Assembled protein nanocarrier cabri for targeted delivery of small interfering nucleic acid, and preparation method therefor and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an assembled protein nanocarrier CABRi for targeted delivery of a small interfering nucleic acid, and a preparation method therefor and a use thereof. The designed assembled protein nanocarrier CABRi (a CABRi series of siRNA protein delivery carriers) can be efficiently loaded with an siRNA having no sequence selectivity, can be mixed with one or more siRNAs at a specific ratio to form a carrier / small nucleic acid complex CABRi-siRNA, and is self-assembled into a nanoparticle having a size of 10-100 nm. A new siRNA delivery carrier is constructed. Three key technical bottlenecks in siRNA drug delivery are broken through, i.e., the problems of extremely low in vivo siRNA delivery efficiency, endosomal escape, and extrahepatic delivery are solved, ultimately achieving efficient siRNA drug delivery, endosomal escape, targeting of extrahepatic organs or tumors, high safety, and ease of industrialization.

Description

CABRi, an assembled protein nanocarrier for targeted delivery of small interfering nucleic acids, its preparation method and applications Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, its preparation method, and its applications. Background Technology

[0002] Since scientists discovered in 1998 that double-stranded RNA can specifically silence gene expression, RNA interference (RNAi), as a post-transcriptional gene silencing technique, has shown great potential and application prospects in the treatment of various diseases. Meanwhile, with the deepening of genomic research and the rapid development of sequencing technology, gene therapy has become one of the revolutionary biological treatment options. Theoretically, siRNA can specifically and effectively inhibit any gene by directly targeting and reducing mRNA expression. Since Alnylam's first siRNA drug, Patisiran, was successfully launched in the United States on August 10, 2018, six other siRNA drugs have been successfully launched. However, no domestically developed siRNA drug has yet been approved for marketing in China.

[0003] Global siRNA drug development is still in its early stages, facing numerous technological bottlenecks, particularly low siRNA delivery efficiency. Currently, commonly used siRNA delivery systems include viral and non-viral vectors. Viral vectors are known for their high siRNA delivery efficiency, but they can integrate into the genome, potentially leading to unpredictable consequences, including immune responses and liver toxicity. Therefore, the application of viral siRNA drugs is significantly limited. Conversely, non-viral vectors, including lipid nanoparticles (LNPs), polymer nanoparticles, peptide complexes, and antibody-oligonucleotide conjugates (AOCs), offer higher safety profiles, but their targeting specificity, delivery efficiency, endosomal escape, and overall safety require improvement. Patisiran, the world's first approved siRNA drug, utilizes an LNP delivery system, encapsulating the siRNA drug within an LNP for intravenous administration. Liposome encapsulation significantly improves drug stability and targeting to liver tissue, ensuring that siRNA is not filtered out by the kidneys and is gradually taken up by target cells in liver tissue during blood circulation. This is key to Patisiran's successful approval despite delivery challenges. Inclisiran, the second small nucleic acid drug approved in December 2020, features a specially designed GalNAc-siRNA delivery system. Its principle is that N-acetylgalactosamine (GalNAc) specifically binds to the desialized glycoprotein receptor (ASGPR) on the hepatocyte membrane, thereby specifically targeting the liver with siRNA. Both of these approved small nucleic acid drugs achieved their market launch thanks to their unique delivery methods.

[0004] While the aforementioned commercially available siRNA delivery methods have been relatively successful, several issues remain. For example, current LNP-siRNA delivery systems often involve the addition of PEG lipids, leading to excessive immune responses and increased toxicity, which is a major limitation to clinical application. Therefore, LNP-mediated siRNA drugs (such as Patisiran) require pretreatment with steroids and antihistamines before intravenous injection to eliminate unnecessary allergic reactions. Furthermore, GalNAc-modified siRNA drugs (such as Inclisiran) can only specifically target the liver and are not suitable for other organs, tissue lesions, or tumors, significantly limiting the application scenarios of siRNA drugs. Currently, siRNA drug delivery faces three major technical bottlenecks: 1) Low in vivo delivery efficiency of siRNA. Designing a stable and effective complex between siRNA and the delivery vector is crucial for drug development; 2) In vivo escape problem. For example, less than 0.1-1% of siRNA from marketed siRNA drug delivery technologies like LNP or GalNAC can escape from the body. Improving siRNA in vivo escape is also critical; 3) Extrahepatic tissue delivery problem. Currently, LNP and GalNAc only provide liver delivery. Solving the problem of specific targeted delivery of siRNA to organs, tissues, or tumors is a critical technical challenge that urgently needs to be addressed.

[0005] Specially designed proteins hold great promise as siRNA delivery vectors. Composed of natural amino acids, these vectors are non-toxic, readily degradable, and unlikely to trigger immune responses. They can be obtained through various methods, including prokaryotic expression, eukaryotic expression, or cell-free in vitro expression. Recombinant protein technology can endow these vectors with cell-targeting capabilities, modifying their siRNA-binding domains to enhance their siRNA delivery capacity. Utilizing mature recombinant protein technology, the complex structure of these vectors can be precisely controlled, enabling them to protect siRNA from RNase degradation, overcome cell membrane barriers, enhance cellular uptake of siRNA, promote endosome escape, and ultimately exert the desired siRNA biological activity.

[0006] It is worth noting that current protein-based siRNA delivery systems are still largely in the theoretical research and animal experiment stages, and many challenges remain to be overcome for the clinical application of siRNA protein delivery vectors. First, currently reported protein-delivered siRNA drugs are typically administered locally or intravenously, with limited oral bioavailability. Second, although the fusion expression of domains with different functions can be achieved using recombinant protein technology, ensuring that the function of individual domains within these recombinant proteins remains unaffected is still an unresolved issue. Finally, protein-delivered siRNA drugs have a short circulating half-life; improving structural stability and prolonging the drug's half-life also requires special attention.

