CN115137745A - RNA delivery system for treating glioblastoma - Google Patents
RNA delivery system for treating glioblastoma Download PDFInfo
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- CN115137745A CN115137745A CN202210329250.4A CN202210329250A CN115137745A CN 115137745 A CN115137745 A CN 115137745A CN 202210329250 A CN202210329250 A CN 202210329250A CN 115137745 A CN115137745 A CN 115137745A
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Abstract
The present application provides an RNA delivery system for treating glioblastoma. The system comprises a viral vector, wherein the viral vector carries an RNA segment capable of treating glioblastoma, the viral vector can be enriched in organ tissues of a host, and endogenously and spontaneously forms a composite structure containing the RNA segment capable of treating glioblastoma in the organ tissues of the host, and the composite structure can deliver the RNA segment to the brain to realize the treatment of glioblastoma. The RNA delivery system provided by the application has the advantages of fully verified safety and reliability, very good druggability, strong universality, and excellent economic benefit and application prospect.
Description
Technical Field
The application relates to the technical field of biomedicine, in particular to an RNA delivery system for treating glioblastoma multiforme.
Background
Glioblastoma is the most malignant glioma among astrocytic tumors. The growth rate of the glioblastoma is high, 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 is increased obviously, all patients have headache, emesis 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, patients have different 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 an organism 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. Among them, the patent of the 3 rd aspect is many, and the root cause thereof is that researchers have recognized that there is a lack of suitable siRNA delivery system for delivering siRNA to target tissues safely, accurately and efficiently, which has become a core problem that restricts RNAi therapy.
A virus (Biological virus) is a noncellular organism which is small in size, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic in living cells and proliferated in a replicative manner. Viral vectors can bring genetic material into cells, and the principle is that viruses have a molecular mechanism for transmitting their genomes into other cells for infection, can occur in whole living organisms (in vivo) or in cell culture (in vitro), and are mainly applied to basic research, gene therapy or vaccines. However, there are currently few studies related to the use of viruses as vectors for the delivery of RNA, in particular siRNA, using specific self-assembly mechanisms.
Chinese patent publication No. CN108624590a discloses a siRNA capable of inhibiting DDR2 gene expression; chinese patent with publication number CN108624591A discloses 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 with publication number CN108250267A discloses a polypeptide, polypeptide-siRNA induction co-assembly body, and uses polypeptide as carrier of siRNA. Chinese patent with publication number 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 the siRNA. Chinese patent publication No. CN108096583A discloses a nanoparticle carrier, which contains chemotherapeutic drugs and can also 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.
So far, the medicine taking exosome as an active ingredient has never been approved by CFDA, and the core problem is that the consistency of exosome products cannot be ensured, and the problem directly causes that the products cannot obtain the medicine 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 loop essential 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 delivery system for treating glioblastoma to solve the technical deficiencies in the prior art.
One aspect of the present invention is to provide an RNA delivery system for treating glioblastoma, the system including a viral vector carrying an RNA fragment capable of treating glioblastoma, the viral vector being capable of being enriched in organ tissues of a host, and endogenously and spontaneously forming a composite structure containing the RNA segment capable of treating the glioblastoma in the host organ tissues, wherein the composite structure is capable of delivering the RNA segment to the brain, thereby treating the glioblastoma.
Further, the viral vector is an adeno-associated virus.
Further, the adenovirus-associated virus is adenovirus-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
Further, the RNA fragment comprises 1, two or more specific RNA sequences of medical significance, which are siRNA, shRNA or miRNA of medical significance.
Further, the viral vector 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 a host, the targeting structure is positioned on the surface of the complex structure, and the complex structure is capable of delivering the RNA segment into the brain of a target tissue through the targeting structure.
Further, the virus vector comprises any one line or combination of lines of the following: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each viral vector comprises at least one RNA segment and one targeting label, wherein the RNA segment and the targeting label are positioned in the same line or different lines.
