RNA medicine for treating kidney cancer, self-assembly delivery system and application of self-assembly delivery system

Abstract

The invention provides an RNA (Ribonucleic Acid) medicine for treating kidney cancer, which comprises a medicine siRNA capable of treating in-situ tumors of the kidney cancer, the nucleotide sequence of the medicine siRNA is shown as SEQ ID No.2, and / or a medicine siRNA capable of treating metastatic tumors of the kidney cancer, and the nucleotide sequence of the medicine siRNA is shown as SEQ ID No.4. The invention also provides an RNA self-assembly delivery system containing the RNA medicine for treating kidney cancer, and stable, efficient and safe production and transmission of siRNA in vivo are realized by utilizing a strategy of self-assembling siRNA in vivo. The RNA drug and the RNA self-assembly delivery system for treating kidney cancer can treat advanced kidney cancer and metastatic kidney cancer, and have a good clinical treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to RNA drugs for treating renal cell carcinoma, self-assembly delivery systems, and their applications. Background Technology

[0002] Renal cell carcinoma (RCC), also known as kidney cancer, is one of the ten most common malignant tumors, accounting for 4% of all adult malignant tumors. Worldwide, more than 250,000 new cases of kidney cancer are diagnosed each year. 1 More than 140,000 people die from kidney cancer each year. The incidence of renal cell carcinoma varies globally, with higher rates in developed countries than in developing countries. It is predominantly male, with a male-to-female ratio of 1.5:1, and peaks in age between 60 and 70 years. 2 .

[0003] Clear cell RCC (CCRCC) is the most common type of RCC (accounting for approximately 75% of all cases). 3 In addition, there are papillary RCC (pRCC) and chromophilic RCC (chRCC). Because clear cell renal cell carcinoma is often only discovered at an advanced stage, ccRCC has the worst disease-specific survival rate.

[0004] Renal cell carcinoma is clinically classified into four stages based on its development within the kidney, local spread, lymph node involvement, and metastatic spread. Intrarenal tumors are classified as stage I; stage II is when the intrarenal tumor extends to Gerota's fascia; stage III is when the tumor spreads beyond Gerota's fascia or extends to the inferior vena cava; and stage IV is when the tumor has metastasized to distant sites. 4 .

[0005] It has been reported that approximately 20-30% of patients with renal cell carcinoma (RCC) have metastases at diagnosis, with the lungs being the most common site of distant metastasis. Nearly one-third of patients with localized renal cell carcinoma who undergo radical nephrectomy develop metastases. Among patients with metastatic RCC, 45-76% have lung metastases. 5 Furthermore, the prognosis for patients with metastasis is uncertain, with a 2-year survival rate of only 10%-30%. 6 Therefore, advanced and metastatic renal cell carcinoma is the leading cause of death among patients with renal cell carcinoma.

[0006] Treatment for early-stage renal cell carcinoma primarily relies on surgery; partial nephrectomy for small tumors and radical nephrectomy for large tumors remain the gold standard treatments. 7 Furthermore, the 5-year survival rates after nephrectomy for stage I and stage II renal cell carcinoma were 81% and 74%, respectively. 8 It can achieve a fairly good curative effect.

[0007] However, for patients with advanced renal cell carcinoma (stages III and IV) and those with metastatic renal cell carcinoma, surgery carries a high risk and a high recurrence rate. 9 Direct surgery is not suitable for these conditions. Therefore, in clinical practice, for patients with locally advanced or large renal cell carcinoma, neoadjuvant therapy is first adopted, mainly through drug therapy or radiotherapy to shrink the tumor size, thereby improving the feasibility and efficacy of surgery; then radical surgery is performed; finally, to achieve better prognosis, targeted drugs or immunotherapies are used for adjuvant therapy. 10 This is done to inhibit tumor metastasis and recurrence. Clinically, for patients with advanced metastatic renal cell carcinoma, where the tumor has spread to other organs or lymph nodes, the curative effect of surgery is low. In this case, other treatment methods such as chemotherapy, targeted therapy, or immunotherapy may be more suitable. However, advanced metastatic renal cell carcinoma exhibits significant resistance to conventional chemotherapy and radiotherapy. 11 Therefore, whether it is neoadjuvant or adjuvant therapy after radical nephrectomy for nephrotic cancer or the treatment of advanced metastatic renal cell carcinoma, the main strategy is still drug therapy.

[0008] Drug therapy and adjuvant therapy for advanced renal cell carcinoma have seen significant development in the past few decades. (1) Cytokine therapy, prior to the era of targeted therapy, using interleukin-2 12 and interferon 13 Cytokine therapy is the standard treatment for advanced renal cell carcinoma. These cytokines can activate the patient's own immune system to fight the tumor. However, its widespread use in clinical practice is limited by limited treatment response and unpredictable side effects. (2) Targeted therapy: With the development of drug therapy for renal cell carcinoma, small molecule targeted drugs have also begun to be used in clinical research. Drugs targeting the VEGF / PDGFR / mTOR pathway are the mainstream treatment for advanced metastatic renal cell carcinoma. 14 Anti-vascular endothelial growth factor (VEGF) drugs and mammalian rapamycin target (mTOR) inhibitors are standard treatment options for patients with metastatic renal cell carcinoma. 15 Current treatment guidelines for metastatic renal cell carcinoma recommend first-line VEGF inhibitors, including sunitinib, with everolimus used for progression. While improved target specificity reduces the risk of toxicities associated with these drugs, durable complete remission remains an exception, and moreover, targeted therapy often leads to disease stabilization rather than substantial reduction in tumor size. 16 In addition, an increasing number of studies have reported adverse reactions to small molecule targeted drugs, such as fatigue, hypertension, nausea, diarrhea, speech disorders, palmoplantar erythematous sensory disturbances, pneumonia, hepatitis, and cardiotoxicity. 17 (3) Immunotherapy, including anti-PD-1 antibodies and anti-PD-L1 antibodies, activates the patient's own immune system to fight tumors by breaking the mechanism by which tumor cells escape immune surveillance.18 These drugs have achieved significant results in improving the survival rate and quality of life of patients with advanced metastatic renal cell carcinoma. However, immunotherapy has also led to serious adverse reactions such as autoimmune reactions, immune-related toxicity, fatigue, and neurotoxicity, which affect the treatment effect. (4) Combination therapy has also begun to be applied to the treatment of advanced metastatic renal cell carcinoma. 19-21 This includes the combined use of targeted therapies, which further improves patient response and survival. However, the combined use of drugs increases the risk of drug interactions and metabolic burden, and may also generate new adverse reactions on top of the side effects of individual drugs, threatening patient safety.

