Circular RNA (Ribonucleic Acid) capable of being used as potential therapeutic target of chronic obstructive pulmonary disease

Abstract

The invention discloses a circular RNA (Ribonucleic Acid) capable of being used as a potential therapeutic target for chronic obstructive pulmonary disease, and relates to the technical field of biological medicines, tests prove that circular RNA circFCHO2 is remarkably and highly expressed in COPD patient tissues and mouse COPD models, so that circFCHO2 can be used as a basis for clinical diagnosis of COPD. According to the invention, circFCHO2 is further utilized to construct an adeno-associated virus AAV knock-down vector, and in-vivo verification finds that circFCHO2 can significantly improve lung function, emphysema and airway remodeling related indexes of a mouse COPD model. Therefore, circFCHO2 can be used as a novel target and a potential drug for clinical treatment of COPD, a new direction is provided for research and development of drugs for treatment of COPD, and the circFCHO2 has clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a circular RNA that can serve as a potential therapeutic target for COPD. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory system disease characterized by chronic respiratory symptoms and airflow limitation. GOLD 2024 defines COPD as a heterogeneous lung disease characterized by chronic respiratory symptoms, usually persistent and progressive, caused by abnormalities in the airways (such as bronchitis, bronchiolitis) or alveoli (such as emphysema).

[0003] Current diagnostic technologies suffer from significant lag; pulmonary function tests only show abnormalities after patients exhibit obvious clinical symptoms and irreversible lung tissue damage, hindering early diagnosis. By the time FEV1 / FVC abnormalities are detected, the patient's lung structure has often already undergone significant damage. Imaging examinations are expensive and involve radiation, making them unsuitable for large-scale screening. Furthermore, there is a lack of molecular markers that can be stably detected in blood and other bodily fluids for early COPD screening, classification, and prognosis. Despite advancements in molecular biology, some studies suggest a possible link between certain linear non-coding RNAs and COPD, research on the role of circular RNAs (circRNAs) in COPD, particularly their specific functional mechanisms and clinical translational value, remains insufficient. Many studies have shown that aberrant expression of circRNAs in various diseases plays a crucial role in gene regulation. Clinical treatments aim to relieve symptoms and control acute exacerbations. Bronchodilators (β2 receptor agonists, anticholinergics) are commonly used to relieve symptoms by relaxing airway smooth muscle but cannot reverse disease progression. Glucocorticoids are used to control inflammation, but their efficacy is limited for many patients, and long-term use has significant side effects. Currently, no therapy can halt or reverse the core pathological processes of emphysema and airway remodeling. Studies have demonstrated that circRNAs can treat and intervene in various diseases through targeted inhibition, exosome delivery, and small molecule drugs. Therefore, there is an urgent need for a circRNA that can serve as a potential therapeutic target for COPD to change this situation. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a circular RNA that can serve as a potential therapeutic target for COPD. Its advantage lies in the fact that circFCHO2 can serve as a novel target and potential drug for the clinical treatment of COPD, providing a new direction for the development of COPD therapeutic drugs and possessing clinical translational potential.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A circular RNA that can serve as a potential therapeutic target for COPD, wherein the circular RNA is used to detect the expression level of circular RNA circFCHO2 in biological samples.

[0006] Preferably, the circular RNA contains an active ingredient capable of reducing the expression level of the intracellular circular RNA circFCHO2.

[0007] Preferably, the nucleotide sequence of circFCHO2 is shown in SEQ ID NO:1.

[0008] Preferably, the circular RNA includes reagents for detecting circFCHO2 expression, the reagents including specific primers, probes, or gene chips.

[0009] Preferably, the biological sample is blood, serum, plasma, sputum, or lung tissue.

[0010] Preferably, the circular RNA is used to diagnose chronic obstructive pulmonary disease (COPD), including early screening, auxiliary diagnosis, or assessment of disease severity.

[0011] Preferably, the active ingredient comprises a nucleic acid molecule that targets the interfering sequence of circFCHO2, the nucleic acid molecule comprising smaller interfering RNA, short hairpin RNA, gRNA, antisense oligonucleotides, or DNA sequences encoding them.

[0012] Preferably, the circular RNA comprises a gene therapy vector that specifically targets the shRNA coding sequence of circFCHO2 and can be formulated for administration by nebulization, nasal instillation, or intravenous injection.

