CN115137834A - RNA plasmid delivery system for treating glioblastoma - Google Patents

Abstract

The present application provides an RNA plasmid delivery system for the treatment of glioblastoma. The system comprises a plasmid carrying an RNA fragment capable of treating glioblastoma, said plasmid being capable of enriching in the host's organ tissues and spontaneously forming endogenously in said host's organ tissues a complex structure containing said RNA fragment, said complex structure being capable of delivering said RNA fragment to the brain in order to treat glioblastoma. The safety and reliability of the RNA delivery system provided by the application are fully verified, and the RNA delivery system has the advantages of very good druggability, strong universality, very good economic benefit and excellent application prospect.

Description

RNA plasmid delivery system for treating glioblastoma

Technical Field

The application relates to the technical field of biomedicine, in particular to an RNA plasmid delivery system for treating glioblastoma multiforme.

Background

Glioblastomas are the most malignant of the astrocytic tumors. Glioblastomas grow rapidly, 70% -80% of patients have a disease course of 3-6 months, and the disease course is only 10% for more than 1 year. Those with longer disease progression may develop from astrocytomas with less malignancy. Because the tumor grows rapidly, the encephaledema is extensive, the intracranial pressure increase symptom is obvious, all patients have headache, vomiting symptom. The optic disc edema may be headache, mental change, weakness of limbs, vomiting, disturbance of consciousness and speech. Tumor infiltrates and destroys brain tissue, causing a series of focal symptoms, with patients with varying degrees of hemiplegia, hemiparesis, aphasia, hemianopsia, and the like. Examination of the nervous system can reveal hemiplegic, cranial nerve damage, hemiparesis and hemianopsia. The incidence of epilepsy is less common than astrocytoma and oligoglioma, some patients have epileptic seizures, and some patients show mental symptoms such as apathy, dementia, and mental retardation.

RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but in clinical practice a number of problems have been encountered, the progress of which has fallen far behind expectations.

It is generally considered that RNA cannot exist stably for a long period outside cells because RNA is degraded into fragments by RNase which is abundant outside cells, and therefore, a method for stably existing RNA outside cells and allowing targeted entry into a specific tissue must be found to highlight the effect of RNAi therapy.

Many patents related to siRNA are focused on the following aspects: 1. siRNA with medical effect is designed. 2. The siRNA is chemically modified, so that the stability of the siRNA in organisms is improved, and the yield is improved. 3. Various artificial carriers (such as lipid nanoparticles, cationic polymers and viruses) are designed to improve the efficiency of siRNA delivery in vivo. Many of these patents in aspect 3 are based on the fact that researchers have recognized the lack of suitable siRNA delivery systems to deliver siRNA to target tissues safely, accurately and efficiently, which has become a central problem in RNAi therapy.

Chinese patent with publication number CN108624590A discloses a siRNA capable of inhibiting DDR2 gene expression; chinese patent with publication number CN108624591A discloses a siRNA capable of silencing ARPC4 gene, and the siRNA is modified by alpha-phosphorus-selenium; chinese patent with publication number CN108546702A discloses siRNA of targeting long-chain non-coding RNA DDX11-AS 1. Chinese patent publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents have designed specific sirnas and are directed to certain diseases caused by genetic changes.

Chinese patent publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as carrier of siRNA. Chinese patent publication No. CN108117585A discloses a polypeptide for promoting breast cancer cell apoptosis by targeted introduction of siRNA, and the polypeptide is also used as a carrier of siRNA. Chinese patent publication No. CN108096583A discloses a nanoparticle carrier, which contains chemotherapeutic drugs and also can be loaded with siRNA with breast cancer therapeutic effect. These patents are all inventions in the aspect of siRNA vector, but the technical scheme has a common feature that the vector and siRNA are pre-assembled in vitro and then introduced into the host. In fact, most of the current designs for delivery technology do so. However, such delivery systems have a common problem in that these artificially synthesized exogenous delivery systems are easily cleared by the host's circulatory system, may elicit an immunogenic response, and may even be toxic to specific cell types and tissues.

The research team of the present invention finds that endogenous cells can selectively encapsulate miRNAs into exosomes (exosomes) which can deliver miRNAs into recipient cells, and the secreted miRNAs can powerfully block the expression of target genes at relatively low concentrations. Exosomes are biocompatible with the host immune system and have the innate ability to protect and transport miRNA across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery. For example, chinese patent publication No. CN110699382A discloses a method for preparing exosomes for delivering siRNA, and discloses a technique for isolating exosomes from plasma and encapsulating siRNA into exosomes by electroporation.

However, such technologies for in vitro separation or preparation of exosomes usually require a large amount of exosomes obtained by cell culture and a step of siRNA encapsulation, which makes the clinical cost of large-scale application of the product very high and cannot be borne by general patients; more importantly, the complex production/purification process of exosomes makes it almost impossible to comply with GMP standards.

The drug taking exosome as an active ingredient has not been approved by CFDA so far, and the core problem is that the consistency of exosome products cannot be ensured, and the problem directly results in that the products cannot obtain the drug production license. If this problem can be solved, it would be of great significance to drive RNAi therapy to treat glioblastoma.

Therefore, the development of a safe, accurate and efficient siRNA delivery system is a crucial part for improving the effect of RNAi therapy and promoting RNAi therapy.

Disclosure of Invention

In view of the above, the present embodiments provide an RNA plasmid delivery system for treating glioblastoma, so as to solve the technical defects existing in the prior art.

One aspect of the present invention is to provide an RNA plasmid delivery system for treating glioblastoma, the system including a plasmid carrying RNA fragments capable of treating glioblastoma, the plasmid being capable of being enriched in organ tissues of a host and forming a composite structure containing the RNA fragments endogenously and spontaneously in the organ tissues of the host, the composite structure being capable of delivering the RNA fragments into the brain to treat glioblastoma.

Optionally, the RNA fragment comprises 1, two or more specific RNA sequences of medical interest, said RNA sequences being siRNA, shRNA or miRNA sequences of medical interest capable of inhibiting or hindering the development of glioblastoma.

It is shown by FIGS. 14-17 that the plasmid does have the effect of enriching in vivo and spontaneously forming a complex structure containing RNA fragments.

Optionally, the plasmid further comprises a promoter and a targeting tag, wherein the targeting tag is capable of forming a targeting structure of the composite structure in the organ tissue of the host, the targeting structure is located on the surface of the composite structure, and the composite structure is capable of finding and binding to the target tissue through the targeting structure to deliver the RNA fragment into the target tissue.

Optionally, the plasmid comprises any one or a combination of several of the following lines: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each plasmid at least comprises an RNA segment and a targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines.

The effect of in vivo enrichment and spontaneous formation of complex structures containing RNA fragments is demonstrated by FIGS. 18-19 for plasmids containing multiple RNA fragments and multiple targeting tags.

Optionally, the plasmid further comprises flanking sequences, compensating sequences and loop sequences capable of folding the lines into the correct structure and expressing, the flanking sequences comprising a 5 'flanking sequence and a 3' flanking sequence;

the plasmid comprises any one line or combination of lines as follows: 5' -promoter-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence, 5' -promoter-targeting tag or 5' -promoter-targeting tag-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence.

Optionally, the 5' flanking sequence is ggatcctggaggcttgctgtaggctgctgtatgctgaattc or a sequence having more than 80% homology thereto;

the loop sequence is gtttggccactgactgac or a sequence with homology more than 80 percent;

<xnotran> 3' accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 80% ; </xnotran>

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted. The base at position 1 to 5 of the reverse complement sequence of RNA is deleted in order to prevent the sequence from being expressed.