[0009] Therefore, it is crucial and urgent to develop drugs that can both prolong the survival of patients with advanced metastatic renal cell carcinoma and those who have undergone radical surgery, while also being safe, reliable, and having few adverse reactions.

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[0011] (2) Levi, F.; Ferlay, J.; Galeone, C.; Lucchini, F.; Negri, E.; Boyle, P.; LaVecchia, C. The changing pattern of kidney cancer incidence and mortality in Europe. BJU International 2008,101(8),949-958.DOI:10.1111 / j.1464-410X.2008.07451.x.

[0012] (3)Chadwick,B.P.;Ricketts,C.J.;Linehan,W.M.Gender Specific MutationIncidenceand Survival Associations in Clear Cell Renal Cell Carcinoma(CCRCC).Plos One 2015,10(10).DOI:10.1371 / journal.pone.0140257.

[0013] (4)Delahunt,B.;Eble,J.N.;Samaratunga,H.;Thunders,M.;Yaxley,J.W.;Egevad,L.Staging of renal cell carcinoma:current progress and potentialadvances.Pathology 2021,53(1),120-128.DOI:10.1016 / j.pathol.2020.08.007.

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[0019] (11)Martinez Chanza,N.;Tripathi,A.;Harshman,L.C.Adjuvant TherapyOptions inRenal Cell Carcinoma:Where Do We Stand?Current Treatment Options inOncology 2019,20(5).DOI:10.1007 / s11864-019-0639-0.

[0020] (12)Dibajnia,P.;Cardenas,L.M.;Lalani,A.-K.A.The emerging landscapeofneo / adjuvant immunotherapy in renal cell carcinoma.Human Vaccines&Immunotherapeutics 2023,19(1).DOI:10.1080 / 21645515.2023.2178217.

[0021] (13)Sun,M.;Abdollah,F.;Bianchi,M.;Trinh,Q.-D.;Jeldres,C.;Thuret,R.;Tian,Z.;Shariat,S.F.;Montorsi,F.;Perrotte,P.;et al.Treatment Management ofSmall Renal Massesin the 21st Century:AParadigm Shift.Annals of SurgicalOncology 2012,19(7),2380-2387.DOI:10.1245 / s10434-012-2247-0.

[0022] (14)Klapper,J.A.;Downey,S.G.;Smith,F.O.;Yang,J.C.;Hughes,M.S.;Kammula,U.S.;Sherry,R.M.;Royal,R.E.;Steinberg,S.M.;Rosenberg,S.High-doseinterleukin-2for the treatment of metastatic renal cell carcinoma.Cancer2008,113(2),293-301.DOI:10.1002 / cncr.23552.

[0023] (15)Christina Canil 1,S.H.,Linda A Mayhew,Tricia S Waldron,EricWinquist.Interferon-alfa in the treatment of patients with inoperable locallyadvanced or metastaticrenal cell carcinoma:a systematic review.2010 Jun,4(3),201-208.DOI:doi:10.5489 / cuaj.853.

[0024] (16)Makhov,P.;Joshi,S.;Ghatalia,P.;Kutikov,A.;Uzzo,R.G.;Kolenko,V.M.Resistance to Systemic Therapies in Clear Cell Renal Cell Carcinoma:Mechanisms andManagement Strategies.Molecular Cancer Therapeutics 2018,17(7),1355-1364.DOI:10.1158 / 1535-7163.Mct-17-1299.

[0025] (17)Bex,A.New options in treatment of advanced renal cancer.TheLancet Oncology2016,17(7),850-852.DOI:10.1016 / s1470-2045(16)30175-9.

[0026] (18)Diederich,S.Imaging beyond RECIST:CT and MRI in moleculartherapies.CancerImaging 2012,12(2),347-350.DOI:10.1102 / 1470-7330.2012.9013.

[0027] (19)Ruiz,J.N.;Belum,V.R.;Creel,P.;Cohn,A.;Ewer,M.;Lacouture,M.E.CurrentPractices in the Management of Adverse Events Associated WithTargeted Therapies forAdvanced Renal Cell Carcinoma:A National Survey ofOncologists.Clinical GenitourinaryCancer 2014,12(5),341-347.DOI:10.1016 / j.clgc.2014.04.001.

[0028] (20)Braun, DA; Bakouny, Z.; Hirsch, L.; Flippot, R.; Van Allen, EM; Wu, CJ; Choueiri, TKBeyond conventional immune-checkpoint inhibition—novel immunotherapies for renal cell carcinoma. Nature Reviews Clinical Oncology2021,18(4),199-214.DOI:10.1038 / s41571-020-00455-z.

[0029] (21)Kuusk, T.; Albiges, L.; Escudier, B.; Grivas, N.; Haanen, J.; Powles, T.; Bex, A. Antiangiogenic therapy combined with immune checkpoint blockade inrenal cancer.Angiogenesis 2017,20(2),205-215.DOI:10.1007 / s10456-017-9550-0. Summary of the Invention

[0030] To address the shortcomings of existing technologies and current treatments for advanced renal cell carcinoma, we propose a drug targeting and inhibiting VEGFR2 and mTOR, along with a multi-target delivery system. These findings have been validated using models of advanced, highly metastatic clear cell renal cell carcinoma and a radical resection model of clear cell renal cell carcinoma. This invention overcomes the safety and efficacy issues associated with existing marketed drugs.

[0031] A first aspect of the present invention provides an RNA drug for treating renal cell carcinoma, the drug comprising a drug capable of treating in situ tumors of renal cell carcinoma and / or a drug capable of treating metastatic tumors of renal cell carcinoma; wherein the drug capable of treating in situ tumors of renal cell carcinoma is a double-stranded RNA, siRNA, or shRNA, the double-stranded RNA, siRNA, or shRNA containing a nucleotide sequence capable of hybridizing with the mTOR gene under stringent conditions, and the target sequence in the corresponding mTOR gene to which the double-stranded RNA, siRNA, or shRNA hybridizes with the mTOR gene is as shown in SEQ ID No. 1; the drug capable of treating metastatic tumors of renal cell carcinoma is a double-stranded RNA, siRNA, or shRNA, the double-stranded RNA, siRNA, or shRNA containing a nucleotide sequence capable of hybridizing with the VEGFR2 gene under stringent conditions, and the target sequence in the corresponding VEGFR2 gene to which the double-stranded RNA, siRNA, or shRNA hybridizes with the VEGFR2 gene is as shown in SEQ ID No. 3.