Further, the viral vector also comprises a flanking sequence, a compensation sequence and a loop sequence which can enable the line to be folded into a correct structure and expressed, wherein the flanking sequence comprises a 5 'flanking sequence and a 3' flanking sequence;
the virus vector comprises any one line or combination of several lines: 5 '-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3 'flanking sequence, 5' -promoter-targeting label-5 'flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence.
Further, the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with more than 80% homology thereto;
the loop sequence is gttttggccactgactgac or a sequence with homology of more than 80 percent;
the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with homology of more than 80%;
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.
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.
Further, in the case where at least two kinds of lanes are present in the viral vector, adjacent lanes are connected by a sequence consisting of sequences 1 to 3 (sequence 1 to sequence 2 to sequence 3);
wherein, the sequence 1 is CAGATC, the sequence 2 is a sequence consisting of 5-80 bases, and the sequence 3 is TGGATC.
Further, in the case where at least two kinds of lanes are present in the viral vector, adjacent lanes are connected to each other through sequence 4 or a sequence having a homology of more than 80% to sequence 4;
wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
Further, the organ tissue is a liver, and the composite structure is an exosome.
Further, the targeting label is selected from targeting peptides or targeting proteins with targeting function.
Further, the targeting peptides include RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, 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.
Further, the RNA sequence is 15-25 nucleotides in length. For example, the RNA sequence may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length. Preferably, the RNA sequence is 18-22 nucleotides in length.
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. It should be noted that the RNA sequence in the "nucleic acid molecule encoding the RNA sequence" also includes RNA sequences having a homology of more than 80% for each RNA.
The siRNA of the EGFR gene comprises UGUUGCUUCUCUUAAUUCCU, AAAUGAUCUUCAAAAGUGCCC, UCUUUAAGAAGGAAAGAUCAU, AAUAUUCGUAGCAUUUAUGGA, UAAAAAUCCUCACAUAUACUU, 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 the TNC gene comprises UAUGAAAUGUAAAAAAAGGGA, AAUCAUAUCCUUAAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAAAUCCUUAGUUUUCAU, AGAAGUAAAAAACUAUUGCGA, other sequences which can inhibit the expression of the TNC gene and sequences with homology of more than 80 percent with the sequences.
The "sequence having a homology of more than 80" may be 85%, 88%, 90%, 95%, 98%, or the like.
Optionally, the RNA fragment comprises an RNA sequence body and a modified RNA sequence obtained by ribose modification of 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 acids also include variants and derivatives thereof. The nucleic acid 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 fragment 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, phosphoester phosphoramidates, carbamates, carbonates, phosphotriesters, acetamide esters, carboxymethyl esters, 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.
In order to prove that the delivery system has the effect of enrichment and self-assembly in vivo, the invention randomly provides experimental data of the RNA sequence of the delivery system after ribose modification, and verifies the enrichment and self-assembly effect of the delivery system through experiments, as shown in FIG. 20 in particular.
Further, the delivery system is a delivery system for use in mammals including humans.
Another aspect of the present invention is to provide a method for treating glioblastoma multiforme, comprising administering an RNA delivery system as described above to a patient.
Further, the administration mode of the medicine comprises oral administration, inhalation, subcutaneous injection, intramuscular injection and intravenous injection.
Optionally, the drug includes the above viral vector, specifically, the viral vector herein refers to a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter a host body, be enriched at a liver site, and self-assemble to form a complex structure exosome, where the complex structure can deliver the RNA fragment to a brain, so that the RNA fragment is expressed in the brain, and further, the expression of a gene matched with the RNA fragment is inhibited, thereby achieving the purpose of treating glioblastoma.