It is shown in FIGS. 20-23 that plasmids containing homologous sequences of different flanking sequences and loop sequences all have in vivo enrichment, self-assembly, and therapeutic effects.

Preferably, the complementing sequence is the reverse complement of the RNA fragment, and any 1-3 bases in the RNA fragment are deleted.

More preferably, the complementary sequence is the reverse complement of the RNA fragment, and any 1-3 consecutive bases in the complementary sequence are deleted.

Most preferably, the complementing sequence is the reverse complement of the RNA fragment, and the 9 th and/or 10 th base is deleted.

Alternatively, in the case where at least two lines are present in the plasmid, adjacent lines are connected by a sequence consisting of sequences 1 to 3;

wherein, the sequence 1 is CAGATC, the sequence 2 is a sequence consisting of 5-80 bases, and the sequence 3 is TGGATC.

FIGS. 24-25 show that the plasmid carries four lines and sequence 2 has multiple bases, all of which have enrichment, self-assembly and therapeutic effects in vivo.

Alternatively, in the case where at least two lines are present in the plasmid, adjacent lines are connected by sequence 4 or a sequence having more than 80% homology to sequence 4;

wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGAC.

FIG. 26 shows that plasmids constructed with SEQ ID No. 4 and homologous sequences both have enrichment and self-assembly effects.

Optionally, the organ tissue is liver and the composite structure is exosome.

Optionally, the targeting tag is selected from a targeting peptide or a targeting protein having a targeting function.

It is shown by FIGS. 27-28 that plasmids containing either a targeting peptide tag or a targeting protein tag all have the effect of enriching in vivo and spontaneously forming a complex structure containing RNA fragments.

Optionally, the targeting peptide comprises an RVG targeting peptide, a GE11 targeting peptide, a PTP targeting peptide, a TCP-1 targeting peptide, an MSP targeting peptide;

the target protein comprises RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein and MSP-LAMP2B fusion protein.

The targeting tag is preferably a RVG targeting peptide or a RVG-LAMP2B fusion protein.

Optionally, the RNA sequence is 15-25 nucleotides in length.

Optionally, the RNA capable of treating glioblastoma is selected from any one or several of the following RNAs: siRNA of EGFR gene, siRNA of TNC gene, or RNA sequence with homology more than 80% with the sequence, or nucleic acid molecule for coding the RNA.

The siRNA of the EGFR gene comprises UGUUGCUUCUUCUAAUUCU, AAAUGAUCUUCAAAGUGCCC, UCUUAAGAAGGAAAGAUCAU, AAUAUUCCGUAGCAUUUAGGA, UAAAUCCUCACAAUACUU, other sequences which can inhibit the expression of the EGFR gene and sequences which have homology of more than 80 percent with the sequences.

The siRNA of TNC gene includes UAUGAAAUGUAAAAAAAGGGA, AAUCAUCCUUAAAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAUCCUUAAGUUCAUCAU, AGAAGUAAAAAACUAUUGCGA, other sequences capable of inhibiting TNC gene expression and sequences with homology more than 80%.

The "sequence having a homology of more than 80" may be 85%, 88%, 90%, 95%, 98%, or the like.

The gene routes of siRNA containing EGFR gene, siRNA containing TNC gene are shown by FIGS. 29-30 to have the effect of enriching in vivo and spontaneously forming complex structures containing RNA fragments.

Optionally, the RNA fragment comprises an RNA sequence body and a modified RNA sequence obtained by ribose-modifying the RNA sequence body. That is, the RNA fragment may consist of only at least one RNA sequence entity, may consist of only at least one modified RNA sequence, or may consist of both the RNA sequence entity and the modified RNA sequence.

In the present invention, the isolated nucleic acid also includes variants and derivatives thereof. The nucleic acids can be modified by one of ordinary skill in the art using conventional methods. Modifications include (but are not limited to): methylation modification, alkyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, alkyl-glycosyl modification, sugar ring modification and the like), nucleic acid modification, peptide segment modification, lipid modification, halogen modification, nucleic acid modification (such as 'TT' modification) and the like. In one embodiment of the invention, the modification is an internucleotide linkage, for example selected from: thiophosphate, 2'-O Methoxyethyl (MOE), 2' -fluoro, alkyl phosphonate, dithiophosphate, alkyl thiophosphonate, phosphoramidate, carbamate, carbonate, phosphotriester, acetamide ester, carboxymethyl ester, and combinations thereof. In one embodiment of the invention, the modification is a modification to a nucleotide, for example selected from: peptide Nucleic Acids (PNA), locked Nucleic Acids (LNA), arabinose-nucleic acids (FANA), analogs, derivatives, and combinations thereof. Preferably, the modification is a 2' fluoropyrimidine modification. The 2 '-fluoropyrimidine modification is to replace 2' -OH of pyrimidine nucleotide on RNA with 2'-F, and the 2' -F can make RNA not be easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment in vivo delivery.

FIG. 31 shows that the delivery system containing RNA sequence modified by ribose has in vivo enrichment and self-assembly effects.

Optionally, the delivery system is a delivery system for use in a mammal, including a human.

The application also provides an RNA delivery system for the treatment of glioblastoma in a medicament.

Optionally, the administration of the drug comprises oral administration, inhalation, subcutaneous injection, intramuscular injection, intravenous injection.

The technical effects of this application do:

the RNA delivery system for treating glioblastoma provided by the application takes the plasmid as a carrier and the plasmid as a mature injectant, so that the safety and reliability of the RNA delivery system are fully verified, and the medicine property is very good. The RNA sequence which finally exerts the effect is encapsulated and transported by endogenous exosomes, there is no immune reaction, and the safety of the exosome does not need to be verified. The delivery system can deliver various small-molecule RNAs and has strong universality. And the preparation of the plasmid is cheaper than that of the exosome or the substances such as protein, polypeptide and the like, and the economy is good. The RNA delivery system for treating glioblastoma provided herein is capable of self-assembling with an AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

The RNA delivery system for treating glioblastoma multiforme is applied to medicines, namely a medicine delivery platform is provided, the treatment effect of glioblastoma multiforme can be greatly improved, the research and development basis of more RNA medicines can be formed through the platform, and the RNA medicine research and development and use are greatly promoted.

Drawings

FIG. 1 is a graph comparing the distribution of plasmids and their metabolism in mice according to one embodiment of the present application;

FIG. 2 is a graph comparing the expression levels of proteins in mice provided by an embodiment of the present application;

FIG. 3 is a graph comparing relevant siRNA levels in mice provided by an example of the present application;

FIG. 4 is a graph comparing absolute siRNA levels in various tissues of a mouse as provided in an example of the present application;

FIG. 5 is a graph comparing the effect of plasmid dose on mouse siRNA levels as provided in one embodiment of the present application;

FIG. 6 is a graph comparing the metabolism of precursors and matures in the liver of mice injected with plasmids, provided by an embodiment of the present application;

FIG. 7 is a graph comparing the kinetics and distribution of siRNA in different tissues of a mouse, as provided by an example of the present application;

FIG. 8 is a graph comparing the effect of different promoters on siRNA provided in one embodiment of the present application;

FIG. 9 is a graph comparing the eGFP fluorescence intensity in different tissues of a mouse, as provided in an example of the present application;

FIG. 10 is a graph comparing the levels of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus weight, spleen weight, and percentage of peripheral blood cells in mice provided by an example of the present application;

FIG. 11 is a graph showing a comparison of the expression levels of siRNA in mice provided in an embodiment of the present application;

FIG. 12 is a graph comparing the treatment of glioblastoma in mice according to one embodiment of the present application;

FIG. 13 is a comparison of mouse brain immunohistological staining provided in an embodiment of the present application.