[0007] Given the aforementioned technical obstacles, it is of particular importance to develop a delivery system that is highly efficient, allows for endosome escape, targets extrahepatic organs or tumors, and is highly safe. Summary of the Invention

[0008] The purpose of this invention is to provide an assembled protein nanocarrier, CABRi, for targeted delivery of small interfering nucleic acids, along with its preparation method and applications, to address the problems existing in the prior art. The assembled protein nanocarrier CABRi provided by this invention has advantages such as efficient delivery, endosome escape, targeting of extrahepatic organs or tumors, and high safety.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides an assembled protein nanocarrier, CABRi, for targeted delivery of small interfering nucleic acids. The assembled protein nanocarrier CABRi comprises an assembled functional peptide (AFP), a linker, and a small RNA binding protein / peptide segment (sRBP). In this invention, the AFP refers to a peptide with the functional properties of a cell-penetrating peptide (CPP) or simultaneously a pH-responsive endosome escape peptide (EEP), or a specific targeting peptide (STP) for organs or tumor tissues.

[0011] The assembled functional peptides include polypeptides (CPP / STP) with membrane-penetrating and pH-responsive functions and / or specific targeting peptides (STP); the amino acid sequence of the polypeptide is any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.13; the amino acid sequence of the specific targeting peptide is any one of the sequences shown in SEQ ID NO.14-SEQ ID NO.24;

[0012] The linker comprises a flexible linker (FL) and / or a rigid linker (RL); the flexible linker has 5 or 15 amino acid residues; the amino acid of the flexible linker is selected from glycine and serine; the flexible linker connects CPP / EEP / STP to sRBP, or connects two sRBPs; the amino acid sequence of the flexible linker is shown in SEQ ID NO. 25 or SEQ ID NO. 26; the rigid linker has 12 or 15 amino acid residues; the amino acid of the rigid linker is selected from alanine, glutamic acid, and lysine; the rigid linker connects CPP / EEP and STP; the amino acid sequence of the rigid linker is shown in SEQ ID NO. 27 or SEQ ID NO. 28.

[0013] The small RNA-binding protein is derived from a double-stranded RNA-binding domain (dsRNA-binding domain, DRBM or dsRBD); the amino acid sequence of the small RNA-binding protein is any one of the sequences shown in SEQ ID NO.29-SEQ ID NO.35.

[0014] The assembled functional peptides described in this invention can achieve three functions: (1) as cell-penetrating peptides (CPPs), enabling them to penetrate the cell membrane; (2) some CPPs also function as pH-responsive endosome escape peptides (EEPs), enabling them to undergo pH-responsive protonation or helical conversion, thereby escaping from the endosome and effectively releasing siRNA into the cytoplasm to perform gene silencing; (3) as short peptides as ligands, or specific targeting peptides (STPs) for organs or tumor tissues, enabling them to bind to specific receptors on the surface of organs, tissues, or tumor cells, and precisely deliver siRNA to these receptors.

[0015] The sRBP described in this invention is a protein-siRNA complex formed by binding to a specific protein tertiary structure, enabling efficient delivery into cells within tissues. The sRBP described in this invention can be two or more repeating copies, connected by a flexible linker with the amino acid sequence SEQ ID NO.25 or SEQ ID NO.26. This ensures the functional independence of each peptide domain and allows for the spontaneous formation of domain dimers or multimers within the protein.

[0016] Preferably, the copy number of the assembled functional peptide, the linker, and the small RNA-binding protein is ≥1; the basic vector of the assembled protein nanocarrier CABRi includes a prokaryotic system expression vector and a eukaryotic system expression vector.

[0017] The assembled protein nanocarrier CABRi of this invention is a complete CABRi protein delivery carrier constructed by assembling functional peptides, linkers (flexible and / or rigid linkers), and small RNA-binding proteins in different assembly arrangements. This includes, but is not limited to, the integration of cell-penetrating peptides or peptides that simultaneously respond to pH and escape from endosomal tissues, organ or tumor tissue-specific targeting peptides, flexible or rigid linkers, and small RNA-binding proteins / peptides, thereby obtaining the CABRi series of recombinant protein carriers capable of efficiently delivering siRNA. This assembled protein nanocarrier CABRi can self-assemble with siRNA into nanoparticles with a size ranging from 10 to 100 nm, exhibiting a high deposition rate in the human respiratory system. It can penetrate capillaries or endothelial cells to achieve highly efficient and specific targeting of organ tissues (such as the lungs and pancreas) or tumor tissues, and can also achieve targeted delivery of siRNA to organ tissues or tumors. Most CABRi-siRNA nanoparticles have an average diameter of approximately 30-40 nm or 50-70 nm, and show good deposition in the lungs after nebulization.

[0018] The assembled protein nanocarrier CABRi of this invention effectively loads various siRNAs via a protein carrier, enabling the simultaneous delivery of one or more siRNAs into cells. The siRNAs can be efficiently released from endosomes and further exert their effects by inducing the siRNA-induced silencing complex (siRISC), which degrades pathogenic mRNAs or inhibits protein translation of mRNAs. Furthermore, the assembled protein nanocarrier CABRi of this invention can efficiently load sequence-insensitive siRNAs while protecting them, achieving highly efficient delivery.

[0019] Furthermore, the siRNA described in this invention can be either unmodified or chemically modified. Chemically modified siRNA exhibits better performance, enhancing its stability, resistance to nucleases, and delivery efficiency; it also reduces the immunogenicity of small nucleic acids. Examples of such chemical modifications include 2'-O-Methyl Base 2-methoxy modified bases (2'-ome), 2'-Fluoro RNA 2'-fluoroRNA (2'-F RNA), and Phosphorothioate modification (PS).