[0032] Renal cell carcinoma, also known as kidney cancer, is a highly malignant tumor of the urinary system and one of the most common tumors. It is a malignant tumor that originates from the epithelial system of the renal parenchyma and urinary tubules. It is also known as renal adenocarcinoma and accounts for 80% to 90% of malignant kidney tumors, of which clear cell renal cell carcinoma accounts for 85%.

[0033] The target sequence in the corresponding mTOR gene of the hybridization can have a nucleotide sequence with ≥75% homology to the nucleotide sequence shown in SEQ ID No. 1, specifically, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0034] In molecular evolution studies, homology generally refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules.

[0035] The target sequence in the corresponding VEGFR2 gene of the hybridization can have a nucleotide sequence with ≥75% homology to the nucleotide sequence shown in SEQ ID No. 3, specifically, it can have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0036] As some embodiments of the present invention, the drug capable of treating in situ tumors of renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 2; the drug capable of treating metastatic tumors of renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0037] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length, with many different uses in biology. siRNA is known to primarily participate in RNA interference (RNAi) to regulate gene expression in a specific manner.

[0038] The drug siRNA capable of treating in situ tumors of renal cell carcinoma can have a nucleotide sequence with ≥75% homology to the nucleotide sequence shown in SEQ ID No. 2, specifically having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0039] The drug siRNA capable of treating metastatic tumors of renal cell carcinoma can have a nucleotide sequence with ≥75% homology to the nucleotide sequence shown in SEQ ID No. 4, specifically having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0040] As some embodiments of the present invention, the siRNA capable of treating in situ tumors of renal cell carcinoma and the siRNA capable of treating metastatic tumors of renal cell carcinoma have nucleotide sequences that exist in the same expression vector or in different expression vectors.

[0041] Expression vectors are vectors that add expression elements (such as promoters, RBS, terminators, etc.) to the basic skeleton of cloning vectors, enabling the target gene to be expressed.

[0042] As some embodiments of the present invention, the nucleotide sequence of the siRNA capable of treating in situ tumors of renal cell carcinoma is located in the miR-155 backbone following the CMV promoter, and / or the nucleotide sequence of the siRNA capable of treating metastatic tumors of renal cell carcinoma is located in the miR-155 backbone following the CMV promoter.

[0043] In some embodiments of the present invention, two siRNAs are expressed in tandem.

[0044] Gene tandem expression refers to linking multiple genes together in a specific order to form a genome in order to achieve a specific function, and it can achieve the simultaneous expression of multiple genes.

[0045] A second aspect of the invention is to provide a self-assembly delivery system for RNA used to treat renal cell carcinoma, the self-assembly delivery system comprising the aforementioned drug for treating renal cell carcinoma.

[0046] The self-assembly delivery system refers to the strategy of using in vivo self-assembly of siRNA to reconstruct the liver into an organ capable of producing and secreting siRNA. Then, by utilizing the body's own exosome secretion and transport pathways, the system can achieve stable, efficient, and safe production and delivery of siRNA in vivo, thus avoiding the bottleneck problem of in vivo siRNA delivery.

[0047] As some embodiments of the present invention, the self-assembly delivery system for the RNA used to treat renal cell carcinoma is an extracellular vesicle.

[0048] Extracellular vesicles are a collective term for various membrane-bound vesicle structures released by cells, and are widely distributed in bodily fluids such as blood, saliva, and urine. Extracellular vesicles include different subgroups such as exosomes, microvesicles, and apoptotic bodies.

[0049] As some embodiments of the present invention, the self-assembly delivery system for the RNA used to treat renal cell carcinoma is an exosome.

[0050] Exosomes are nanoscale lipid-containing structures with a diameter of 30–100 nm, containing various proteins, lipids, DNA, RNA, and even viral / prion biomolecules. Released by cells, exosomes spread in bodily fluids such as blood, and can then be phagocytosed by other cells, serving as important mediators of intercellular communication.

[0051] A third aspect of the present invention provides a pharmaceutical composition comprising a drug capable of treating an in situ tumor of clear cell renal cell carcinoma and a drug capable of treating a metastatic tumor of clear cell renal cell carcinoma; wherein the drug capable of treating the in situ tumor of clear cell renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 2; and the drug capable of treating a metastatic tumor of clear cell renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0052] A pharmaceutical composition refers to a complex consisting of two or more drugs or active molecules that work together or have a synergistic effect to achieve the purpose of treating a disease or improving health.

[0053] A fourth aspect of the present invention provides a pharmaceutical combination comprising a drug capable of treating an in situ tumor of clear cell renal cell carcinoma and a drug capable of treating a metastatic tumor of clear cell renal cell carcinoma; wherein the drug capable of treating the in situ tumor of clear cell renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 2; and the drug capable of treating a metastatic tumor of clear cell renal cell carcinoma is siRNA, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0054] Drug combination therapy refers to the combined use of different drugs to achieve better results. It is a common treatment strategy that can improve treatment efficacy and reduce side effects and adverse reactions. Drug combination therapy is influenced by factors such as drug interactions, synergistic effects, complementary effects, and contraindications, as well as individual patient differences.

[0055] A fifth aspect of the invention provides the aforementioned medicament for treating renal cell carcinoma, the aforementioned self-assembly delivery system for renal cell carcinoma RNA, or the use of the aforementioned pharmaceutical composition or combination in the preparation of a medicament for treating renal cell carcinoma, wherein the renal cell carcinoma is clear cell renal cell carcinoma.

[0056] As some embodiments of the present invention, the renal cell carcinoma is advanced renal cell carcinoma and metastatic renal cell carcinoma.