FIG. 14 is a graph showing the effect of the plasmid delivery system provided in one embodiment of the present application on in vivo enrichment and spontaneous formation of complex structure in the case of carrying 1 RNA fragment alone; wherein A is the enrichment effect of plasmids containing different RNA fragments in vivo, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.

FIG. 15 is a graph showing the effect of the plasmid delivery system provided in one embodiment of the present application on the in vivo enrichment and spontaneous formation of complex structure in the case of carrying any 2 RNA fragments; wherein A is the enrichment effect of plasmids containing different combination RNA fragments in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of the different combination RNA fragments.

FIG. 16 is a diagram illustrating the effect of the plasmid delivery system provided in one embodiment of the present application on enrichment in vivo and spontaneous formation of complex structure when carrying any 3 RNA fragments; wherein A is the enrichment effect of plasmids containing different combinations of RNA fragments in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of the different combinations of RNA fragments.

FIG. 17 is a graph showing the effect of the plasmid delivery system provided in another embodiment of the present application on the in vivo enrichment and spontaneous formation of complex structure in the case of carrying any 2 RNA fragments; wherein A is the enrichment effect of plasmids containing different combination RNA fragments in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of the different combination RNA fragments.

FIG. 18 is a verification that the plasmid delivery system provided in one embodiment of the present application has in vivo enrichment effect when carrying random 1-2 RNA fragments and 1-2 targeting tags in the same line. FIG. 19 is a verification that the plasmid delivery system provided in another embodiment of the present application has in vivo enrichment effect in the case of carrying random 1-2 RNA fragments and 1-2 targeting tags, which are located in different lines.

FIG. 20 is a demonstration of the effect of a plasmid delivery system provided in one embodiment of the present application on in vivo enrichment and spontaneous formation of complex structures, carrying well-defined 5' flanking sequences and at least 2 defined sequences with greater than 80% homology thereto; wherein A is the enrichment effect in vivo of plasmids containing different 5 'flanking sequences, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different 5' flanking sequences.

FIG. 21 is a graph showing the effect of in vivo enrichment and spontaneous formation of complex structures in a plasmid delivery system provided in one embodiment of the present application, carrying a defined loop sequence and at least 2 defined sequences having greater than 80% homology thereto; wherein A is the enrichment effect of plasmids containing different loop sequences in vivo, and B is the in vivo self-assembly effect displayed by the expression level of RNA fragments of different loop sequences.

FIG. 22 is a demonstration of the effect of a plasmid delivery system provided in one embodiment of the present application on in vivo enrichment and spontaneous formation of complex structures, carrying well-defined 3' flanking sequences and at least 2 defined sequences with greater than 80% homology thereto; wherein A is the enrichment effect in vivo of plasmids containing different 3 'flanking sequences, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different 3' flanking sequences.

FIG. 23 is a verification of the effect of the plasmid delivery system provided in one embodiment of the present application on the in vivo enrichment and spontaneous formation of complex structure of RNA sequence carrying reverse complementary sequence after deletion of any 1, 2, 3, 4, 5 bases; wherein A is the enrichment effect of plasmids containing different compensation sequences in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of RNA fragments of different compensation sequences.

FIG. 24 is a graph showing the effect of the plasmid delivery system provided in one embodiment of the present application in spontaneously forming a complex structure when four of the lines are carried and adjacent lines are connected with each other by the sequences 1 to 2 to 3.

FIG. 25 shows a verification of the effect of the plasmid delivery system of the present application on spontaneous formation of complex structure when the plasmid delivery system carries four of the above-mentioned lines, and the adjacent lines are connected by the sequences 1-2-3, and the sequences 2 are respectively composed of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases.

FIG. 26 is a diagram showing the effect of spontaneously forming a complex structure when a plasmid delivery system provided in one embodiment of the present application contains a sequence having a linker sequence of SEQ ID No. 4 and at least 2 sequences having a homology of more than 80% to SEQ ID No. 4.

Figure 27 is a demonstration of the efficacy of the plasmid delivery system provided in one embodiment of the present application with in vivo enrichment when only targeting peptide tags are present.

FIG. 28 is a graph demonstrating the effect of in vivo enrichment when the plasmid delivery system provided in one embodiment of the present application contains only the targeting protein tag.

FIG. 29 is a diagram illustrating in vivo enrichment and spontaneous formation of complex structures of siRNA containing EGFR gene in a genetic circuit according to an embodiment of the present application; wherein A is the enrichment effect of different gene circuits containing EGFR gene siRNA sequences in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of different siRNA sequences containing EGFR gene.

FIG. 30 is a graph showing the effect of in vivo enrichment and spontaneous formation of a complex structure when siRNA containing TNC gene is contained in the gene line provided in an embodiment of the present application; wherein A is the enrichment effect of different gene circuits containing TNC gene siRNA sequences in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of different TNC gene siRNA containing sequences.

FIG. 31 is a graph showing the effect of in vivo enrichment and spontaneous formation of complex structures when a delivery system containing 2 different RNA sequences modified with ribose is provided according to an embodiment of the present application; wherein A is the effect of the delivery system of RNA modified by different ribose on enrichment in vivo, and B is the effect of self-assembly in vivo shown by the expression level of RNA modified by different ribose.

Detailed Description

The following description of specific embodiments of the present application refers to the accompanying drawings.

First, terms, test methods, and the like according to the present invention will be explained.

Hematoxylin-eosin staining (HE staining) is short for hematoxylin-eosin staining. HE staining is one of the most basic and widely used technical methods in histology, pathology teaching and scientific research.

The hematoxylin staining solution is alkaline, and can stain basophilic structures (such as ribosome, nucleus, ribonucleic acid in cytoplasm and the like) of tissues into bluish purple; eosin is an acid dye that stains tissue eosinophils (e.g., intracellular and intercellular proteins, including lewy bodies, alcosomes, and most of the cytoplasm) pink, making the morphology of the entire tissue clearly visible.

The HE staining method comprises the following specific steps: fixing and slicing sample tissues; deparaffinizing the tissue sample; hydrating the tissue sample; staining tissue sections with hematoxylin, differentiating and turning blue; eosin staining and dehydrating the tissue section; air-drying the tissue sample slice and sealing; finally, the film was observed under a microscope and photographed.

Masson staining gives collagen fibers either a blue (stained with aniline blue) or green (stained with brilliant green) color and muscle fibers a red (stained with acid fuchsin and ponceau red) color, depending on the size of the anionic dye molecules and the permeability of the tissue. Fixed tissue is stained with a series of anionic water-soluble dyes, either sequentially or in combination, and it is found that red blood cells are stained with the smallest anionic dye, muscle fibers and cytoplasm are stained with the medium-sized anionic dye, and collagen fibers are stained with the larger anionic dye. This demonstrates that the permeability of erythrocytes to anionic dyes is minimal, the muscle fibers are inferior to the cytoplasm, and collagen fibers have the greatest permeability. Type I and type III collagen are green (GBM, TBM, mesangial matrix, and renal interstitium are green), and rhodopsin, tubule cytoplasm, and erythrocytes are red.