The dosage form of the medicine can be tablets, capsules, powder, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
The technical effects of the application are as follows:
the RNA delivery system for treating glioblastoma provided by the application takes the virus as a vector and the virus vector as a mature injectant, the safety and reliability of the RNA delivery system are fully verified, and the RNA delivery system is very good in drug performance. The final RNA sequence exerting effect is from the outside of the endogenous sourceThe exosome is packed and delivered, 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, the universality is strong. Moreover, the preparation of viral vectors is much cheaper than that of exosomes or substances such as proteins and polypeptides, and is economical. 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 survival and tumor assessment of mice provided in an embodiment of the present application.
FIG. 2 is a graph showing in vivo enrichment and self-assembly effects of 3 other viral vectors provided in an example of the present application, in which A is the in vivo enrichment results of the other viral vectors 1, B is the in vivo enrichment results of the other viral vectors 2, C is the in vivo enrichment results of the other viral vectors 3, and D is the in vivo self-assembly results of the three other viral vectors.
FIG. 3 is a graph showing in vivo enrichment and self-assembly effects of a viral vector provided in an embodiment of the present application, each carrying one of 6 RNA fragments; wherein A is the in vivo enrichment effect of vectors containing different RNA fragments, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
FIG. 4 is a graph showing that the virus vector provided in one embodiment of the present application has in vivo enrichment and self-assembly effects under the condition that the virus vector carries 4 sets of RNA fragments containing any 2 RNA sequences; wherein A is the in vivo enrichment effect of vectors containing different RNA fragments, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
FIG. 5 shows verification of in vivo enrichment and self-assembly effects of a viral vector provided in one embodiment of the present application, wherein the viral vector carries 3 sets of RNA fragments containing any 3 RNA sequences; wherein A is the in vivo enrichment effect of vectors containing different RNA fragments, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
FIG. 6 is a graph showing in vivo enrichment and self-assembly effects of 2 groups of RNA fragments containing any other 2 RNA sequences, respectively, in a viral vector provided in another embodiment of the present application; wherein A is the in vivo enrichment effect of vectors containing different RNA fragments, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
FIG. 7 is a graph showing that the viral vector provided in one embodiment of the present application has in vivo enrichment and self-assembly effects under the condition that the viral vector carries random 1-2 RNA fragments and 1-2 targeting tags which are located on the same line; wherein A is the in vivo enrichment effect of the vector containing different RNA fragments and the targeting tag, and B is the in vivo self-assembly effect displayed by the expression levels of the different RNA fragments.
FIG. 8 is a graph showing in vivo enrichment and self-assembly of viral vectors carrying random 1-2 RNA fragments and 1-2 targeting tags in different routes; wherein A is the in vivo enrichment effect of the vector containing different RNA fragments and the targeting tag, and B is the in vivo self-assembly effect displayed by the expression levels of the different RNA fragments.
FIG. 9 is a verification that the viral vector provided in one embodiment of the present application has the effect of in vivo enrichment and self-assembly, carrying the defined 5' flanking sequence and at least 2 defined sequences having greater than 80% homology thereto; wherein A is the in vivo enrichment effect of vectors 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. 10 shows verification of the in vivo enrichment and self-assembly effect of a viral vector provided in an 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 vectors containing different loop sequences in vivo, and B is the in vivo self-assembly effect displayed by the expression levels of RNA fragments of different loop sequences.
FIG. 11 is a diagram showing the verification of the in vivo enrichment and self-assembly effect of a viral vector provided in one embodiment of the present application, carrying a defined 3' flanking sequence and at least 2 defined sequences having a homology of more than 80% with the flanking sequence; wherein A is the enrichment effect in vivo of vectors 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. 12 is a verification of the in vivo enrichment and self-assembly effects of the viral vector provided in one embodiment of the present application on the RNA sequence carrying the reverse complementary sequence after deleting any 1, 2, 3, 4, 5 bases therein; wherein A is the enrichment effect of vectors containing different compensating sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different compensating sequences.
FIG. 13 is a verification of the effect of self-assembly of a viral vector provided in an embodiment of the present application when the viral vector carries four lanes and adjacent lanes are connected with each other by the sequences 1-2-3.
