL4A on the inflammatory response of arthritis mice.

[0064] See also Figure 9 Figure A, 8-week-old female C57BL / 6 mice were adaptively fed for one month and then orthotopically injected with CUL4A knockdown adenovirus into K / BxN STA mice. The mice were randomly divided into two groups, a control group (injected with Nc-shRNA adenovirus) and a model group (Cul4a-shRNA). Arthritis was induced by injection of K / BxN mouse serum on the 2nd and 4th days after adenovirus injection. The modeling lasted for 14 days. After the modeling, the ankle swelling of the mice was observed and photographed. The ankle swelling degree and joint swelling score of the mice were recorded every 2 days during the modeling period to reflect the inflammatory response of CUL4A knockdown in arthritis mice. The serum of the mice was collected during the modeling period, and the expression of inflammatory factors (Il-Iβ, Il-6, and Tnf-α) in the serum was evaluated by ELISA. The ankle joints of the mice were collected, and the effect of CUL4A knockdown on the inflammatory response of arthritis mice was evaluated by micro-CT, HE staining, and immunohistochemistry.

[0065] Joint swelling score: The forelimb score ranges from 0 to 3, with 0 for no swelling, 1 for mild redness and swelling, 2 for moderate redness and swelling, and 3 for severe redness and swelling or even deformity; the hindlimb score ranges from 0 to 4, with 0 for no swelling, 1 for redness without swelling, 2 for mild redness and swelling, 3 for moderate redness and swelling, and 4 for severe redness and swelling or even deformity.

[0066] Swelling Measurement: Vernier calipers were used to measure the diameter of the left and right hind paws of each group of mice 0.5 mm below the ankle joint. Swelling measurements were performed every two days starting on day 0 of the first immunization. Mice were considered ill if a clinical score of 1 or higher was observed in one paw.

[0067] The results of visual images, X-rays and micro-CT scans of mice showed that the ankle swelling and bone damage in the model group were significantly higher than those in the control group. Figure 9 Middle B

[0068] HE staining results showed that the inflammatory cell infiltration in the model group was significantly improved after knockdown of CUL4A. Figure 10 The line graph of ankle swelling and joint swelling score of mice and the results of ELISA experiments showed that knocking down CUL4A could significantly alleviate the inflammatory response of mice. Figure 11 Middle A; Immunohistochemistry results showed that the secretion of CUL4A, IL-1β and IL-6 in the model group mice was significantly reduced after knockdown of CUL4A. Figure 11 Middle B.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the CUL4A gene or CUL4A protein as a molecular marker for screening, diagnosis, or prognosis assessment of rheumatoid arthritis.

2. Use of the CUL4A gene or CUL4A protein in the preparation of products for screening, diagnosis or prognosis assessment of rheumatoid arthritis.

3. The use according to claim 2, characterized in that The products include reagents and kits.

4. A product for screening, diagnosis or prognosis assessment of rheumatoid arthritis, characterized in that: The product is a reagent for detecting the mRNA expression level of CUL4A and / or the protein expression level of CUL4A, or the product comprises a reagent for detecting the mRNA expression level of CUL4A and / or the protein expression level of CUL4A.

5. The product according to claim 4, characterized in that The object of detection is selected from at least one of the following: peripheral blood, peripheral serum, synovial cells, synovial tissue, bone tissue, rheumatoid arthritis bone tissue, CUL4A protein or its mRNA.

6. The product according to claim 5, characterized in that When the test results show that the CUL4A mRNA expression level and / or the CUL4A protein expression level are significantly higher than the preset threshold of the healthy control group, it indicates that the test subject has a risk of rheumatoid arthritis.

7. A method for treating rheumatoid arthritis, characterized in that: Inhibit the expression or activity of CUL4A gene in rheumatoid arthritis fibroblast-like synoviocytes or synovial tissues.

8. The method according to claim 7, wherein An inhibitory element targeting the CUL4A gene is introduced into fibroblast-like synoviocytes or synovial tissues, wherein the inhibitory element includes an RNA interference molecule targeting the CUL4A gene or the inhibitory element is a CRISPR gene editing system targeting the CUL4A gene.

9. The method according to claim 8, wherein The RNA interference molecule targeting the CUL4A gene is siRNA or shRNA.

10. The method according to claim 8, wherein The CRISPR gene editing system targeting the CUL4A gene includes a gRNA that specifically recognizes the CUL4A gene, a Cas9 protein bound to the gRNA, and a pharmaceutically acceptable delivery vector.

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