The Masson staining method comprises the following specific steps:

fixing the tissue in Bouin's fluid, flushing with running water for one night, and conventionally dehydrating and embedding; slicing and dewaxing to water (dewaxing in xylene for 10min × 3 times, blotting liquid with absorbent paper, 100% ethanol for 5min × 2 times, blotting liquid with absorbent paper, 95% ethanol for 5min × 2 times, blotting liquid with absorbent paper, flowing for 2min, blotting water with absorbent paper); weiger's ferrohematoxylin staining for 5-10min; slightly washing with running water; differentiating with 0.5% hydrochloric acid alcohol for 15s; flushing with running water for 3min; dyeing the ponceau acid fuchsin liquid for 8min; slightly washing with distilled water; treating with 1% phosphomolybdic acid water solution for about 5min; without washing, the fabric is directly re-dyed with aniline blue solution or brilliant green solution for 5min; treating with 1% glacial acetic acid for 1min; dehydrating with 95% ethanol for 5min × 2 times, and drying with absorbent paper; 100% ethanol for 5min × 2 times, and sucking off the liquid with absorbent paper; transparent in xylene for 5min × 2 times, and sucking the liquid with absorbent paper; and (5) sealing the neutral gum.

The Western immunoblotting (Western Blot) is carried out by transferring the protein to a membrane and detecting the protein with an antibody.

Western Blot was performed by polyacrylamide gel electrophoresis, and the test substance was a protein, "probe" was an antibody, "and" secondary antibody for color development "was labeled. Transferring the protein sample separated by PAGE to a solid phase carrier (such as nitrocellulose film), adsorbing the protein by the solid phase carrier in a non-covalent bond form, keeping the type and biological activity of the electrophoretically separated polypeptide unchanged, taking the protein or polypeptide on the solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then reacting with an enzyme or isotope labeled second antibody, and carrying out substrate chromogenic or autoradiography to detect the protein component expressed by the specific target gene separated by electrophoresis. The method mainly comprises the following steps: protein extraction, protein quantification, glue preparation and electrophoresis, membrane transfer, immune labeling and development.

Immunohistochemistry, which is the principle of antigen-antibody reaction, i.e., the specific binding of antigen and antibody, determines the antigens (polypeptides and proteins) in tissue cells by developing color-developing agents (fluorescein, enzyme, metal ions, isotopes) of labeled antibodies through chemical reaction, and performs localized, qualitative and relatively quantitative studies on the antigens, is called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).

The main steps of immunohistochemistry include: soaking the slices, airing overnight, dewaxing xylene, dewaxing gradient alcohol (100%, 95%, 90%, 80%, 75%, 70%, 50%, 3min each time), double-distilling with water, dropping 3% hydrogen peroxide solution to remove catalase, washing with water, repairing antigen, dropping 5% BSA, sealing for 1h, diluting primary antibody, washing with PBS buffer solution, incubating secondary antibody, washing with PBS buffer solution, developing with developing solution, washing with water, dyeing with hematoxylin, dehydrating with gradient ethanol, and sealing with neutral gum.

The detection of siRNA level, protein content and mRNA content in the invention is realized by injecting RNA delivery system into mouse body to establish mouse stem cell in vitro model. mRNA and siRNA expression levels in cells and tissues were examined by qRT-PCR. The absolute quantification of siRNA was determined by plotting a standard curve against the standard. The expression amount of each siRNA or mRNA relative to the internal reference can be expressed by 2- Δ CT, where Δ CT = C sample-C internal reference. The internal reference gene is U6 snRNA (in tissues) or miR-16 (in serum and exosomes) molecules when siRNA is amplified, and the time base of mRNA is GAPDH or 18s RNA. And detecting the expression level of the protein in the cells and tissues by using a Western blotting experiment, and carrying out protein quantitative analysis by using ImageJ software.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those that are widely used in the corresponding fields.

Example 1

The present embodiment provides an RNA plasmid delivery system for treating glioblastoma, the system comprising a plasmid carrying RNA fragments capable of treating glioblastoma, the plasmid being capable of being enriched in organ tissues of a host and spontaneously forming a composite structure containing the RNA fragments endogenously in the organ tissues of the host, the composite structure being capable of delivering the RNA fragments into the brain to treat glioblastoma.

In this example, the plasmid also includes a promoter and a targeting tag. The plasmid comprises any one line or combination of lines as follows: the plasmid comprises a promoter-RNA sequence, a promoter-targeting label and a promoter-RNA sequence-targeting label, wherein each plasmid at least comprises an RNA segment and a targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines. In other words, the plasmid may include only the promoter-RNA sequence-targeting tag, or may include a combination of the promoter-RNA sequence, the promoter-targeting tag, or a combination of the promoter-targeting tag, the promoter-RNA sequence-targeting tag.

In order to prove that the plasmid has the effects of in vivo enrichment and spontaneous formation of a composite structure containing RNA fragments, 1-2 RNA fragments and 1-2 targeting labels are randomly adopted in the invention, the RNA fragments and the targeting labels are respectively positioned in the same or different lines, and the enrichment and self-assembly effects of the plasmid are verified through experiments, and are shown in figures 18-19. The grouping is listed as follows:

1. in the same lines (both including the promoter) (fig. 18):

1) RNA segment 1+ targeting label 1, RNA segment 2+ targeting label 2, RNA segment 1+ targeting label 2, RNA segment 2+ targeting label 1;

2) RNA fragment 1+ RNA fragment 2+ targeting tag 1, RNA fragment 1+ RNA fragment 2+ targeting tag 2, RNA fragment 1+ targeting tag 2, RNA fragment 2+ targeting tag 1+ targeting tag 2;

3) RNA fragment 1+ RNA fragment 2+ targeting tag 1+ targeting tag 2;

2. in different gene lines (all including promoters) (fig. 19):

1) RNA segment 1+ targeting label 1, RNA segment 2+ targeting label 2, RNA segment 1+ targeting label 2, RNA segment 2+ targeting label 1;

2) RNA fragment 1+ RNA fragment 2+ targeting tag 1, RNA fragment 1+ RNA fragment 2+ targeting tag 2, RNA fragment 1+ targeting tag 2, RNA fragment 2+ targeting tag 1+ targeting tag 2;

3) RNA fragment 1+ RNA fragment 2+ targeting tag 1+ targeting tag 2.

Further, the plasmid may also include flanking sequences including 5 'flanking sequences and 3' flanking sequences, compensating sequences and loop sequences that enable the lines to be folded into the correct structure and expressed; the plasmid comprises any one line or combination of lines as follows: 5 '-promoter-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 'flanking sequence, 5' -promoter-targeting tag-5 'flanking sequence-RNA sequence-loop sequence-compensating sequence-3' flanking sequence.

In order to prove that the plasmids have the effects of in vivo enrichment and spontaneous formation of a composite structure containing RNA fragments, the invention randomly provides 4 groups of plasmids containing different sequences, and the enrichment and self-assembly effects of the plasmids are verified through experiments, which are shown in figures 20-23. The grouping is listed as follows:

1. the above-identified 5' flanking sequences and at least 2 identified sequences having greater than 80% homology thereto;

2. the defined loop sequence and at least 2 defined sequences having more than 80% homology thereto;

3. the above-mentioned already defined 3' flanking sequences and at least 2 defined sequences having more than 80% homology thereto;

4. 2 to 3 of the RNA sequences are selected, and any of the 1, 2, 3, 4, and 5 bases is deleted to obtain a reverse complementary sequence.

Wherein, the 5' flanking sequence is preferably ggatcctggaggcttgctgagaggctgtatgctgactgaattc or a sequence with homology of more than 80 percent with the ggatcctggaggcttgctgagagctgctgtatgctgaattc, including a sequence with homology of 85 percent, 90 percent, 92 percent, 95 percent, 98 percent, 99 percent with the ggatcctggaggctgaattc, and the like.

The loop sequence is preferably gttttgggccactgactgac or a sequence with homology of more than 80 percent, and comprises sequences with homology of 85 percent, 90 percent, 92 percent, 95 percent, 98 percent, 99 percent and the like with the gtttggccactgactgac.

<xnotran> 3' accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 80% , accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 85%, 90%, 92%, 95%, 98%, 99% . </xnotran>

The sequences are specifically shown in table 1 below.