FIG. 14 shows the effect of self-assembly when the viral vector provided in one embodiment of the present invention carries four lanes, and the adjacent lanes are connected by the sequences 1 to 2 to 3, and the sequences 2 are composed of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases, respectively.
FIG. 15 is a verification of the effect of self-assembly of a viral vector provided in one embodiment of the present application when the vector contains a linker sequence of SEQ ID NO. 4 and at least 2 sequences with greater than 80% homology to SEQ ID NO. 4.
FIG. 16 is a graph demonstrating the effect of in vivo enrichment of viral vectors provided in one embodiment of the present application when they contain different targeting peptide tags.
FIG. 17 is a graph demonstrating the effect of in vivo enrichment of viral vectors provided in one embodiment of the present application when they contain different targeting protein tags.
FIG. 18 shows the effect of in vivo enrichment and self-assembly when siRNA of EGFR gene is contained in the gene circuit provided in one 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. 19 is a diagram illustrating in vivo enrichment and self-assembly effects of siRNA containing TNC gene in the gene circuit 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. 20 is a verification that the viral vector delivery system provided in one embodiment of the present application has in vivo enrichment and self-assembly effects when it contains 2 different RNA sequences modified by ribose; wherein A is the effect of in vivo enrichment of viral vector delivery systems of RNA modified by different ribose sugars and B is the effect of in vivo self-assembly shown by the expression levels of RNA modified by different ribose sugars.
Detailed Description
The following description of specific embodiments of the present application refers to the accompanying drawings.
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. Western blotting experiment is used for detecting the expression level of protein in cells and tissues, and ImageJ software is used for carrying out quantitative analysis on the protein.
In the illustrations provided herein, "+" indicates P <0.05, "+" indicates P <0.01, and "+" indicates P <0.005.
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 delivery system for treating glioblastoma, the system comprising a viral vector carrying RNA fragments capable of treating glioblastoma, the viral vector being capable of being enriched in organ tissues of a host and forming a composite structure containing the RNA fragments capable of treating glioblastoma endogenously and spontaneously in the organ tissues of the host, the composite structure being capable of delivering the RNA fragments into the brain to achieve treatment of glioblastoma.
In addition to adenovirus, other viral vectors also had the effect of in vivo enrichment and self-assembly (FIG. 2).
The viral vector is preferably an adeno-associated virus, more preferably an adeno-associated virus type 5, adeno-associated virus type 8 or adeno-associated virus type 9.
In this embodiment, the viral vector further comprises a promoter and a targeting tag. The virus vector comprises any one line or combination of lines of: the virus vector comprises a promoter-RNA sequence, a promoter-targeting label and a promoter-RNA sequence-targeting label, wherein each virus vector 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 viral vector 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 and the promoter-RNA sequence-targeting tag.
In order to prove that the virus vector really has the effects of in vivo enrichment and self-assembly, the invention randomly adopts 1-2 RNA segments and 1-2 targeting labels, the RNA segments and the targeting labels are respectively positioned in the same or different lines, and the enrichment and self-assembly effects of the virus vector are verified through experiments, which are specifically shown in figures 7-8. The grouping is listed as follows:
1. in the same lines (both including the promoter) (fig. 7):
1) RNA fragment 1+ targeting label 1, RNA fragment 2+ targeting label 2, RNA fragment 1+ targeting label 2, RNA fragment 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. 8):
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.
The specific sequences (precursors) are shown in table 1 below.
Further, the viral vector may further comprise flanking sequences including 5 'flanking sequences and 3' flanking sequences, a complementing sequence and a loop sequence which enable the lines to be folded into the correct structure and expressed; the virus vector comprises any one line or combination of lines as follows: 5 '-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3 'flanking sequence, 5' -promoter-targeting label-5 'flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence.
Wherein, the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with homology of more than 80 percent, including a sequence with homology of 85 percent, 90 percent, 92 percent, 95 percent, 98 percent, 99 percent and the like with ggatcctggaggcttgctgaaggctgtatgctgaattc.