[0057] The present invention also provides a nucleic acid molecule comprising: a. a double-stranded RNA, siRNA, or shRNA; b. the double-stranded RNA, siRNA, or shRNA containing a nucleotide sequence capable of hybridizing with the mTOR gene under stringent conditions, wherein the target sequence in the corresponding mTOR gene to which the double-stranded RNA, siRNA, or shRNA hybridizes with the mTOR gene is shown in SEQ ID No. 1.

[0058] As some embodiments of the present invention, the nucleotide sequence of the siRNA is shown in SEQ ID No. 2.

[0059] The present invention also provides a nucleic acid molecule comprising: a. a double-stranded RNA, siRNA, or shRNA; b. the double-stranded RNA, siRNA, or shRNA containing a nucleotide sequence capable of hybridizing with the VEGFR2 gene under stringent conditions, wherein the double-stranded RNA, siRNA, or shRNA corresponds to a target sequence in the VEGFR2 gene as shown in SEQ ID No. 3.

[0060] As some embodiments of the present invention, the nucleotide sequence of the siRNA is shown in SEQ ID No. 4.

[0061] As described above, the RNA drug for treating renal cell carcinoma, the self-assembly delivery system, and their applications of the present invention have the following advantages:

[0062] Beneficial effects:

[0063] The VEGFR2 siRNA self-assembly delivery system of this invention effectively inhibits the occurrence of distant lung metastases, and the mTORsiRNA self-assembly delivery system effectively inhibits the growth of in situ clear cell renal tumors. The VEGFR2 siRNA and mTORsiRNA tandem delivery system simultaneously inhibits the growth of in situ tumors and the occurrence of distant lung metastases in advanced highly metastatic clear cell renal tumor models and radical resection models of clear cell renal tumors, demonstrating a very good therapeutic effect. Compared with the combination of targeted drugs (the first-line clinical drug sunitinib and the second-line clinical drug everolimus), the therapeutic effect is slightly better or comparable.

[0064] The VEGFR2 siRNA and mTOR siRNA tandem delivery system of this invention offers enhanced safety. It leverages the unparalleled advantages of in vivo self-assembled nucleic acid drug delivery systems, including high efficacy, multi-target therapy, low toxicity, and non-immunogenicity. Furthermore, this invention can be combined with fermentation processes, significantly reducing the production cost of targeted drugs. Attached Figure Description

[0065] Figure 1 This is a schematic diagram illustrating the construction of a mouse model of advanced highly metastatic clear cell renal cell carcinoma according to the present invention.

[0066] Figure 2 In vivo imaging can be used to detect the growth of tumors in situ, as well as the organ sites and time of distant metastasis in advanced renal cell carcinoma. Figure 2 a represents the result of in vivo imaging. Figure 2 Figure b shows the quantitative statistics of the in vivo imaging results;

[0067] Figure 3 The generation of metastatic lesions in a model of advanced, highly metastatic clear cell renal cell carcinoma was determined by hematoxylin and eosin staining of tissue layers. Figure 3 a represents the HE result of the in situ tumor. Figure 3 b represents the HE result of the metastatic foci;

[0068] Figure 4 The diagram shows the construction of a mouse model of radical resection of clear cell renal cell carcinoma according to the present invention;

[0069] Figure 5 In vivo imaging is used to detect the growth of the in situ tumor before surgery and to investigate the occurrence of distant metastases after radical surgery. Figure 5 a represents the result of in vivo imaging. Figure 5 b represents the quantitative statistics of the in vivo imaging results;

[0070] Figure 6 The occurrence of metastatic lesions after radical surgery was determined by hematoxylin and eosin staining of tissue layers. Figure 6 a represents the HE result of the in situ tumor. Figure 6 b represents the HE result of the metastatic foci;

[0071] Figure 7 The diagram shown illustrates the construction of the in vivo self-assembly delivery system of this invention. Figure 7 a is a gene loop sequence map of VEGFR2 siRNA constructed into the miR-155 backbone with CMV as the promoter. Figure 7 b is the gene loop sequence map of mTOR siRNA constructed into the miR-155 backbone with CMV as the promoter;

[0072] Figure 8 This is shown as an in vitro screening of an in vivo self-assembly delivery system. Figure 8 a) Detection of 3 CMV-siRs via qPCR experiment. v The effect of gene loops on VEGFR2 expression in EOMA cells at the transcriptomic level; Figure 8 bc detected three CMV-siRs using Western blotting and quantitative statistical analysis. v The effect of gene loops on VEGFR2 protein expression in EOMA cells at the transcriptome level, and the selection of optimal CMV-siR. v Gene loops. Figure 8 d represents the detection of three CMV-siRs via qPCR. m The effect of gene loops on the inhibition of mTOR expression in Renca cells at the transcriptomic level; Figure 8 ef detected three CMV-siRs using western blot and quantitative statistical analysis. m The effect of gene loops on mTOR protein expression in Renca cells at the transcriptome level, and selection of the optimal CMV-siR. m Gene loops;

[0073] Figure 9 This demonstrates the in vivo efficacy validation of an in vivo self-assembly delivery system. Figure 9 a. Detection of CMV-siR by mouse in vivo imaging v The inhibitory effect of gene loops on in situ tumors before radical surgery; Figure 9 b. Detection of CMV-siR by in vivo lung imaging in mice v The impact of in vivo self-assembly delivery systems on distant metastases after radical surgery. Figure 9 c detects CMV-siR m The effect of gene loops on downstaging of in situ tumors before radical surgery; Figure 9 d. Detection of CMV-siR by in vivo imaging of mouse lungs m Interference of in vivo self-assembly delivery systems with distant metastases after radical surgery;

[0074] Figure 10 This demonstrates the construction of in vivo self-assembled tandem gene loops and the verification of their in vitro function. Figure 10 a is CMV-siR m+v Schematic diagram of tandem gene loop construction; Figure 10 bc used RT-qPCR to detect the effects of tandem gene loops on VEGFR2 mRNA expression in EOMA cells and on mTOR mRNA expression in Renca cells, respectively. Figure 10 dg used Western blot and quantitative statistical analysis to detect the effect of tandem gene loops on the expression of VEGFR2 and mTOR proteins; Figure 10 hi represents the effect of tandem gene loops on the proliferation of EOMA and Renca cells;