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence from which any 1-5 bases are deleted.

Preferably, the complementing sequence is a reverse complement of the RNA fragment, and any 1-3 bases in the complementing sequence are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence from which any 1 to 3 bases are deleted.

More preferably, the complementary sequence is the reverse complement of the RNA fragment, and any 1-3 consecutive bases in the complementary sequence are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence from which any of the bases arranged consecutively at positions 1 to 3 are deleted.

Most preferably, the complementing sequence is the reverse complement of the RNA fragment, and the 9 th and/or 10 th base is deleted. When only one RNA sequence is included in the RNA fragment, the complementing sequence may be a reverse complement of the RNA sequence from which the 9 th and/or 10 th position is deleted. The deletion of the 9 th and 10 th bases is most effective.

The flanking sequence, the compensating sequence and the loop sequence are not randomly selected, but are determined based on a large amount of theoretical research and experiments, and the expression rate of the RNA fragment can be improved to the maximum extent under the coordination of the specific flanking sequence, the compensating sequence and the loop sequence.

In the case of plasmids carrying two or more strands, adjacent strands may be connected by the sequences 1-2-3; among them, the sequence 1 is preferably CAGATC, the sequence 2 may be a sequence consisting of 5 to 80 bases, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases, preferably a sequence consisting of 10 to 50 bases, more preferably a sequence consisting of 20 to 40 bases, and the sequence 3 is preferably TGGATC.

In order to prove that the plasmids have the effects of in vivo enrichment and spontaneous formation of a composite structure containing RNA fragments, experimental data that when a group of plasmids carry four lines, adjacent lines are connected by a sequence 1-a sequence 2-a sequence 3 are randomly provided, and the enrichment and self-assembly effects of the plasmids are verified through experiments, and are particularly shown in FIG. 24.

Meanwhile, in order to prove that the plasmids have the effects of in vivo enrichment and spontaneous formation of a complex structure containing RNA fragments, the invention randomly provides experimental data consisting of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases and 80 bases respectively when a group of plasmids carry four lines, and the sequences 2 are respectively connected by the sequences 1-2-3, and the experimental data verify the enrichment and self-assembly effects of the plasmids, as shown in FIG. 25.

Sequence 2 is specifically shown in table 2 below.

More preferably, in the case of plasmids carrying two or more lines, the adjacent lines are connected by sequence 4 or a sequence having more than 80% homology to sequence 4; wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACGACCAGTGGAC.

In order to prove that the plasmids have the effects of in vivo enrichment and spontaneous formation of a composite structure containing RNA fragments, the invention randomly provides a group of corresponding experimental data of the plasmids containing a connecting sequence of sequence 4 and at least 2 sequences with homology of more than 80 percent with the sequence 4, and verifies the enrichment and self-assembly effects of the plasmids through experiments, and the experimental data is shown in figure 26.

The sequence is specifically shown in Table 3 below.

Name (R)	Sequence of
		Sequence 4	CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
Sequence 4-1	CAGATCTGCTCTAACTCGATTTAGTGAGTCGACCAGTGGATC
		Sequence 4-2	CAGATCTGGTTTCACTCATTCTAGTGAGTCGACCAGTGGATC

The RNA fragments described above comprise 1, two or more specific RNA sequences of medical interest, which are capable of being expressed in the target receptor and the complementing sequences are not capable of being expressed in the target receptor. The RNA sequence can be an siRNA sequence, an shRNA sequence or an miRNA sequence, and is preferably an siRNA sequence.

An RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, 22 nt. The range of the sequence length is not arbitrarily selected, but determined by trial and error. A large number of experiments prove that under the condition that the length of the RNA sequence is less than 18nt, particularly less than 15nt, the RNA sequence is mostly ineffective and can not play a role, and under the condition that the length of the RNA sequence is more than 22nt, particularly more than 25nt, the cost of a line is greatly improved, the effect is not better than that of the RNA sequence with the length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15 to 25nt, particularly 18 to 22nt, both cost and action can be achieved, and the effect is the best.

The RNA segment capable of treating the glioblastoma is selected from any one or more of the following: siRNA of EGFR gene, siRNA of TNC gene, or nucleic acid molecules encoding the above RNAs.

The number of effective sequences of the RNA to be delivered is 1, 2 or more. For example, if glioblastoma needs to be treated, the siRNA of the EGFR gene and the siRNA of the TNC gene may be used in combination on the same plasmid vector, or the siRNA of the EGFR gene or the siRNA of the TNC gene may be used alone.

Taking the combined use of "siRNA1" and "siRNA2" on the same plasmid vector as an example, the functional structural region of the plasmid vector can be represented as: (promoter-siRNA 1) -joining sequence- (promoter-siRNA 2) -joining sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA 1) -joining sequence- (promoter-targeting tag-siRNA 2), or (promoter-siRNA 1) -joining sequence- (promoter-targeting tag-siRNA 2), and the like.

More specifically, the functional structural region of the plasmid vector can be represented as: (5 ' -promoter-5 ' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag) -5' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), or (5 ' -promoter-5 ' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), (5 ' -promoter-targeting tag-5 ' flanking sequence-1-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), etc. In other cases, the same can be analogized, and the description is omitted here. The above linker sequence may be "sequence 1-sequence 2-sequence 3" or "sequence 4", and a bracket indicates a complete line (circuit).

Preferably, the RNA may be obtained by modifying RNA sequences (siRNA, shRNA, or miRNA) thereof with ribose, preferably 2' fluoropyrimidine. The 2 '-fluoropyrimidine modification is to replace 2' -OH of pyrimidine nucleotide on siRNA, shRNA or miRNA with 2'-F, wherein 2' -F can make RNA enzyme in human body not easily recognize siRNA, shRNA or miRNA, thus being capable of increasing the stability of RNA transmission in vivo.

In order to prove that the delivery system has the effects of enriching in vivo and spontaneously forming a composite structure containing RNA fragments, the invention randomly provides experimental data of the RNA sequence of the delivery system after ribose modification, and verifies the enriching and self-assembling effects of the delivery system through experiments, as shown in FIG. 31 in particular.

Specifically, the liver phagocytoses exogenous plasmids, and up to 99% of the exogenous plasmids enter the liver, so that when the plasmids are used as a vector, the exogenous plasmids can be enriched in liver tissues without specific design, and then the exogenous plasmids are opened to release RNA molecules (siRNA, shRNA or miRNA), and the liver tissues spontaneously wrap the RNA molecules into exosomes, which become RNA delivery mechanisms.

Preferably, in order to make the RNA delivery mechanism (exosome) have the ability of "precise targeting", we design a targeting tag in the plasmid injected into the body, and the targeting tag will also be assembled into the exosome by the liver tissue, especially when selecting some specific targeting tags, the targeting tag can be inserted into the exosome surface, thereby becoming a targeting structure capable of guiding exosome, which greatly improves the accuracy of the RNA delivery mechanism of the present invention, on one hand, the amount of exogenous plasmid to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.

The targeting label is selected from one of peptides, proteins or antibodies with targeting functions, the selection of the targeting label is a process requiring creative labor, on one hand, an available targeting label needs to be selected according to target tissues, and on the other hand, the targeting label is ensured to be stably present on the surface of exosomes, so that the targeting function is achieved. The currently screened targeting peptides include, but are not limited to, RVG targeting peptide (nucleotide sequence is shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence is shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence is shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence is shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence is shown in SEQ ID No: 5); the target protein includes, but is not limited to, RVG-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 7), PTP-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 9), MSP-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 10).