[0020] This invention provides a method for preparing the above-mentioned assembled protein nanocarrier CABRi, comprising the following steps:

[0021] The CABRi protein was obtained by linking the assembled functional peptide, the linker, and the small RNA-binding protein using seamless cloning technology.

[0022] The CABRi protein was cloned into a base vector to obtain the assembled protein nanocarrier CABRi.

[0023] Preferably, the copy number of the assembled functional peptide, the adapter, and the small RNA binding protein is ≥1; the basic vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0024] More preferably, the purification of the assembled protein nanocarrier CABRi is carried out by a two-step method, including two of the following: His affinity purification method, GST affinity purification method, ion exchange chromatography, and gel filtration chromatography (molecular sieve).

[0025] This invention provides the application of the above-mentioned assembled protein nanocarrier CABRi in the preparation of siRNA drug delivery systems, siRNA nanocomplexes, and targeted drugs.

[0026] This invention provides an siRNA drug delivery system comprising the aforementioned assembled protein nanocarrier CABRi.

[0027] The present invention provides a siRNA nanocomplex, wherein the siRNA nanocomplex comprises the above-mentioned assembled protein nanocarrier CABRi and siRNA.

[0028] The CABRi-siRNA nanocomposite provided by this invention consists of appropriately sized (10-100 nm) and highly stable nanoparticles suitable for in vivo delivery of siRNA. This composite exhibits high safety and low toxicity; after releasing siRNA within 12-48 hours of cell entry, the CABRi protein itself undergoes rapid degradation. Furthermore, this composite is easy to produce and low in cost.

[0029] The present invention provides a method for preparing the above-mentioned siRNA nanocomplex, comprising the steps of mixing the above-mentioned assembled protein nanocarrier CABRi and siRNA, incubating, and obtaining the siRNA nanocomplex.

[0030] More preferably, the molar ratio of the assembled protein nanocarrier CABRi protein to siRNA is 4:1.

[0031] More preferably, the pH of the system obtained by mixing the assembled protein nanocarrier CABRi and siRNA is 7.4; the incubation time is 30 min, and the temperature is room temperature.

[0032] This invention provides a targeted drug comprising the above-described assembled protein nanocarrier CABRi and siRNA.

[0033] Preferably, the siRNA includes one or more of siKRAS, siSHP2, and siSOS1;

[0034] The positive chain sequence of siKRAS is shown in SEQ ID NO.36, and the negative chain sequence is shown in SEQ ID NO.37; the positive chain sequence of siSHP2 is shown in SEQ ID NO.40, and the negative chain sequence is shown in SEQ ID NO.41; the positive chain of siSOS1 is shown in SEQ ID NO.42, and the negative chain sequence is shown in SEQ ID NO.43.

[0035] More preferably, the drug comprises the above-described assembled protein nanocarrier CABRi and an effective amount of siRNA.

[0036] More preferably, the drug is administered via nebulization, intravenous injection, or subcutaneous administration.

[0037] The present invention discloses the following technical effects:

[0038] The CABRi series of siRNA protein delivery vectors designed in this invention can efficiently load sequence-free siRNAs. These vectors can be mixed with one or more siRNAs in a specific ratio to form a vector / small nucleic acid complex, CABRi-siRNA, which self-assembles into a nanoparticle of 10-100 nm in size. The AFP fused to the CABRi terminal possesses three functional properties: membrane penetration, ligand binding to specific cell surface receptors, and endosomal escape. This enables strong resistance to nucleases and high delivery efficiency in vivo; specific targeting of organs and tissues (such as the lungs and pancreas) or tumor tissues; and pH-responsive protonation or α-helix conversion facilitates efficient siRNA escape from endosomals, effectively releasing the siRNA into the cytoplasm for gene silencing. Regarding the safety of CABRi-siRNA, the CABRi protein itself has low immunogenicity and is rapidly degraded; the siRNA undergoes chemical modification to reduce immunogenicity. The drug formulations of the CABRi-siRNA designed in this invention can be administered intravenously, subcutaneously, or orally, effectively avoiding glomerular and circulatory clearance. This invention constructs a novel siRNA delivery vector, overcoming three major technical bottlenecks in siRNA drug delivery. It also overcomes the problems of extremely low in vivo delivery efficiency, in vivo escape, and extrahepatic delivery of siRNA, ultimately achieving efficient delivery of siRNA drugs, in vivo escape, targeting of extrahepatic organs or tumors, high safety, and ease of industrialization. Furthermore, this invention represents a novel method for siRNA drug delivery, applicable to the treatment of various diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (IF), lung cancer, pancreatic cancer, and colorectal cancer. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 shows the CABRi-3 protein after Ni 2+-NTA affinity chromatography with Coomassie Brilliant Blue staining; where Marker is protein standard; Cell pellets are cell pellets; Cell lysate is cell lysate supernatant; Flow through is flow-through buffer; Wash is wash buffer; Elution-first is the first elution buffer; Elution-second is the second elution buffer; Elution-third is the third elution buffer; Elution-fourth is the fourth elution buffer; Elution-fifth is the fifth elution buffer; Elution-last is the last elution buffer; Elution-total is the total elution buffer.

[0041] Figure 2 shows the CABRi-8 protein after Ni 2+ -NTA affinity chromatography with Coomassie brilliant blue staining; where Marker is a protein standard; Cell lysate is cell lysate supernatant; Flow through is flow-through buffer; Wash is wash buffer; Elution-first is first elution buffer; Elution-last is last elution buffer; Elution-total is total elution buffer;

[0042] Figure 3 shows the CABRi-12 protein after Ni 2+ -NTA affinity chromatography with Coomassie brilliant blue staining; where Marker is a protein standard; Cell lysate is cell lysate supernatant; Flow through is flow-through buffer; Wash is wash buffer; Elution-first is first elution buffer; Elution-last is last elution buffer; Elution-total is total elution buffer;

[0043] Figure 4 shows the purification curve of CABRi-8 after separation by cation exchange column;

[0044] Figure 5 shows the separation and purification of CABRi-8 via cation exchange column and Coomassie Brilliant Blue staining; where Marker is protein standard; Purified sample is purified sample; Flow through is flow-through solution; Peak 1 is the first peak, and A05-A09 are collected in tubes A05, A06, A07, A08, and A09 respectively; Peak 2 is the second peak, and A10 is collected in tube A10, and B05-B10 are collected in tubes B05, B06, B07, B08, B09, and B10 respectively.