The loop sequence is preferably gttttggccactgactgac or a sequence with homology of more than 80%, including 85%, 90%, 92%, 95%, 98%, 99% with gttttggccactgactgac, and the like.
The 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence having greater than 80% homology thereto, including sequences having 85%, 90%, 92%, 95%, 98%, 99% homology to accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, and the like.
The specific sequences are shown in Table 2 below.
| Name (R) | Sequence of |
| 5' flanking sequence-1 | CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG |
| 5' flanking sequence-2 | CTGGAGGCAGCCTGAAGGCTTTATGCTGAATTCG |
| loop-1 | GTTTTGGCCACTGACTGAC |
| loop-2 | GTTTATCCCACTGACTGAC |
| 3' flanking sequence-1 | CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC |
| 3' flanking sequence-2 | CACCGGTTGACACACAAGGCCTGTTACTAGCACTCACATGAGGCAAATGGCC |
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 in which any 1-5 bases are deleted.
Preferably, the complementing sequence is the reverse complement of the RNA fragment, and any 1-3 bases in the RNA fragment 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 in which any of the bases arranged in sequence at positions 1 to 3 is 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 the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence in which position 9 and/or position 10 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 number of theoretical researches 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 order to prove that the virus vector has the effect of in vivo enrichment and self-assembly, the invention randomly provides 4 groups of virus vectors containing different sequences, and the enrichment and self-assembly effects of the virus vectors are verified through experiments, and are shown in figures 9-12. The grouping is listed as follows:
1. the above-mentioned already defined 5' flanking sequences and at least 2 defined sequences having more 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.
In the case of viral vectors carrying two or more strands, adjacent strands may be connected by sequence 1-sequence 2-sequence 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.
The specific sequence diagram of sequence 2 is shown in Table 3 below.
In order to prove that the viral vector has the effects of in vivo enrichment and self-assembly, the invention randomly provides experimental data of connecting adjacent lines by a sequence 1-a sequence 2-a sequence 3 when a group of viral vectors carry four lines, and the enrichment and self-assembly effects of the viral vector are verified through experiments, and are particularly shown in figure 13.
Meanwhile, also in order to prove that the viral vector has the effects of in vivo enrichment and self-assembly, the invention randomly provides experimental data consisting of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases and 80 bases respectively for the sequences 2 when a group of viral vectors carries four lines, and the sequences 1-sequence 2-sequence 3 are connected between adjacent lines, and the experimental data proves the enrichment and self-assembly effects of the viral vectors through experiments, and is particularly shown in fig. 14.
More preferably, in the case of a viral vector carrying two or more strands, adjacent strands are connected to each other via sequence 4 or a sequence having a homology of greater than 80% to sequence 4; wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
In order to prove that the virus vector has the effect of in vivo enrichment and self-assembly, the invention randomly provides a group of virus vectors containing corresponding experimental data of a connecting sequence of sequence 4 and at least 2 sequences with homology of more than 80 percent with the sequence 4, and the enrichment and self-assembly effects of the virus vectors are verified through experiments, and particularly shown in figure 15.
The sequence is shown in Table 4 below.
| Name (R) | Sequence of |
| Sequence 4 | CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC |
| Sequence 4-1 | CAGATCTGGCCGAGCTCGAGGTAGTGAGTCGGAAAGTGGTAA |
| Sequence 4-2 | CAGATCTGGCCGCATACGAGGTAGTGAGTTTACCAGACCATC |
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 recipient, and the complementing sequences are not capable of being expressed in the target recipient. 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 capable of treating the 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.
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 viral vector, or the siRNA of the EGFR gene or the siRNA of the TNC gene may be used alone.
In order to prove that the gene circuit has the effects of in vivo enrichment and self-assembly, the invention randomly provides a group of experimental data of siRNA of EGFR gene and siRNA of TNC gene in the gene circuit, and verifies the enrichment and self-assembly effects of the gene circuit through experiments, as shown in figures 18-19.