In order to prove that the plasmids have the effects of enrichment in vivo and spontaneous formation of a complex structure containing RNA fragments, the invention randomly provides a group of experimental data that the plasmids only contain a targeting peptide tag or a targeting protein tag, and verifies the enrichment and self-assembly effects of the plasmids through experiments, as shown in figures 27-28.

In addition, for the purpose of precise delivery, we tested various plasmid vector loading schemes, and developed another optimized scheme: the plasmid vector may also be composed of multiple plasmids with different structures, wherein one plasmid contains a promoter and a targeting tag, and the other plasmid contains a promoter and an RNA fragment. The targeting effect of the two plasmid vectors is not inferior to that generated by loading the same targeting tag and RNA fragment in one plasmid vector.

More preferably, when two different plasmid vectors are injected into a host, the plasmid vector with the RNA sequence can be injected first, and the plasmid vector containing the targeting tag can be injected after 1-2 hours, so that a better targeting effect can be achieved.

The delivery systems described above may be used in mammals including humans.

As shown in FIG. 1A, in order to understand the distribution of plasmids in vivo, we performed a plate test on mice, sampled at time points (1 h, 3h, 6h, 9h, 12h, 24h, 72h, 168h, 720 h) after injection of the plasmids, transformed with plasmids extracted with spectinomycin, and observed the number of clones in liver, plasma, lung, brain, kidney, spleen, and as a result, as shown in FIGS. 1B, 1C, and 1D, it can be seen that the plasmids were distributed most in the liver of the mice, and reached a peak around 3h after injection, and were substantially metabolized after 12h after injection.

C57BL/6J mice were injected intravenously with CMV eGFP siRE circuit co-expressing eGFP protein and EGFR siRNA, and as shown in FIG. 2, the eGFP fluorescence in the mouse liver gradually increased with the passage of time, reaching a peak at about 12 hours, decreasing to a background level at 48 hours, and no significant eGFP signal was observed in other tissues.

Mice were injected with control plasmid (CMV-scrR), plasmid expressing EGFR siRNA (CMV-siR), respectively ^E ) And establishing a mouse hepatocyte in vitro model, and respectively detecting the injected CMV-scrR and CMV-siR ^E The results are shown in FIG. 3A, and it can be seen that CMV-siR was injected ^E The mouse hepatocyte exosomes of (a) present expression of siRNA.

We generally consider that binding to Ago2 protein is a necessary condition for siRNA function, i.e. siRNA in exosomes can bind to Ago2 protein, so we performed Ago2 immunoprecipitation experiments, and the results are shown in fig. 3B, fig. 3C. Wherein, input represents a sample which directly cracks and detects the exosome without immunoprecipitation, and represents a positive control.

The distribution of mature siRNA in different tissues after intravenous injection of plasmid into mice is shown in FIG. 4. As can be seen from FIG. 4A, EGFR-siRNA levels in plasma, exosomes, plasma without exosomes are time-dependent; as can be seen from FIG. 4B, the accumulation of mouse EGFR-siRNA in liver, lung, pancreas, spleen, kidney is time-dependent.

Mice were injected with control plasmid (CMV-scrR), 0.05mg/kg CMV-siR ^E Plasmid, 0.5mg/kg CMV-siR ^E Plasmid, 5mg/kg CMV-siR ^E Plasmid, detecting mouse liver, spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, CD4 ⁺ Absolute siRNA (EGFR siRNA) levels in cells, results are shown in FIG. 5AAs can be seen, no siRNA expression was observed in the tissues of mice injected with the control plasmid, CMV-siR was injected ^E Level of siRNA expression and CMV-siR in mouse tissues of plasmid ^E Plasmid concentrations were positively correlated. As shown in FIG. 5B, fluorescence In Situ Hybridization (FISH) also confirmed the level of siRNA expression and CMV-siR ^E The plasmid concentration is positively correlated, i.e., the tissue distribution of EGFR siRNA is dose dependent.

Since the plasmid will express the Precursor (Precurror) after entering into the body and then be processed into the mature body (siRNA), the metabolism of the Precursor (Precurror) and the mature body (siRNA) in the liver after the plasmid is injected into the mouse is detected, and the result is shown in FIG. 6. It can be seen that the expression levels of the Precursor (precorsor) and the mature body (siRNA) in the mouse liver reached the peak at the time node of 6 hours after the injection of the plasmid, the metabolism of the mature body (siRNA) in the mouse liver was completed 36 hours after the injection of the plasmid, and the metabolism of the Precursor (precorsor) in the mouse liver was completed 48 hours after the injection of the plasmid.

After the mice were injected with exogenous siRNA into the common bile duct, the absolute siRNA levels in the plasma (exosome-free), exosome (exosome) and plasma of the mice were measured, respectively, and the results are shown in fig. 7A. After the mice are injected with exogenous siRNA in common bile duct, the spleen, heart, lung, kidney, pancreas, brain, skeletal muscle and CD4 of the mice are respectively detected ⁺ Levels of siRNA in cells, the results are shown in FIG. 7B. These two graphs reflect that the kinetics of siRNA are almost the same in different tissues, and the distribution of siRNA is significantly different in different tissues.

The results of intravenous injection of siRNA using albumin ALB as a promoter, siRNA using CMV as a promoter, and siRNA without any promoter into mice were shown in fig. 8, in which absolute siRNA levels in mice were measured at 0h, 3h, 6h, 9h, 12h, 24h, 36h, and 48h after injection. As can be seen, the level of siRNA using CMV as a promoter in mice is the highest, namely the CMV as the promoter has the best effect.

We observed the inhibition of eGFP levels in mice by self-assembled eGFP sirnas by fluorescence assay as follows: eGFP transgenic mice were injected intravenously with PBS or 5mg/kg CMV-siR ^G Or CMV-RVG-siR ^G Plasmid, mice sacrificed 24 hours after treatment and eGFP fluorescence levels detected in frozen sections, fig. 9A shows representative fluorescence microscopy images, where green indicates a positive eGFP signal, blue indicates DAPI stained nuclei, scale bar: 100 μm, visible as CMV-RVG-siR ^G The plasmid has more obvious inhibition effect on the mouse eGFP; intravenous injection of PBS or CMV-scrR or CMV-siR into eGFP transgenic mice ^E Plasmid, 24 hours after treatment mice were sacrificed and eGFP fluorescence levels were measured in frozen sections, FIG. 9B is PBS, CMV-siR injections ^E 、CMV-RVG-siR ^E The Fluorescence intensity (Fluorescence intensity) column comparison graphs of the heart, the lung, the kidney, the pancreas, the brain and the skeletal muscle of the mice show that the Fluorescence intensity comparison of the mice at the parts of the liver, the spleen, the lung and the kidney is more obvious.

For injections of PBS, CMV-scrR, CMV-siR, respectively ^E The mice (a) were tested for their glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), total Bilirubin (TBIL), blood Urea Nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and peripheral blood cell percentage (percent of peripheral blood cells), and the results are shown in FIG. 10, in which 10A-F are PBS, mouse CMV-scrR, CMV-siR cells, and 10A-F are injected into the mice, respectively ^E The comparison graphs of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase and creatinine content are shown in the figure 10G, the comparison graphs of tissues of liver, lung, spleen and kidney of mice are shown in the figure 10H-I, the comparison graphs of tissues of thymus and spleen of mice are shown in the figure 10J, and the comparison graphs of percentage of peripheral blood cells (peripheral in peripheral blood cells) of mice are shown in the figure 10J.

The results show that PBS, CMV-scrR, CMV-siR were injected ^E The contents of ALT, AST and the like of mice, the weight of thymus gland, the weight of spleen and the percentage of peripheral blood cells are almost the same, and CMV-siR is injected ^E The mice (2) had no tissue damage to the liver, lung, spleen and kidney as compared with the mice injected with PBS.