[0045] Figure 6 shows the particle size and potential of the self-formed CABRi-8 nanoparticles; the three curves represent the curves from three measurements.

[0046] Figure 7 shows the particle size and potential of the self-formed CABRi-12 nanoparticles; the three curves represent the curves from three measurements.

[0047] Figure 8 shows the particle size and potential of CABRi-8-siRNA nanoparticles; the three curves represent the three measurement curves respectively.

[0048] Figure 9 shows the particle size and potential of CABRi-12-siRNA nanoparticles; the three curves represent three measurement curves respectively.

[0049] Figure 10 shows the binding efficiency of CABRi-8 to siRNA as detected by EMSA.

[0050] Figure 11 shows the binding efficiency of CABRi-12 to siRNA as detected by EMSA.

[0051] Figure 12 shows the EMSA experiment used to detect the protection of siRNA by CABRi-8-siRNA nanoparticles.

[0052] Figure 13 shows the results of Northern blotting detection of siRNA delivery into cells via the CABRi-8 vector;

[0053] Figure 14 shows the detection of CABRi-8-siRNA nanoparticles escaping from endosomes / lysosomes and releasing siRNA into the cytoplasm using confocal microscopy live cell imaging technology.

[0054] Figure 15 shows the Western blotting experiment used to detect how CABRi-8-siKRAS nanoparticles reduced the expression level of the intracellular target gene KRAS.

[0055] Figure 16 shows the plate clone assay to detect the inhibition of H1975 cell proliferation by CABRi-12-siRNAs; where A is the culture plot and B is the H1975 cell statistical plot.

[0056] Figure 17 shows the fluorescence imaging experiment used to detect the delivery of siRNA by the CABRi-12 vector to tumor tissue; where Lung represents the lung; Heart represents the heart; Tumor represents the tumor; Liver represents the liver; Spleen represents the spleen; and Kidney represents the kidney.

[0057] Figure 18 shows the inhibition of malignant tumor growth in mice by CABRi-12-siRNAs; where A is a statistical graph of tumor volume and B is a statistical graph of tumor mass.

[0058] Figure 19 shows the H&E staining results for detecting the toxic side effects of the CABRi-12 delivery system on normal tissues and organs. In Figure 19, A shows the H&E staining results of different treatments on Lung, Liver, Brain, and Intestine; B shows the H&E staining results of different treatments on Spleen, Kidney, Heart, and Muscle. Detailed Implementation

[0059] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0060] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0061] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0062] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0063] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0064] The partial sequences involved in this invention are shown below:

[0065] The sequence of CPP / EEP is selected from one of the sequences shown in SEQ ID NO.1-13:

[0066] SEQ ID NO.1: VSRRRRGGRRRRRR;

[0067] SEQ ID NO.2: RRRRRRRRKKR;

[0068] SEQ ID NO.3: KVVVVKVVVVKVVVVKVVVVK;

[0069] SEQ ID NO.4: KLLLLKLLLLKLLLLKLLLLK;

[0070] SEQ ID NO.5: KLALKLALKALKAALKLA;

[0071] SEQ ID NO.6: KKALLAHALHLLALLALHLAHALKKA;

[0072] SEQ ID NO.7: KKALLALALHHLAHLALHLALALKKA;

[0073] SEQ ID NO.8: WEAKLAKALAKALAKHLAKALAKALKACEA;

[0074] SEQ ID NO.9: LIRLWSHLIHIWFQNRRLKWKKK;

[0075] SEQ ID NO.10: GLWRALWRLLRSLWRLLWRA;

[0076] SEQ ID NO.11: LLRLLRWWWRLLRLL;

[0077] SEQ ID NO.12: WEAALAEALAEALAEHLAEALAEALEALAA;

[0078] SEQ ID NO.13: HHEHHEHHEHHEHHEHHEHHEHHEHHE;

[0079] The sequence of STP is selected from one of those shown in SEQ ID NO.14 - 24:

[0080] SEQ ID NO.14: HLNILSTLWKYR;

[0081] SEQ ID NO.15: KLLLLKLLLLKLLLLKLLLLK;

[0082] SEQ ID NO.16: KVVVVKVVVVKVVVVKVVVVK;

[0083] SEQ ID NO.17: VVVVVVKKGRGDS;

[0084] SEQ ID NO.18: GRGDSGRGDS;

[0085] SEQ ID NO.19: CDCRGDCFC;

[0086] SEQ ID NO.20: SHSFSVGSGDHSPFT;

[0087] SEQ ID NO.21: GRFLTGGTGRLLRIS;

[0088] SEQ ID NO.22: KPVSLSYRSPSRFFESH;

[0089] SEQ ID NO.23:APWHLSSQYSRT;

[0090] SEQ ID NO.24: DMPGTVLP;

[0091] The sequence of FL is selected from one of the sequences shown in SEQ ID NO.25-26:

[0092] SEQ ID NO.25: GSSGG;

[0093] SEQ ID NO.26: GGGGSGGGGGSSSGG;

[0094] The amino acid sequence of RL is selected from one of the sequences shown in SEQ ID NO: 27-28:

[0095] SEQ ID NO.27: AEAAAKEAAAKA;

[0096] SEQ ID NO.28: EAAAKEAAAKAAAK;