[0075] Figure 11 The in vivo therapeutic effect is shown as an in vivo self-assembled tandem gene loop; Figure 11 ab demonstrated CMV-siR through in situ in vivo imaging of mice and lung in vivo imaging results. m+v The therapeutic effect of tandem gene loops on a model of advanced highly metastatic clear cell renal cell carcinoma; Figure 11 cd demonstrated CMV-siR through preoperative and postoperative in vivo imaging results of radical surgery. m+v The therapeutic effect of tandem gene loops on a model of radical resection of clear cell renal cell carcinoma;

[0076] Figure 12 The in vivo safety assessment of the self-assembled tandem gene loop is shown. Figure 12 a represents the pathological section of liver tissue stained with hematoxylin and eosin (HE). Figure 12 b represents the detection of serum aspartate aminotransferase levels; Figure 12 c represents the detection of serum alanine aminotransferase levels; Figure 12d represents the detection of alkaline phospholipase levels in serum;

[0077] Figure 13 This study demonstrated the safety profile of in vivo self-assembled tandem gene loops compared to existing technologies, specifically sunitinib monotherapy and sunitinib in combination with everolimus. Figure 13 Image a shows an image of immune cell infiltration in mouse liver tissue as indicated by HE (hematologic resection) results, illustrating whether liver inflammation is present. Figure 13 The results of serum total cholesterol, triglycerides, and free fatty acids were analyzed to show that lipid metabolism abnormalities occurred with the use of everolimus alone and in combination with sunitinib, while the VEGFR2 siRNA and mTOR siRNA tandem delivery systems did not show this side effect. Detailed Implementation

[0078] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0079] [Experimental Materials]

[0080] Renca cells were purchased from Pronosei, catalog number CL-0568.

[0081] Balb / c mice were purchased from the Model Animal Research Center of Nanjing University.

[0082] The Matrigel Matrix was purchased from Corning, item number: 354248.

[0083] Sumu was purchased from YEASEN, 60502ES60, Shanghai, China.

[0084] Erythromycin was purchased from Seville Biotechnology, product code G1001-100ML.

[0085] The liveness detection system was purchased from IVIS Lumina XR System.

[0086] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0087] Example 1: Construction of a mouse model of advanced highly metastatic clear cell renal carcinoma

[0088] Figure 1 This diagram illustrates the construction of a mouse model of advanced, highly metastatic clear cell renal cell carcinoma according to the present invention. The specific construction method is as follows:

[0089] Renca cells equipped with luciferase were cultured to a confluent level (80% cell coverage) in cell culture flasks. The luciferase-equipped Renca cells were then resuspended in a 2:1 mixture with Matrigel. Matrigel's main components are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), and nestin, and it also contains various growth factors such as TGF-β, EGF, IGF, and FGF. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane, thus facilitating cell culture and differentiation in vitro.

[0090] Renca cells containing luciferase were implanted into the subcapsular region of Balb / c mice using a suspension mixed with matrix gel. Six-week-old male Balb / c mice were intraperitoneally injected with 10% sodium pentobarbital solution during surgery, and their body temperature was maintained using a heating block. On a sterile operating table, the hair on the left subcostal region was shaved and fixed laterally. After routine disinfection with povidone-iodine, an incision was made approximately 1-2 cm along the left midline to separate the left kidney from the left costal margin, exposing the left kidney. 25 μL of a suspension of Renca cells containing luciferase was implanted into the subcapsular capsule of the left kidney. After the surgery, the incision was sutured, and the mice were allowed to recover. Tumor cell colonization was detected by in vivo imaging on day 3.

[0091] Subsequently, the growth of the orthotopic tumor in mice was examined on days 10, 13, and 16 post-tumor transplantation. Simultaneously, the heart, liver, spleen, lungs, and non-tumor-transplanted kidneys of the mice were removed. In vivo imaging detected fluorescent signals in the lungs on day 13 (see [link to original text]). Figure 2 -a live imaging results and Figure 2 (Statistics on the time of metastasis shown in Figure b) indicate that distant lung metastases occurred on day 13 at the time of initial diagnosis. Furthermore, the histopathological HE sections also revealed the morphology of the in situ tumor and adjacent tissues, with the same tumor morphology appearing on the lung foci (see...). Figure 3 ,in Figure 3 a represents the HE result of the in situ tumor. Figure 3 (b represents the HE results of metastatic lesions), confirming the occurrence of distant lung metastases from clear cell renal cell carcinoma. These results demonstrate the successful invention and construction of a model for advanced, highly metastatic clear cell renal cell carcinoma.

[0092] This application successfully constructed a mouse model of advanced, highly metastatic clear cell renal cell carcinoma by implanting Renca cell suspension into the renal capsule of Balb / c mice. This model can simulate the distant metastasis that occurs in clinical renal cancer patients at the initial diagnosis. The construction method of this application has high reproducibility, is simple to operate, and is easy to apply in industry.

[0093] Example 2: Construction of a mouse model of radical resection for clear cell renal cell carcinoma

[0094] Figure 4 The diagram shown illustrates the construction of a mouse model for radical resection of clear cell renal cell carcinoma according to the present invention. The specific construction method is as follows:

[0095] Renca cells with luciferase were cultured to a confluent level (80% cell coverage) in cell culture flasks. The luciferase-containing Renca cells were then resuspended in a 2:1 mixture with Matrigel. This suspension was then implanted subcapsularly into Balb / c mice. Six-week-old male Balb / c mice were intraperitoneally injected with 10% sodium pentobarbital solution during surgery, and their body temperature was maintained using a heating block. On a sterile operating table, the hair on the left subcostal region was shaved and fixed laterally. After routine disinfection with povidone-iodine, an incision was made approximately 1-2 cm along the left midline to separate the left kidney from the left costal margin, exposing the left kidney. 25 μL of a luciferase-containing Renca cell suspension was implanted into the subcapsular capsule of the left kidney. After the surgery, the incision was sutured, and the mice were allowed to recover. Tumor cell colonization was detected by in vivo imaging on day 3.