The above experiments are sufficient to show that the RNA delivery system for treating glioblastoma provided in this example uses plasmid as the vector and plasmid as the mature injectant, which is safeThe property and the reliability are fully verified, and the druggability is very good. The RNA sequence which finally exerts the effect is encapsulated and conveyed by the endogenous exosome, no immune reaction exists, and the safety of the exosome does not need to be verified. The delivery system can deliver various small-molecule RNAs and has strong universality. Moreover, the preparation of the plasmid is much cheaper than that of exosome or substances such as protein, polypeptide and the like, and the economy is good. The RNA delivery system for treating glioblastoma provided in this example is capable of self-assembling with AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

Example 2

On the basis of example 1, this example provides a drug. The drug comprises a plasmid, wherein the plasmid carries RNA capable of treating glioblastoma, after the drug enters a human body, the plasmid can be enriched in host organ tissues, a composite structure which contains the RNA capable of treating glioblastoma and has a targeting structure is formed in the host organ tissues endogenously and spontaneously, the composite structure searches for and is combined with target tissues through the targeting structure, the RNA capable of treating glioblastoma is sent to the brain, and the glioblastoma is treated.

Further, the RNA capable of treating glioblastoma is one or more of siRNA, shRNA and miRNA having medical significance capable of inhibiting or hindering glioblastoma development.

In order to prove that the plasmid has the effects of in vivo enrichment and spontaneous formation of a composite structure containing RNA fragments, the invention randomly adopts 2 siRNAs, 2 shRNAs and 2 miRNAs and is named as siRNA1, siRNA2, shRNA1, shRNA2, miRNA1 and miRNA2, and verifies the enrichment and self-assembly effects of the plasmid through experiments under the condition that the plasmid contains the RNA alone or the plasmid contains any of the RNAs, and the experiments are particularly shown in figures 14-17. The groupings are listed below:

1) siRNA1 alone, siRNA2 alone, shRNA1 alone, shRNA2 alone, miRNA1 alone and miRNA2 alone;

2) 4 sets of RNA fragments containing any 2 RNA sequences in 1) above;

3) The group 3 of RNA fragments comprising any 3 RNA sequences in 1) above;

4) The above 1), 2 sets of RNA fragments comprising 2 additional RNA sequences.

The specific sequences (precursors) are shown in Table 4 below.

Further, the plasmid comprises a promoter sequence and an RNA sequence capable of treating glioblastoma.

Further, the plasmid further comprises a targeting tag that forms a targeting structure of the complex structure in an organ tissue of the host.

Further, the functional structural regions of the plasmid are arranged in any one of the following orders: 5' -promoter-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence, 5' -promoter-targeting tag or 5' -promoter-targeting tag-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence;

wherein the RNA sequence comprises 1, two or more specific RNA sequences of therapeutic interest, said RNA sequences being capable of being expressed in the target recipient, and said complementing sequences being incapable of being expressed in the target recipient.

Further, the plasmid is composed of multiple plasmids with different structures, wherein one plasmid contains a promoter and a targeting tag, and the other plasmid contains a promoter and an RNA sequence.

Further, the organ tissue is liver.

Further, the composite structure is an exosome.

Further, the targeting tag is selected from one of a peptide, a protein or an antibody having a targeting function, and the targeting structure is located on the surface of the composite structure.

Further, the targeting tag is RVG-LAMP2B fusion protein or GE11-LAMP2B fusion protein.

Further, the number of the desired delivery RNA effective sequences is 1, 2 or more.

Further, the delivery system may be used with mammals, including humans.

Further, the RNA capable of treating glioblastoma is selected from any one or more of the following RNAs: siRNA of EGFR gene, siRNA of TNC gene, or nucleic acid molecules encoding the above RNAs.

In order to prove that the gene circuit has the effects of in vivo enrichment and spontaneous formation of a complex structure containing RNA fragments, the invention randomly provides experimental data of a group of gene circuits containing siRNA of EGFR gene and siRNA of TNC gene, and verifies the enrichment and self-assembly effects of the gene circuit through experiments, as shown in figures 29-30.

The drug can be delivered to the brain through the RNA delivery system for treating glioblastoma described in example 1 after entering the human body by oral, inhalation, subcutaneous, intramuscular or intravenous injection, and thus can exert a therapeutic effect.

The drug provided by the embodiment can also be used in combination with other drugs for treating glioblastoma to enhance the therapeutic effect, such as temozolomide and the like.

The medicament provided in this embodiment may further comprise a pharmaceutically acceptable carrier, including but not limited to diluents, buffers, emulsions, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorbent carriers, and the like.

The dosage form of the drug provided in this embodiment may be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.

The safety and reliability of the drug provided in this example, which uses plasmid as the vector and plasmid as the mature injectant, have been fully verifiedThe patent medicine property is very good. The RNA sequence which finally exerts the effect is encapsulated and conveyed by the endogenous exosome, no immune reaction exists, and the safety of the exosome does not need to be verified. The medicine can deliver various small molecular RNAs and has strong universality. Moreover, the preparation of the plasmid is much cheaper than that of exosome or substances such as protein, polypeptide and the like, and the economy is good. The drugs provided by the present application can be self-assembled with AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

Example 3

Based on example 1 or 2, the present example provides the use of an RNA delivery system for treating glioblastoma in a medicament for treating glioblastoma. This example describes the application of the RNA delivery system in the treatment of glioblastoma in conjunction with the following two assays.

In the first experiment, we set up 5 test groups and 3 control groups. The test groups are CMV-siR respectively ^E Group CMV-siR ^T Group CMV-RVG-siR ^E+T Group, CMV-siR ^E+T Group, CMV-Flag-SiR ^E+T Groups, wherein "E" represents EGFR, "T" represents TNC, and the control groups are PBS group, CMV-scrR group, and CMV-Flag-scrR group, respectively, and the specific experimental process is shown in FIG. 11A.

The results of the separate detection of CD63 protein expression level and siRNA expression level in different groups of mice are shown in FIGS. 11B-11D, which indicates that intravenous CMV-RVG-siR injection ^E+T The wire may deliver siRNA to the brain.

In the second experiment, we set up 2 experimental groups and 2 control groups. The test groups are CMV-RVG-siR respectively ^E Group CMV-RVG-siR ^E+T The group and the control group are respectively a PBS group and a CMV-scrR group.

The specific test process is shown in fig. 12A, selecting a mouse, injecting glioblastoma cells (U-87 MG-Luc cells) into the mouse, and injecting PBS into the mouse once every two days from day 7 to day 21buffer/CMV-scrR/CMV-RVG-siR ^E /CMV-RVG-siR ^E+T (5 mg/kg) were treated and mice were subjected to survival analysis and tumor assessment, respectively. BLI in vivo imaging assays were performed on mice on days 7, 14, 28, and 35, respectively.

As shown in FIG. 12B, which is a comparison graph of BLI live imaging tests of mice at 7 days, 14 days, 28 days, and 35 days, it can be seen that CMV-RVG-siR ^E+T The mice in the group had the most significant glioblastoma inhibiting effect.

As shown in FIG. 12C, which is a graph comparing survival rates of various groups of mice, CMV-RVG-siR ^E+T The mice in the group had the longest survival time.

As shown in FIG. 12D, which is a fluorescence contrast plot of each group of mice, the plot was obtained by luciferase biological in vivo imaging, and the ordinate reflects the intensity of lucifer fluorescence signal. Since the gene has been artificially integrated into the implanted tumor, the map reflects the progression of the tumor. It can be seen that the tumor development of the control mice is rapid, while the tumor development of the test mice is greatly inhibited.