[0097] The amino acid sequence of sRBP is selected from one of the sequences shown in SEQ ID NO.29-35:

[0098] SEQ ID NO.29(Human PKR / E2AK2 DRBM1 / 2):

[0099] SEQ ID NO.30 (RNA silencing suppressor 1):

[0100] SEQ ID NO.31 (RNA silencing suppressor 2):

[0101] SEQ ID NO.32(Human TARBP2 DRBM1):

[0102] SEQ ID NO.33(Human TARBP2 DRBM2):

[0103] SEQ ID NO.34 (Human Dicer dsRNA-binding fold):

[0104] SEQ ID NO.35(Human Dicer DRBM):

[0105] Example 1: Construction of the CABRi series of siRNA-targeted protein nanocarriers

[0106] The overall construction scheme of the CABRi series vectors: The CABRi series of siRNA protein delivery vectors provided by this invention utilizes seamless cloning technology and artificial gene synthesis technology to assemble the corresponding nucleotide sequences of FL / RL, sRBP, and AFP (CPP / EEP / STP) together and insert them into prokaryotic expression vectors such as pET-28a and pGEX-4T-1, or into eukaryotic expression vectors such as pcDNA3.1 V5-His A, pcDNA3.1-3xFlag, and pcDNA3.1-3xHA. From these different combinations, a series of vectors can be constructed, named CABRi-1 vector, CABRi-2 vector, ..., CABRi-99 vector. Through a series of screenings based on protein expression levels, ease of purification, and final yield, the optimal expression plasmid for the desired CABRi siRNA protein delivery vector is finally determined.

[0107] In this embodiment, CABRi-3, CABRi-8 and CABRi-12 vectors are used as examples. The designed CABRi nucleotide sequence is cloned into the prokaryotic system expression vector pET-28a(+) with insertion sites of NheI and BamHI. The N-terminus of the CABRi-3 vector is 6×His / HA, the former used for protein purification, and the latter for protein vector identification and in vivo recognition; the middle region contains an sRBP (SEQ ID NO. 31); the C-terminus is AFP, composed of CPP / EEP (SEQ ID NO. 6); the N-terminal region and the middle sRBP are linked by FL (SEQ ID NO. 25), while the middle sRBP and the C-terminal CPP / EEP are linked by FL (SEQ ID NO. 26). The amino acid sequence of the CABRi-3 protein in the CABRi-3 vector is obtained by seamlessly linking the following parts (no base insertion is required between sequences): FL (SEQ ID NO. 25)-sRBP (SEQ ID NO. 31)-FL (SEQ ID NO. 26)-AFP (SEQ ID NO. 6). The CABRi-8 vector is based on CABRi-3, utilizing... The Ultra One Step Cloning Kit inserts another copy of sRBP (SEQ ID NO. NO. 31)-FL (SEQ ID NO. NO. 26) into the middle region, thus forming two sRBPs. The amino acid sequence of the CABRi-8 protein in the CABRi-8 vector is obtained by seamlessly linking the following parts (no bases need to be inserted between the sequences), specifically: FL (SEQ ID NO. 25)-sRBP (SEQ ID NO. 31)-FL (SEQ ID NO. 26)-sRBP (SEQ ID NO. 31)-FL (SEQ ID NO. 26)-AFP (SEQ ID NO. 6). The CABRi-12 vector is based on CABRi-8 and is obtained by seamless cloning technology, adding STP (SEQ ID NO.18) after CPP / EEP (SEQ ID NO.6), and linking them together with RL (SEQ ID NO.27). The amino acid sequence of CABRi-12 in the CABRi-12 vector is obtained by seamlessly linking the following parts (no bases need to be inserted between the sequences), specifically: FL (SEQ ID NO.25)-sRBP (SEQ ID NO.31)-FL (SEQ ID NO.26)-sRBP (SEQ ID NO.31)-FL (SEQ ID NO.26)-AFP (SEQ ID NO.6)-RL (SEQ ID NO.27)-STP (SEQ ID NO.18).

[0108] Example 2: Expression and purification of the CABRi series of siRNA-targeted protein nanocarriers

[0109] Taking the CABRi-3, CABRi-8, and CABRi-12 vectors constructed in Example 1 as examples, these expression plasmids were transformed into Escherichia coli expression strain BL21(DE3) and cultured. When the bacteria grew to an OD value of 0.6, they were induced at 16°C for 16-20 hours with a final concentration of 0.2 mM IPTG.

[0110] After collecting the bacterial culture, it was sequentially ultrasonically disrupted and lysed, followed by the first round of Ni... 2+Purification of histidine-tagged proteins using NTA affinity chromatography, followed by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. The Coomassie Brilliant Blue staining results are shown in Figures 1-3, corresponding to the Coomassie Brilliant Blue staining results of CABRi-3, CABRi-8, and CABRi-12 proteins, respectively. The components shown in the figures are as follows: Cell pellets are the supernatant after cell collection, lysis with lysis buffer, high-speed centrifugation, and resuspending of the pellets with an equal volume of lysis buffer; Cell lysate is the supernatant after cell collection, lysis with lysis buffer, and high-speed centrifugation; Flow through is the supernatant from cell lysis buffer passed through a Ni... 2+ -NTA followed by the permeate solution; Wash is the rinsing solution, used to rinse Ni. 2+ - The rinsing solution after NTA; Elution-first is the first elution solution, using 6 ml of Elution buffer to elute Ni. 2+ - The eluent collected after NTA; Elution-second is the second eluent, which is 6 ml of Elution buffer used to elute Ni. 2+ - The eluent collected after NTA; Elution-third is the third eluent, which is 8 ml of Elution buffer used to elute Ni. 2+ - The eluent collected after NTA; Elution-fourth is the fourth eluent, which is eluted with 10 ml of Elution buffer to remove Ni. 2+ - The eluent collected after NTA; Elution-fifth is the fifth eluent, which is eluted with 10 ml of Elution buffer to remove Ni. 2+ - The eluent collected after NTA; Elution-last is the final eluent, which is eluted with 10 ml of Elution buffer to remove Ni. 2+ - The eluent collected after NTA; Elution-total is the total eluent, which is the combined eluent collected above.