[0096] A radical nephrectomy was performed on day 10: the surgical area was strictly disinfected, mice were anesthetized, and the tumor-bearing left kidney was exposed through a lateral ventral incision. The renal artery, vein, and ureter were bound with 4-0 silk sutures, and the kidney was removed. After blood clotted, the surgical incision was sutured. The mice maintained normal body temperature throughout the procedure. After recovery from anesthesia, the mice were returned to the rearing room, and postoperative lung metastasis was detected by in vivo imaging on day 28 after tumor grafting (see...). Figure 5 ,in, Figure 5 a represents the result of in vivo imaging. Figure 5 b represents the quantitative statistics of the in vivo imaging results.

[0097] Meanwhile, the histopathological HE sections also showed that the lesions in the lung tissue had the same tumor morphology as the in situ tumor (see...). Figure 6 , Figure 6 a represents the HE result of the in situ tumor. Figure 6 b represents the HE result for the metastatic foci, from... Figure 6 The results confirmed the occurrence of metastatic lesions after radical resection surgery, and determined that distant lung metastases occurred after radical resection. In summary, these results demonstrate the successful invention and construction of a lung metastasis model after radical resection of clear cell renal cell carcinoma.

[0098] This application successfully constructed a mouse model of clear cell renal cell carcinoma radical resection by implanting Renca cell suspension into the renal capsule of Balb / c mice. This model simulates the metastatic situation that occurs in clinical patients with renal cancer after curative surgical resection. The construction method of this application has high reproducibility, is simple to operate, and is easy to apply in industry.

[0099] Example 3: Constructing in vivo self-assembly delivery systems targeting the VEGFR2 and mTOR target genes, respectively.

[0100] First, we constructed a delivery system that can inhibit VEGFR2 expression, targeting the VEGFR2 gene of first-line targeted drugs in clinical practice. This delivery system can secrete extracellular vesicles expressing VEGFR2 siRNA in vivo and deliver them to the target tissue via blood circulation, thereby inhibiting the expression of VEGFR2 in the target tissue.

[0101] We designed three VEGFR2 siRNA sequences and selected the one with the best inhibitory effect for our subsequent use in disease treatment.

[0102]

[0103]

[0104] We inserted our designed three VEGFR2 siRNA sequences into the miR-155 backbone following the CMV promoter element to construct three plasmids targeting the VEGFR2 target gene, which were named CMV-siRNA and CMV-siRNA, respectively. v-1 CMV-siR v-2 CMV-siR v-3 Additionally, the nucleotide sequence of the target sequence for the third VEGFR2 siRNA binding is shown in SEQ ID No. 3.

[0105] Then we constructed a delivery system that can inhibit mTOR expression for the target gene mTOR of second-line targeted drugs in clinical practice. This delivery system can secrete extracellular vesicles expressing mTOR siRNA in vivo and deliver them to the target tissue through blood circulation, thereby inhibiting the expression of mTOR in the target tissue.

[0106] We designed three mTOR siRNA sequences and selected the one with the best inhibitory effect for our subsequent use in disease treatment.

[0107] Serial Number sequence Serial Number 1 5'-TTCAGTTTCAAAATTAAAGCC-3' SEQ ID No.7 2 5'-TTCTTCAAGATCTTGTTGGCT-3' SEQ ID No. 8 3 5'-CGATCATCTCGATTCATACCC-3' SEQ ID No.2 4 5'-GGGTATGAATCGAGATGATCG-3' SEQ ID No.1

[0108] The three designed mTOR siRNA sequences were then inserted into the miR-155 backbone following the CMV promoter element, constructing three gene loops targeting mTOR target genes, which were named CMV-siRNA. m-1 CMV-siR m-2 CMV-siR m-3 . ( Figure 7 In addition, the target sequence to which the third mTOR siRNA binds has the nucleotide sequence shown in SEQ ID No. 1.

[0109] Example 4: In vivo self-assembly delivery system CMV-siR v and CMV-siR m In vitro screening

[0110] We will build the CMV-siR v-1 CMV-siR v-2 CMV-siR v-3 Three gene loops targeting VEGFR2 target genes were transfected into the mouse hemangioendothelioma cell line EOMA. Cells were harvested 36 hours later, and VEGFR2 mRNA expression in EOMA cells was detected using RT-qPCR. VEGFR2 protein expression levels were detected 48 hours later using Western blotting. The results showed that CMV-siR... v-3 It has the best inhibitory effect on VEGFR2 protein. Figure 8 Similarly, we will use CMV-siR m-1 CMV-siR m-2 CMV-siR m-3 Three gene loops targeting the mTOR target gene were transfected into the mouse renal cell carcinoma line Renca. After 36 hours, the mTOR mRNA expression level was detected by qRT-PCR, and after 48 hours, the mTOR protein expression level was detected by Western blotting. The results showed that CMV-siR... m-3 It has the highest interference efficiency against mTOR protein. Figure 8 (df). In summary, we selected CMV-siR, which showed the best inhibitory effect on VEGFR2 expression. v-3 CMV-siR, which has the best inhibitory effect on mTOR expression m-3 For use in research on subsequent treatments.

[0111] Example 5 CMV-siR v and CMV-siR m The therapeutic effects of an in vivo self-assembled delivery system on a model of radical resection of clear cell renal cell carcinoma.

[0112] The VEGFR2 siRNA self-assembly delivery system and the mTOR siRNA self-assembly delivery system were used for the treatment of advanced highly metastatic clear cell renal cell carcinoma models and models of clear cell renal cell carcinoma after radical resection.

[0113] We first implanted Renca cells stably transfected with luciferase under the renal capsule of Balb / c mice. Three days later, mice with successfully colonized tumors underwent CMV-siR. v In vivo self-assembly systems were used in neoadjuvant therapy before and after surgery. Tumor-bearing mice were randomly divided into four groups and intravenously injected with PBS, CMV-scrR, CMV-siR, etc., at a dose of 10 mg / kg, respectively. v The mice were administered sunitinib via gavage. The medication was given every two days for one week, and in vivo imaging was used to detect changes in the volume of the in situ tumors in the mice. Then, radical nephrectomy was performed, and postoperative adjuvant therapy was initiated, with continued intravenous injection of the gene loop every two days. Distal lung tumor metastasis was assessed after 18 days. The results showed that intravenous injection of CMV-siR during preoperative neoadjuvant therapy was effective. v The volume of kidney tumors in the group of mice was only slightly reduced compared to the control group, and the effect on tumor proliferation was not significant. Figure 9 a) However, in postoperative adjuvant therapy, compared with the control group, CMV-siR injection... v The gene loop significantly inhibited lung metastasis in mice, demonstrating the same therapeutic effect as first-line targeted drugs. Figure 9 b).