[0013] The beneficial effects of this invention are as follows: 1. circFCHO2 has a circular structure, making it less susceptible to degradation by RNases and more stable than linear RNA in clinical samples (such as blood and tissue fluid). This means that detection methods for circFCHO2 are more reliable, have lower requirements for sample preservation and transportation, and are more suitable for routine clinical testing. 2. A gene therapy vector that stably knocks down circFCHO2 was successfully constructed, and the therapeutic effects of targeting circFCHO2 were demonstrated in animals to be significant and multifaceted, improving the core pathological features of COPD (improving lung function, reducing emphysema, and inhibiting airway remodeling). By targeting key regulatory molecules upstream of the disease, a source intervention in the disease process was achieved, rather than merely alleviating symptoms. Attached Figure Description

[0014] Figure 1The nucleotide sequence of circFCHO2 is shown, with the nucleotides linked end-to-end to form a circular structure. Figure 2 The images show qPCR identification of circFCHO2 expression levels in human peripheral blood plasma and lung tissue (A, B) and in situ fluorescence hybridization identification (C). Figure 3 Image (A) showing the qPCR identification of circFCHO2 knockdown in human bronchial epithelial cells and the shRNA sequence (B). Figure 4 A flowchart of the method for establishing a mouse COPD model and the experimental procedure for intrabronchial injection of adeno-associated virus (AAV-sh-circFCHO2) (A), and micro-CT identification of the mouse COPD model in small animals (B). Figure 5 A fluorescence image showing the expression of adeno-associated virus (AAV-sh-circFCHO2) in mouse lung tissue (A), and a qPCR identification image showing the expression level of circFCHO2 in mouse lung tissue (B). Figure 6 A schematic diagram of mouse lung function testing (A) and a statistical chart of lung function-related test indicators (B); Figure 7 Histological sections of mouse lung tissue are shown in the staining diagram. Hematoxylin and eosin (H&E) staining quantifies emphysematous alveolar enlargement (A), and bar graphs show the mean linear intercept and disruption index in each group (B). Simultaneously, H&E staining quantifies the ratio of epithelial cell thickness to basement membrane perimeter in small airways (C, D). Masson's trichrome histochemical staining is used to assess the degree of collagen deposition around small airways in lung sections (E), and bar graphs show the quantitative ratio of collagenous regions around small airways to basement membrane perimeter (F). Detailed Implementation

[0015] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0016] The embodiments of this patent are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this patent, and should not be construed as limiting this patent.

[0017] Reference Figure 1-7 A circular RNA that can serve as a potential therapeutic target for COPD was discovered. The core of this study is to reveal the key role of circular RNA circFCHO2 in chronic obstructive pulmonary disease (COPD) and to systematically demonstrate its dual value as a clinical diagnostic biomarker and a therapeutic target.

[0018] The first part focuses on the identification and diagnostic value of circFCHO2. First, the specific nucleotide sequence of circFCHO2 was determined, confirming its covalently linked circular structure. This demonstrates that the molecule is a true circular RNA, not a splicing variant of nonlinear RNA. Subsequently, qPCR was used to precisely quantify and compare the expression levels of circFCHO2 in the lung tissues of COPD patients and normal controls. The results showed that the expression level of circFCHO2 in the COPD group was significantly higher than that in the normal group, preliminarily establishing its association with the disease. In situ fluorescence hybridization further confirmed the qPCR results. This indicates that circFCHO2 is specifically and highly expressed in the lung tissue of COPD patients; therefore, detecting its expression level can serve as a molecular basis for diagnosing COPD.

[0019] The second part focuses on the development and validation of a therapeutic tool targeting circFCHO2. First, a specific shRNA sequence targeting circFCHO2 was designed. The effectiveness of gene silencing was validated; after introducing the shRNA into human bronchial epithelial cells, qPCR detection revealed a significant decrease in circFCHO2 expression levels. This demonstrated the effectiveness and specificity of the shRNA sequence, laying the foundation for subsequent in vivo therapeutic experiments. In in vivo experiments, a mouse COPD model was successfully established using cigarette smoke exposure, and the model was confirmed by non-invasive observation of changes in the lung-to-chest ratio using micro-CT. Two months after model establishment, an adeno-associated virus (AAV) vector carrying the shRNA was delivered to the mouse lungs via intrabronchial injection. Fluorescence imaging directly observed a strong fluorescent signal in the mouse lung tissue after injection of AAV carrying the reporter gene, demonstrating that the AAV vector could efficiently infect and stably express in mouse lung tissue. qPCR detection confirmed that the expression level of circFCHO2 in the lung tissue of mice treated with AAV-sh-circFCHO2 was successfully knocked down. This indicates that the therapeutic tool is also effective in vivo.

[0020] Part III validated the therapeutic effect of circFCHO2 on COPD. Pulmonary function indicators in mice were assessed using a pulmonary function analyzer. Compared to normal mice, COPD model mice showed significantly worse pulmonary function indicators, which significantly improved after AAV-sh-circFCHO2 treatment. The treatment effect was then evaluated using pathological indicators. H&E staining revealed alveolar structural destruction and fusion expansion in COPD model mice (typical features of emphysema). Quantitative analysis showed a significant increase in the mean linear intercept and destruction index. After treatment, these indicators significantly decreased, indicating that alveolar structural destruction was effectively inhibited. H&E staining also revealed hyperplasia and increased thickness of small airway wall epithelial cells in COPD model mice. Quantitative analysis showed a significantly increased epithelial thickness / basement membrane perimeter ratio. After treatment, this ratio decreased, demonstrating that pathological airway remodeling was alleviated. Masson staining stained collagen fibers blue, visually revealing abundant blue collagen deposition around the small airways in COPD model mice (airway fibrosis). Quantitative analysis showed a significantly increased collagen area / basement membrane perimeter ratio. The ratio decreased significantly after treatment, demonstrating that targeted knockdown of circFCHO2 can reduce peri-airway collagen deposition and alleviate pathological airway remodeling.