[0111] As shown in Figures 1-3, after the first step Ni 2+ After separation by NTA affinity chromatography, the target protein with a purity greater than 50% can be obtained.

[0112] Taking CABRi-8 as an example, after the first round of Ni 2+ After NTA affinity chromatography separation, a protein purification instrument (model UEV-25M Yonglian Biotechnology) and a cation exchange column (HiTrap) were used. TM The second round of separation and purification was performed using an SP / HP column, and the purification curves using the cation exchange column are shown in Figure 4. As shown in Figure 4, the CABRi-8 protein exhibited two elution peaks (Peak 1 and Peak 2). Simultaneously, the collection tubes corresponding to the CABRi-8 protein elution peaks were subjected to SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. The results are shown in Figure 5. Finally, CABRi-8 protein with a purity greater than 95% was obtained and stored at -80℃ for later use.

[0113] Example 3: Preparation and characteristics of a mixture of nanoscale targeted delivery protein carrier CABRi and siRNA

[0114] The CABRi series can form nanoparticles themselves. For example, the particle size and potential of nanoparticles formed by CABRi-8 and CABRi-8-siRNA are shown in Figures 6 and 8, respectively; the particle size and potential of nanoparticles formed by CABRi-12 and CABRi-12-siRNA are shown in Figures 7 and 9, respectively. The above values ​​are summarized in Table 1.

[0115] Table 1. Particle size and potential of nanoparticles formed by CABRi-8 carrier

[0116] The synthesized siRNA is a specific siRNA designed with human KRAS as the target (denoted as siKRAS). The siRNA is dissolved in DEPC water to a final concentration of 20 μM and stored at -20℃.

[0117] The sequences of the justice and antisense chains of siKRAS and siControl are shown below:

[0118] siKRAS Justice Chain: 5'-GUUGAUUACUUCUUAUUUUUC-3', SEQ ID NO.36;

[0119] siKRAS antisense strand: 5'-GAAAAAUAAGAAGUAAUCAACUG-3', SEQ ID NO.37;

[0120] siControl Justice Chain: 5'-CAUCAAGCUGGAGUGUCUCGC-3', SEQ ID NO.38;

[0121] siControl antisense chain: 5'-GCGAGACACUCCAGCUUGAUGUG-3', SEQ ID NO.39.

[0122] The prepared CABRi protein was mixed with siRNA at different molar ratios (e.g., siRNA:CABRi = 1:4 or 1:8). In this example, the prepared CABRi protein and siRNA were mixed at a molar ratio of 4:1. The reaction system was at pH 7.4, and the reaction was carried out at room temperature for 30 min to obtain CABRi-siRNA mixture molecules (CABRi-8-siRNA and CABRi-12-siRNA), forming nanoparticles of suitable size and high stability. As shown in Figures 8 and 9 and Table 1, the particle sizes of the CABRi-8-siRNA nanoparticles and CABRi-12-siRNA nanoparticles are approximately 42.21 nm and 54.03 nm, respectively, and their potentials are approximately -7.258 and -19.31, respectively, which can be used for in vivo delivery of siRNA.

[0123] CABRi's affinity for siRNA. Taking the CABRi-8 vector as an example, the proteins corresponding to the two peaks obtained by purification using a cation exchange column were used to prepare CABRi-siRNA nanoparticles with biotin-labeled siKRAS at molar ratios of 1:1, 2:1, 4:1, and 8:1, respectively. The binding ability of CABRi to siRNA was detected by electrophoretic mobility shift assay (EMSA), and the results are shown in Figure 10. As shown in Figure 10, at a molar ratio of siKRAS to CABRi-8 protein of 1:2 or 1:4, CABRi-8 protein can bind more than 90% of siKRAS. Similarly, as shown in Figure 11, CABRi-12 protein can also bind siKRAS efficiently.

[0124] CABRi-siRNA nanoparticles exhibit strong resistance to RNase degradation. Taking the CABRi-8 carrier as an example, CABRi-8 protein and siKRAS were mixed at a molar ratio of 4:1, and the reaction system was at pH 7.4, reacting at room temperature for 30 min. Then, different concentrations (0 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL) of RNase were added to the reaction system, and the reaction was carried out at 37℃ for 40 min. The protective effect of CABRi on siKRAS was detected by electrophoretic mobility shift assay (EMSA), and the results are shown in Figure 12. Figure 12 shows that under the protection of CABRi-8 protein, siRNA can resist high concentrations of RNase without being degraded.

[0125] With the total number of siRNA moles remaining constant, one or more siRNAs can be simultaneously packaged into the protein delivery vector CABRi. Both unmodified and chemically modified siRNAs are acceptable. Chemically modified siRNAs exhibit better performance, enhancing stability, resistance to nucleases, and delivery efficiency; they also reduce the immunogenicity of small nucleic acids. Examples of such chemical modifications include 2'-O-Methyl Base 2-methoxy modified bases (2'-ome), 2'-Fluoro RNA 2'-fluoroRNA (2'-F RNA), and Phosphorothioate modification (PS).

[0126] Example 4: CABRi-siRNA delivery efficiency and its mediating effect of silencing endogenous target genes

[0127] The delivery efficiency of CABRi protein delivery vectors for siRNA in cells was investigated. Taking the CABRi-8 vector as an example, biotin-labeled siKRAS was first mixed with CABRi-8 protein at a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles. Then, the CABRi-8-siRNA nanoparticles were added to cultured H1299 lung adenocarcinoma cells to be transfected, with a final siKRAS concentration of 60 nM. After 6 h, 12 h, and 24 h of transfection, the transfected cancer cells were collected, washed three times with PBS buffer, and RNA was extracted. Northern blotting analysis was performed, and the results are shown in Figure 13. Figure 13 shows that siKRAS was detectable at 6 h of transfection; the signal increased at 12 h; and the signal became strongest at 24 h. This result indicates that CABRi-8 protein can efficiently deliver siRNA and gradually accumulate within a certain time range.