[0114] We then implanted Renca cells stably transfected with luciferase under the renal capsule of Balb / c mice. Three days later, mice with successfully colonized tumors underwent CMV-siR. m In vivo self-assembly systems were used in neoadjuvant therapy before and after surgery. Tumor-bearing mice were randomly divided into four groups and intravenously injected with PBS, CMV-scrR, CMV-siR, etc., at a dose of 10 mg / kg, respectively. m The mice were administered everolimus via gavage. The medication was given every two days for one week, and changes in tumor volume were assessed using small animal in vivo imaging. Radical nephrectomy was then performed, followed by adjuvant therapy. The gene loop was continued intravenously every two days, and distal lung tumor metastasis was assessed after 18 days. The results showed that intravenous injection of CMV-siR during preoperative neoadjuvant therapy was effective. m The kidney tumor volume in the mice in the first group was significantly reduced, and the treatment effect was similar to that in the second-line targeted drug group. Figure 9c). However, during postoperative adjuvant therapy, compared with the control group, the injection of CMV-siR... m Lung metastasis was not improved in mice with the gene loop. Figure 9 d).

[0115] Example 6 CMV-siR m+v Construction of in vivo self-assembled tandem gene loops and verification of their in vitro functions

[0116] To achieve the combined therapeutic effect, we inserted the VEGFR2 siRNA and mTOR siRNA sequences, which showed the best efficacy, into the tandem miR-155 backbone following the CMV promoter element, respectively, to construct the CMV-siRNA. m+v Tandem gene loops. Figure 10 a) An in vivo self-assembled tandem gene loop delivery system was constructed targeting the VEGFR2 and mTOR target genes. This gene loop can simultaneously express VEGFR2 siRNA and mTOR siRNA, as well as extracellular vesicles that deliver siRNA, thereby delivering siRNA to the target tissue and reducing the expression of the target genes.

[0117] CMV-siR m+v EOMA and Renca cells were transfected with tandem gene loops, respectively. After 36 hours, the mRNA expression levels of mTOR and VEGFR2 genes in the cells were detected using qRT-PCR. Figure 10 bc); and after 48 hours, the expression of mTOR VEGFR2 protein in cells was detected by Western blotting and quantitatively analyzed. Figure 10 The results showed that this gene loop could effectively inhibit the expression of mTOR and VEGFR2 genes. Simultaneously, we used CMV-siR... m+v The tandem gene loop was transfected into EOMA and Renca cells, respectively, and digested and counted 6 hours post-transfection. 5000 cells were seeded into 96-well plates at 100 μL of suspension per well, with six replicates per sample and five identical 96-well plates. CCK-8 proliferation was assessed at 0, 12, 24, 36, and 48 hours, and the OD value at 450 nm was measured using a microplate reader. A proliferation curve was plotted for each sample using the average OD value over 12 hours as the baseline. These results show that CMV-siR… m+v The tandem gene loop can effectively inhibit the proliferation of EOMA cells and Renca cells in vitro.

[0118] ( Figure 10 fi)

[0119] Example 7: In vivo self-assembled CMV-siR m+v The therapeutic effects of tandem gene loops on advanced highly metastatic clear cell renal cell carcinoma models and radical resection models of clear cell renal cell carcinoma.

[0120] A VEGFR2 siRNA and mTOR siRNA tandem delivery system was used to treat advanced highly metastatic clear cell renal cell carcinoma models and a radical renal cell carcinoma model. In the constructed advanced highly metastatic clear cell renal cell carcinoma mouse model, Renca cells stably transfected with luciferase were implanted subcapsularly into Balb / c mice, and distant lung metastases appeared on day 13, simulating the metastatic situation at initial diagnosis in clinical practice. Then, drug administration was started on day 13, with groups receiving PBS, CMV-scrR, and CMV-siR. m+v Three groups of tandem gene loops were intravenously injected with PBS, CMV-scrR, and CMV-siR at a dose of 10 mg / kg, respectively. m +v The drug was administered every two days for 15 consecutive days. In vivo imaging was then used to detect the size of the in situ tumor and the extent of distant lung metastasis in the tumor-bearing mice. These results demonstrate the efficacy of CMV-siR. m+v The tandem gene loop effectively inhibited the growth of in situ tumors and the occurrence of lung metastases in a mouse model of advanced highly metastatic clear cell renal carcinoma, demonstrating a good therapeutic effect. Figure 11 ab)

[0121] We then implanted Renca cells stably transfected with luciferase under the renal capsule of Balb / c mice. Three days later, mice with successfully colonized tumors underwent neoadjuvant therapy before surgery and adjuvant therapy after surgery to utilize the in vivo self-assembly system tandem gene loop. We randomly divided the tumor-bearing mice into four groups, which were intravenously injected with PBS, CMV-scrR, CMV-siR, etc., at a dose of 10 mg / kg, respectively. m+v The mice were treated with a combination of intravenous targeted therapy and gavage (sunitinib as a first-line drug and everolimus as a second-line drug). Administered every two days for one week, and tumor volume changes in mice were assessed using small animal in vivo imaging. Radical nephrectomy was then performed, followed by adjuvant therapy, with continued intravenous injection of the tandem gene loop every two days. Distal lung tumor metastasis was assessed after 18 days. Results showed that intravenous injection of CMV-siR during preoperative neoadjuvant therapy was effective. m+v The kidney tumors in the mice in this group were significantly reduced in size, and the treatment effect was stronger than that of the combination of targeted drugs. Figure 11 c) This achieved a good preoperative downstaging effect. Furthermore, during postoperative adjuvant therapy, compared with combined targeted therapy, CMV-siR injection... m+v Tandem gene loops effectively inhibited the occurrence of lung metastasis in mice. Figure 11d) It has a good auxiliary therapeutic effect.