As shown in FIG. 12E, which is a comparative plot of relative siRNA for each group of mice, CMV-RVG-siR can be seen ^E Group mice with higher levels of EGFR siRNA, CMV-RVG-siR ^E+T The EGFR siRNA and TNC siRNA of the mice in the group have higher levels.

As shown in FIG. 12F, which is a comparison of the western blot of each group of mice, it can be seen that the PBS group, CMV-scrR group, CMV-RVG-siR group ^E The mice in the group have higher contents of EGFR and TNC genes.

The above experimental data illustrate intravenous CMV-RVG-siR ^E+T The plasmid is capable of delivering siRNA to the brain and inhibiting the growth of glioblastoma.

The brain of each mouse group was subjected to immunohistological staining treatment, and the staining ratios of EGFR, TNC, and PCNA in each field were counted, and the results are shown in fig. 13. It can be seen that CMV-RVG-siR ^E+T Mice of the group had minimal brain EGFR, TNC, PCNA content, CMV-RVG-SiR ^E The mice of the group have lower contents of EGFR and PCNA in brain. Visible injections of CMV-RVG-siR ^E The plasmid can inhibit brain EGFR,Expression of PCNA CMV-RVG-siR ^E+T The plasmid can inhibit the expression of EGFR, TNC and PCNA in brain.

In order to verify that the RNA plasmid delivery system of the present invention has the practical effect of in vivo enrichment and spontaneous formation of complex structure (self-assembly), for different RNAs that can be carried in the plasmid delivery system, the RNA possible flanking sequence, loop sequence, compensation sequence (fig. 20-fig. 23), different number of lines, connection sequence between lines, number of bases of connection sequence (fig. 24-26), targeting peptide or targeting protein tag (fig. 27-fig. 28), siRNA containing EGFR gene or TNC gene (fig. 29-30), different ribose modification RNA sequence (fig. 31), and seven angles provide corresponding experimental effect verification, which fully demonstrates that the plasmid delivery system provided by the present invention has the corresponding effect of treating glioblastoma, and is safe, reliable and has a wide application prospect.

In this document, "upper", "lower", "front", "rear", "left", "right", and the like are used only to indicate relative positional relationships between relevant portions, and do not limit absolute positions of the relevant portions.

In this document, "first", "second", and the like are used only for distinguishing one from another, and do not indicate the degree and order of importance, the premise that each other exists, and the like.

In this context, "equal", "same", etc. are not strictly mathematical and/or geometric limitations, but also include tolerances as would be understood by a person skilled in the art and allowed for manufacturing or use, etc.

Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.

The preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the embodiments and examples described above, and various changes can be made within the knowledge of those skilled in the art without departing from the concept of the present application.

Claims (18)

1. An RNA plasmid delivery system for treating glioblastoma comprising a plasmid carrying RNA fragments capable of treating glioblastoma, said plasmid being capable of being enriched in the host's organ tissues and spontaneously forming endogenously in said host's organ tissues a composite structure containing said RNA fragments capable of delivering said RNA fragments to the brain for treatment of glioblastoma.

2. The RNA plasmid delivery system for treating glioblastoma according to claim 1, wherein said RNA fragments comprise 1, two or more specific RNA sequences of medical interest, said RNA sequences being siRNA, shRNA or miRNA sequences of medical interest capable of inhibiting or hindering the development of glioblastoma.

3. The RNA plasmid delivery system of claim 2 wherein said RNA sequence is 15 to 25 nucleotides in length.

4. The RNA plasmid delivery system for the treatment of glioblastoma according to claim 3, wherein said RNA capable of treating glioblastoma is selected from any one or several of the following RNAs: siRNA of EGFR gene, siRNA of TNC gene, or RNA sequence with homology more than 80% with the sequence, or nucleic acid molecule for coding the RNA.

5. The RNA plasmid delivery system of claim 4 for the treatment of glioblastoma according to claim,

siRNA of EGFR gene comprises UGUUGCUUCUUCUAAUUCU, AAAUGAUCUUCAAAGUGCCC, UCUUAAGAAGGAAAGAUCAU, AAUAUUCCGUAGCAUUUAGGA, UAAAUCCUCACAAUACUU, other sequences with EGFR gene expression inhibition and sequences with homology more than 80 percent with the sequences;

the siRNA of the TNC gene comprises UAUGAAAUGUAAAAAAAGGGA, AAUCAUCCUUAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAUCCUUAGUUCAUCAU, AGAAGUAAAACUAUUGCGA, other sequences with TNC gene expression inhibition and sequences with homology of more than 80 percent with the sequences.

6. The RNA plasmid delivery system of claim 1, wherein the plasmid further comprises a promoter and a targeting tag, wherein the targeting tag is capable of forming a targeting structure of the complex structure in an organ tissue of the host, wherein the targeting structure is located on the surface of the complex structure, and wherein the complex structure is capable of finding and binding to a target tissue through the targeting structure to deliver the RNA fragment into the target tissue.

7. The RNA plasmid delivery system for the treatment of glioblastoma according to claim 6, wherein said plasmid comprises any one or a combination of the following lines: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each plasmid at least comprises an RNA segment and a targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines.

8. The RNA plasmid delivery system of claim 7, wherein the plasmid further comprises flanking sequences, compensating sequences, and loop sequences that enable the lines to fold into the correct structure and be expressed, the flanking sequences comprising a 5 'flanking sequence and a 3' flanking sequence;

the plasmid comprises any one line or combination of lines as follows: 5' -promoter-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence, 5' -promoter-targeting tag or 5' -promoter-targeting tag-5 ' flanking sequence-RNA sequence-loop sequence-compensating sequence-3 ' flanking sequence.

9. The RNA plasmid delivery system of claim 8, wherein the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence having greater than 80% homology thereto;

the loop sequence is gtttggccactgactgac or a sequence with homology more than 80 percent;

<xnotran> 3' accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 80% ; </xnotran>

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted.

10. The RNA plasmid delivery system for the treatment of glioblastoma according to claim 6, wherein, in the presence of at least two lines in the plasmid, adjacent lines are connected by a sequence consisting of sequences 1 to 3;

wherein, the sequence 1 is CAGATC, the sequence 2 is a sequence consisting of 5-80 bases, and the sequence 3 is TGGATC.

11. The RNA plasmid delivery system for the treatment of glioblastoma according to claim 10, wherein, in the presence of at least two lines in the plasmid, adjacent lines are connected by sequence 4 or a sequence with greater than 80% homology to sequence 4;

wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACGACCAGTGGAC.

12. The RNA plasmid delivery system of claim 1 wherein said organ tissue is liver and said complex structure is an exosome.

13. The RNA plasmid delivery system for the treatment of glioblastoma according to claim 6, wherein said targeting tag is selected from the group consisting of targeting peptides or targeting proteins with targeting function.

14. The RNA plasmid delivery system for treating glioblastoma of claim 13, wherein said targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, MSP targeting peptides;

the target protein comprises RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein and MSP-LAMP2B fusion protein.

15. The RNA plasmid delivery system for the treatment of glioblastoma of claim 14, wherein the targeting tag is selected from the RVG targeting peptide or RVG-LAMP2B fusion protein.

16. The RNA plasmid delivery system for treating glioblastoma according to claim 1, wherein said delivery system is a delivery system for use in mammals, including humans.

17. Use of an RNA delivery system according to any one of claims 1 to 16 for the treatment of glioblastoma in medicine.

18. The use of claim 17, wherein the medicament is administered orally, by inhalation, subcutaneously, intramuscularly or intravenously.

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