[0128] CABRi-siRNA nanoparticles escape endosomes / lysosomes to release siRNA. Taking the CABRi-8 vector as an example, to detect siRNA escape from endosomes and avoid degradation in lysosomes, FAM'-labeled siKRAS was mixed with CABRi-8 protein at a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles. Then, the CABRi-8-siRNA nanoparticles were added to cultured H1299 lung adenocarcinoma cells awaiting transfection, bringing the final concentration of siKRAS to 60 nM. Lyso-Tracker Red was added half an hour before confocal microscopy observation of live cells, and the results are shown in Figure 14. As shown in Figure 14, at 6 h after transfection, siKRAS can be seen entering the cells and mostly colocalizing in endosomes / lysosomes (bright spots indicated by arrows in Figure 14); while at 16 h and 24 h after transfection, the pH-responsive endosome escape peptide plays a role, causing endosomes / lysosomes to rupture (diffuse spots indicated by circles in Figure 14). At the same time, siKRAS dissociates from CABRi and releases siRNA from endosomes into the cytoplasm.

[0129] The knockdown effect of CABRi-siRNA on target genes in cells. Taking the CABRi-8 vector as an example, siKRAS and CABRi-8 protein were mixed at a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles. A549 cells were then used at a cell density of 1.5 × 10⁻⁶ cells / year. 5 Cells were seeded into 12-well plates. After overnight growth, cells were treated with CABRi-8-siKRAS nanoparticles or an equal amount of CABRi-8 nanoparticles alone, with a final concentration of 240 nM. After culturing for 48 or 72 hours, cells were collected, washed with PBS, and added with lysis buffer. The same amount of protein was analyzed by Western blotting, and the results are shown in Figure 15. Figure 15 shows that the protein delivery vector CABRi-8 entered cells efficiently at 48 hours. Interestingly, by 72 hours, most of the CABRi-8 protein had degraded; the protein expression level of the target gene KARS was significantly reduced, and the phosphorylation level of the downstream signaling molecule pERK1 / 2 was also significantly downregulated. This indicates that CABRi-8-siKRAS nanoparticles can effectively reduce the intracellular expression level of the target gene KRAS, and CABRi-8 is rapidly degraded within 72 hours.

[0130] CABRi-12-siRNAs effectively inhibited tumor cell proliferation. Three effective siRNAs, including siKRAS, siSHP2, and siSOS1, were simultaneously packaged into the delivery protein carrier CABRi-12 at a constant total molar ratio (siRNA and CABRi-12 protein were mixed at a molar ratio of 1:4) to obtain CABRi-12-siRNA nanoparticles. Cells were treated with the CABRi-12-siRNA nanoparticles and CABRi-12 nanoparticles at a concentration of 240 nM. An untreated group, consisting of the untreated malignant lung cancer cell line H1975, was also included. After 24 hours of treatment, the treated H1975 cell line was cultured in plates at a seeding density of 500 cells / well. After 11 days of culture, crystal violet staining was performed, and the results were statistically analyzed, as shown in Figure 16. As shown in Figure 16, compared with the untreated group and the CABRi-12 treated group, CABRi-12-siRNAs effectively inhibited the cell proliferation ability of malignant tumor cells.

[0131] The sequences of siSHP2 and siSOS1 are shown below:

[0132] siSHP2 Justice Chain: 5'-GUUAGGAACGUCAAAGAAAGC-3', SEQ ID NO.40;

[0133] siSHP2 antisense chain: 5'-GCUUUCUUUGACGUUCCUAACAC-3', SEQ ID NO.41.

[0134] siSOS1 Justice Chain: 5'-CCCACAGUUGAGUGGCAUAUA-3', SEQ ID NO.42;

[0135] siSOS1 antisense chain: 5'-UAUAUGCCACUCAACUGUGGGAG-3', SEQ ID NO.43.

[0136] Example 5: CABRi-12-siRNAs specifically target tumor tissue

[0137] First, pancreatic cancer PANC1 tumor cells were subcutaneously inoculated into male nude mice (BALB / c-nu) until the tumors reached a diameter of 8 mm. Cy5-labeled siKRAS was mixed with the delivery protein carrier CABRi-12 to prepare CABRi-12-siRNA nanoparticles (preparation method was the same as that for CABRi-8-siRNA nanoparticles in Example 4), which were then injected via the tail vein. Mice were sacrificed 24 hours after injection, and major organ tissues, including the lungs, heart, liver, kidneys, spleen, and tumor masses, were isolated. The fluorescence intensity of siRNA-Cy5 in each organ was observed using a small animal fluorescence imaging system, and the results are shown in Figure 17. As shown in Figure 17, most of the siRNA was released primarily into the tumor tissue. Although a small amount of signal was observed in the liver tissue, no signal was observed in other organ tissues, including the lungs, heart, kidneys, and spleen. Therefore, the CABRi-12 carrier successfully, efficiently, and specifically targeted the tumor tissue.

[0138] Example 6: CABRi-12-siRNAs effectively treat lung cancer cell xenografts

[0139] When the density of lung adenocarcinoma A549 cell line reaches 80%-90% and the cells are in good condition, it can be used for experimental research. Before the experiment, the experimental cells in the logarithmic growth phase were washed once with PBS, 0.05% trypsin was added, and the cells were digested at 37°C for 1 min. The cells were then dispersed and collected into 15 mL centrifuge tubes, centrifuged at 800g for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. The cells were counted three times using a hemocytometer, and the average value was taken to prepare 2×10⁻⁶ cells. 7 A single-cell suspension of cells per mL (the cells must be thoroughly dispersed to form a single-cell suspension).