[0122] Example 8 CMV-siR m+v In vivo safety evaluation of self-assembled tandem gene loops

[0123] After comparing the efficacy of VEGFR2 siRNA and mTOR siRNA tandem delivery systems with targeted monotherapy and combination therapy, we also evaluated their safety. The main focus was on evaluating liver toxicity in model mice. Following continuous administration, paraffin sections of liver tissue were prepared from the mice, and hematoxylin and eosin (HE) staining was used to assess liver tissue damage. Figure 12 a). Serum biochemical indicators were also tested, including the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum. Figure 12 bd), thus providing feedback on liver function. The above results indicate that CMV-siR m+v The tandem gene loop did not cause liver damage and has advantages such as safety, non-toxicity, and non-immunogenicity.

[0124] Compared with existing technologies, sunitinib monotherapy and sunitinib combined with everolimus, HE results from mouse liver tissue showed that sunitinib monotherapy and its combination with everolimus caused immune cell infiltration and liver inflammation in the liver tissue, while the VEGFR2 siRNA and mTOR siRNA tandem delivery system did not cause liver damage (see...). Figure 13 a). Results from the detection of serum total cholesterol, triglycerides, and free fatty acids, among other lipid metabolites, showed that lipid metabolism abnormalities occurred with everolimus alone and in combination with sunitinib, while these side effects were not observed with the VEGFR2 siRNA and mTOR siRNA tandem delivery systems (see [link to relevant documentation]). Figure 13 This further demonstrates that the VEGFR2 siRNA and mTOR siRNA tandem delivery system has better safety compared to first- and second-line targeted therapies (sunitinib, everolimus).

[0125] In summary, the VEGFR2 siRNA and mTOR siRNA tandem delivery system not only demonstrated excellent therapeutic effects in advanced highly metastatic clear cell renal cell carcinoma models and radical resection models of clear cell renal cell carcinoma, but also inhibited the growth of the in situ tumor and the occurrence of distant lung metastases; moreover, it exhibited better safety compared to the combination of targeted drugs (the first-line clinical drug sunitinib and the second-line clinical drug everolimus).

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An RNA drug for treating renal cell carcinoma, characterized in that, The drugs include drugs that can treat in situ tumors of renal cell carcinoma, and / or drugs that can treat metastatic tumors of renal cell carcinoma. The drug for treating in situ tumors of renal cell carcinoma is a double-stranded RNA, siRNA, or shRNA, wherein the double-stranded RNA, siRNA, or shRNA contains a nucleotide sequence that can hybridize with the mTOR gene under stringent conditions, and the target sequence in the corresponding mTOR gene that the double-stranded RNA, siRNA, or shRNA hybridizes with the mTOR gene is shown in SEQ ID No. 1; and The drug capable of treating metastatic tumors of renal cell carcinoma is a double-stranded RNA, siRNA, or shRNA, wherein the double-stranded RNA, siRNA, or shRNA contains a nucleotide sequence that can hybridize with the VEGFR2 gene under strict conditions, and the target sequence in the corresponding VEGFR2 gene that the double-stranded RNA, siRNA, or shRNA hybridizes with the VEGFR2 gene is as shown in SEQ ID No.

3.

2. The drug for treating renal cell carcinoma according to claim 1, characterized in that: The drug that can treat in situ tumors of renal cell carcinoma is siRNA, and its nucleotide sequence is shown in SEQ ID No. 2; The drug that can treat metastatic tumors of renal cell carcinoma is siRNA, and its nucleotide sequence is shown in SEQ ID No.

4.

3. The drug for treating renal cell carcinoma according to claim 2, characterized in that: The siRNAs that can treat in situ tumors of renal cell carcinoma and those that can treat metastatic tumors of renal cell carcinoma have nucleotide sequences that exist in the same expression vector or in different expression vectors.

4. The medicament for treating renal cell carcinoma according to claim 3, characterized in that: The nucleotide sequence of the siRNA capable of treating in situ tumors of renal cell carcinoma is located in the miR-155 backbone following the CMV promoter. And / or the nucleotide sequence of the siRNA capable of treating metastatic tumors of renal cell carcinoma is located in the miR-155 backbone following the CMV promoter.

5. The medicament for treating renal cell carcinoma according to claim 3, characterized in that: Two siRNAs are expressed in tandem.

6. A self-assembly delivery system for RNA in the treatment of renal cell carcinoma, characterized in that, The self-assembly system of RNA for treating renal cell carcinoma comprises the drug for treating renal cell carcinoma as described in any one of claims 1 to 5; Preferably, the self-assembly delivery system for the RNA used to treat renal cell carcinoma is an extracellular vesicle; Preferably, the self-assembly delivery system for the RNA used to treat renal cell carcinoma is an exosome.

7. A pharmaceutical composition or combination of pharmaceuticals, characterized in that, The pharmaceutical composition or combination of drugs includes drugs capable of treating in situ tumors of clear cell renal cell carcinoma and drugs capable of treating metastatic tumors of clear cell renal cell carcinoma. The drug for treating in situ tumors of clear cell renal cell carcinoma is siRNA, whose nucleotide sequence is shown in SEQ ID No. 2; and The drug that can treat metastatic tumors of clear cell renal cell carcinoma is siRNA, and its nucleotide sequence is shown in SEQ ID No.

4.

8. The use of the medicament for treating renal cell carcinoma according to any one of claims 1 to 5, the self-assembly delivery system for RNA for treating renal cell carcinoma according to claim 6, or the pharmaceutical composition or combination according to claim 7 in the preparation of a medicament for treating renal cell carcinoma, wherein, The renal cell carcinoma mentioned is clear cell renal cell carcinoma; Preferably, the renal cell carcinoma is advanced renal cell carcinoma or metastatic renal cell carcinoma.

9. A nucleic acid, characterized in that, The nucleic acids include: a. Double-stranded RNA, siRNA, or shRNA b. The double-stranded RNA, siRNA, or shRNA contains a nucleotide sequence that can hybridize with the mTOR gene under strict conditions, and the target sequence in the corresponding mTOR gene that the double-stranded RNA, siRNA, or shRNA hybridizes with the mTOR gene is shown in SEQ ID No. 1; Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID No.

2.

10. A nucleic acid molecule, characterized in that, The nucleic acid molecules include: a. Double-stranded RNA, siRNA, or shRNA b. The double-stranded RNA, siRNA, or shRNA contains a nucleotide sequence that can hybridize with the VEGFR2 gene under strict conditions, and the double-stranded RNA, siRNA, or shRNA corresponds to the target sequence in the VEGFR2 gene as shown in SEQ ID No.

3. Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID No. 4.

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