[0021] This invention provides a product concept for the diagnosis and treatment of COPD. The core of this concept is to diagnose COPD by measuring the expression level of circFCHO2 in biological samples, and to treat COPD through gene therapy by delivering an active ingredient that reduces intracellular circFCHO2 expression levels. Details are as follows: Products for diagnosing COPD 1. Sample Collection and Pretreatment: Biological sample types include non-invasive samples and tissue samples. Non-invasive samples include peripheral blood (which can be further separated into serum or plasma) and sputum. Tissue samples are lung biopsy tissue; this sample is more invasive, but the results are the most direct and accurate, especially when obtained during interventional procedures or surgeries. Samples are preserved in a preservation solution containing RNase inhibitors to prevent RNA degradation.

[0022] 2. RNA Extraction: Total RNA was extracted from the above samples using commercially available RNA extraction kits (such as the TRIzol method or centrifugation column method). To improve the extraction efficiency of circular RNA, a DNase I digestion step can be added to the process to completely remove interference from genomic DNA.

[0023] 3. Expression Level Detection: To specifically detect circular RNA rather than parental linear mRNA, primers containing a backsplicing site need to be designed. Based on the intermolecular junction sequence of circFCHO2, a cross-junction "divergent primer" is designed. This type of primer cannot effectively amplify linear RNA, thus achieving specific amplification and quantification of circFCHO2. The extracted RNA is reverse transcribed into cDNA, and qPCR amplification is performed using the aforementioned specific primers. The expression level of circFCHO2 is quantified by the CT value. The 2^(-ΔΔCT) method is typically used for calculation, with the relative circFCHO2 expression level standardized using internal reference genes (such as GAPDH, β-actin).

[0024] Products for preventing / treating COPD 1. Design and preparation of active ingredients: Nucleic acid molecules capable of mediating RNA interference were designed, and highly efficient and specific shRNA sequences were designed and screened targeting the circular region of circFCHO2. Active ingredients are not limited to shRNA, but also include siRNA, antisense oligonucleotides, gRNA, etc.

[0025] 2. Construction of the delivery system: Adeno-associated virus (AAV) was used as the delivery vector. The AAV serotype AAV6, which exhibits high tropism for lung tissue, was selected. The selected high-efficiency shRNA coding sequence was cloned into an AAV expression plasmid containing a promoter suitable for expression in lung cells. Recombinant AAV virus particles were packaged and produced in HEK293 cells using a three-plasmid co-transfection system. The virus was then purified and concentrated using ultracentrifugation and column chromatography. The purified AAV virus particles were mixed with sterile phosphate-buffered saline (PBRS) to prepare a drug formulation. In mice, the viral solution was directly instilled into the lungs via endotracheal intubation. For future clinical translation, the AAV virus formulation can be formulated as an aerosol for nebulization. For patients who cannot undergo nebulization, nasal instillation or intravenous injection can be used.

[0026] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A circular RNA that can serve as a potential therapeutic target for COPD, characterized in that, The circular RNA is used to detect the expression level of circular RNA circFCHO2 in biological samples.

2. The circular RNA that can serve as a potential therapeutic target for COPD according to claim 1, characterized in that, The circular RNA contains an active ingredient that can reduce the expression level of the intracellular circular RNA circFCHO2.

3. The circular RNA that can serve as a potential therapeutic target for COPD according to claim 2, characterized in that, The nucleotide sequence of circFCHO2 is shown in SEQ ID NO:

1.

4. A circular RNA that can serve as a potential therapeutic target for COPD according to claim 3, characterized in that, The circular RNA includes reagents for detecting circFCHO2 expression, including specific primers, probes, or gene chips.

5. A circular RNA that can serve as a potential therapeutic target for COPD according to claim 1, characterized in that, The biological sample may be blood, serum, plasma, sputum, or lung tissue.

6. A circular RNA that can serve as a potential therapeutic target for COPD according to claim 1, characterized in that, The circular RNA is used to diagnose chronic obstructive pulmonary disease (COPD), including early screening, auxiliary diagnosis, or assessment of disease severity.

7. A circular RNA that can serve as a potential therapeutic target for COPD according to claim 2, characterized in that, The active ingredient comprises a nucleic acid molecule that targets the interfering sequence of circFCHO2, including smaller interfering RNA, short hairpin RNA, gRNA, antisense oligonucleotides, or DNA sequences encoding them.

8. A circular RNA that can serve as a potential therapeutic target for COPD according to claim 7, characterized in that, The circular RNA contains a gene therapy vector that specifically targets the shRNA coding sequence of circFCHO2 and can be formulated for administration via nebulization, nasal drops, or intravenous injection.

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