[0140] As required by the experiment, 6-7 week old male BALB / c-nu nude mice were used. No anesthesia was required; 0.1 mL of a 2×10⁻⁶ concentration was dispensed using a 1 mL syringe. 7 A single-cell suspension in logarithmic growth phase is administered via a needle inserted subcutaneously at a depth of approximately 0.5-1.0 cm into the back or armpit (to prevent leakage of the cell suspension). After creating a wheal under the skin, the needle is withdrawn, and the implantation is successful.

[0141] Mice injected with tumor cells were fed continuously for 2 weeks, and tumor growth was measured regularly. When the tumor diameter reached 5 mm, the mice were randomly assigned to two groups to begin drug treatment. The experiment consisted of two groups of 10 mice each.

[0142] Control group: PBS administered via tail vein injection for 4 days;

[0143] The CABRi-12-siRNA group: 0.5 nmol siKRAS, 0.5 nmol siSHP2, and 0.5 nmol siSOS1 were mixed with CABRi-12 protein at a molar ratio of 1:4. After standing at room temperature for 30 min, CABRi-12-siRNA nanoparticles containing the three siRNAs were obtained. These CABRi-12-siRNA nanoparticles were then injected into experimental mice via the tail vein, with treatment occurring every 4 days for a total of 3 treatments.

[0144] After treatment, the mice were observed for 8 days, then sacrificed, and the tumors were dissected, measured, and weighed. The results are shown in Figure 18. As shown in Figure 18, after three treatments, the tumor volume and weight in the treatment group were significantly reduced compared to the control group, indicating that CABRi-12-siRNA nanoparticles can significantly inhibit tumor growth in vivo.

[0145] Example 7 Safety assessment of the CABRi-siRNA delivery system

[0146] In Example 6, PBS-treated mice were used as controls to compare the in vivo physiological toxicity of the CABRi-12 delivery system. The mice's food intake, body weight, and activity levels were consistent with the control mice, and no toxic side effects were observed. After treatment, the heart, liver, spleen, lungs, kidneys, brain tissue, intestinal tissue, and muscle tissue of the mice were collected and stained with H&E. The results are shown in Figure 19. As shown in Figure 19, compared with the PBS control group, the tissues in the CABRi-12-siRNA treatment group were consistent with normal tissues, and no tissue lesions or other side effects were observed. This indicates that the assembled protein nanocarrier CABRi has good in vivo safety and no toxic side effects on normal tissues and organs.

[0147] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A CABRi, an assembled protein nanocarrier for targeted delivery of small interfering nucleic acids, characterized in that, The assembled protein nanocarrier CABRi includes assembled functional peptides, linkers, and small RNA-binding proteins. The assembled functional peptides include polypeptides with membrane-penetrating and pH-responsive functions and / or specific targeting peptides; the amino acid sequence of the polypeptide is any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.13; the amino acid sequence of the specific targeting peptide is any one of the sequences shown in SEQ ID NO.14-SEQ ID NO.24; The connector includes a flexible connector and / or a rigid connector; the amino acid sequence of the flexible connector is shown in SEQ ID NO.25 or SEQ ID NO.26; the amino acid sequence of the rigid connector is shown in SEQ ID NO.27 or SEQ ID NO.28; The amino acid sequence of the small RNA binding protein is any one of the sequences shown in SEQ ID NO.29-SEQ ID NO.

35.

2. The assembled protein nanocarrier CABRi according to claim 1, characterized in that, The copy number of the assembled functional peptide, the linker, and the small RNA binding protein is ≥1; the basic vector of the assembled protein nanocarrier CABRi includes prokaryotic expression vectors and eukaryotic expression vectors.

3. The method for preparing the assembled protein nanocarrier CABRi according to claim 1, characterized in that, Includes the following steps: The CABRi protein was obtained by linking the assembled functional peptide, the linker, and the small RNA-binding protein using seamless cloning technology. The CABRi protein was cloned into a base vector to obtain the assembled protein nanocarrier CABRi.

4. The preparation method according to claim 3, characterized in that, The copy number of the assembled functional peptide, the adapter, and the small RNA binding protein is ≥1; the basic vector includes prokaryotic expression vectors and eukaryotic expression vectors.

5. The application of the assembled protein nanocarrier CABRi according to claim 1 or 2 in the preparation of siRNA drug delivery systems, siRNA nanocomposites and targeted drugs.

6. An siRNA drug delivery system, characterized in that, The siRNA drug delivery system includes the assembled protein nanocarrier CABRi as described in claim 1 or 2.

7. A siRNA nanocomposite, characterized in that, The siRNA nanocomposite comprises the assembled protein nanocarrier CABRi and siRNA as described in claim 1 or 2.

8. The method for preparing the siRNA nanocomposite according to claim 7, characterized in that, The method includes the step of mixing the assembled protein nanocarrier CABRi as described in claim 1 with siRNA and incubating it to obtain the siRNA nanocomplex.

9. A targeted drug, characterized in that, The drug comprises the assembled protein nanocarrier CABRi and siRNA as described in claim 1 or 2.

10. The targeted drug according to claim 9, characterized in that, The siRNA includes one or more of siKRAS, siSHP2, and siSOS1; The positive chain sequence of siKRAS is shown in SEQ ID NO.36, and the negative chain sequence is shown in SEQ ID NO.37; the positive chain sequence of siSHP2 is shown in SEQ ID NO.40, and the negative chain sequence is shown in SEQ ID NO.41; the positive chain of siSOS1 is shown in SEQ ID NO.42, and the negative chain sequence is shown in SEQ ID NO.43.

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