Taking the combined use of "siRNA1" and "siRNA2" on the same viral vector as an example, the functional structural region of the viral vector can be represented as: (promoter-siRNA 1) -linker- (promoter-siRNA 2) -linker- (promoter-targeting tag), or (promoter-targeting tag-siRNA 1) -linker- (promoter-targeting tag-siRNA 2), or (promoter-siRNA 1) -linker- (promoter-targeting tag-siRNA 2), etc.
More specifically, the functional domains of the viral 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. The rest 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 also be obtained by modifying the RNA sequence (siRNA, shRNA or miRNA) in RNA, preferably 2' fluoropyrimidine. The 2 'fluoropyrimidine modification is that 2' -OH of pyrimidine nucleotide on siRNA, shRNA or miRNA is replaced by 2'-F, 2' -F can make the siRNA, shRNA or miRNA difficult to be recognized by RNase in human body, thus increasing the stability of RNA transmission in the body.
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 the 2' -F can make RNA enzyme in human body not easily recognize the siRNA, shRNA or miRNA, so that the stability of RNA in vivo transmission can be increased.
Specifically, the liver phagocytoses exogenous viruses, up to 99% of the exogenous viruses enter the liver, so that when the viruses are used as vectors, the viruses can be enriched in liver tissues without specific design, then the viral vectors are opened to release RNA molecules (siRNA, shRNA or miRNA), the liver tissues spontaneously wrap the RNA molecules into exosomes, and the exosomes become RNA delivery mechanisms.
Preferably, in order to enable the RNA conveying mechanism (exosome) to have the capability of 'precise guidance', a targeting label is designed in a viral vector injected into a body, the targeting label can be assembled into the exosome by liver tissues, and particularly when certain specific targeting labels are selected, the targeting label can be inserted into the surface of the exosome, so that the targeting structure capable of guiding the exosome is formed, the accuracy of the RNA conveying mechanism is greatly improved, the using amount of the viral vector required to be introduced can be greatly reduced, and the potential drug delivery efficiency is 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. Targeting tags that have been selected so far include: targeting peptides, targeting proteins, antibodies. Wherein, the targeting peptide includes but is not limited to RVG targeting peptide (the nucleotide sequence is shown in SEQ ID No. 1), GE11 targeting peptide (the nucleotide sequence is shown in SEQ ID No. 2), PTP targeting peptide (the nucleotide sequence is shown in SEQ ID No. 3), TCP-1 targeting peptide (the nucleotide sequence is shown in SEQ ID No. 4), MSP targeting peptide (the 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), and MSP-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No. 10). The targeting tag is preferably RVG targeting peptide, RVG-LAMP2B fusion protein.
Furthermore, for the purpose of precise delivery, we tested various viral vector loading schemes, and developed another optimized scheme: the viral vector may also be composed of multiple viruses with different structures, one virus containing a promoter and targeting tag, and the other virus containing a promoter and an RNA fragment. The targeting effect of the two viral vectors is not inferior to that generated by loading the same targeting tag and RNA segment in one viral vector.
More preferably, when two different viral vectors are injected into a host, the viral vector with the RNA sequence can be injected first, and then the viral vector with the targeting tag is 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.
The RNA delivery system for treating glioblastoma provided in this example uses a virus as a vector and a viral vector as a mature injectant, and has well-proven safety and reliability and excellent drug efficacy. 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. And the preparation of the virus vector is 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 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 isLow.
Example 2
On the basis of example 1, this example provides a drug. The drug comprises a viral vector, wherein the viral vector carries an RNA segment capable of treating the glioblastoma, the viral vector can be enriched in organ tissues of a host, and endogenously and spontaneously forms a composite structure containing the RNA segment capable of treating the glioblastoma in the organ tissues of the host, and the composite structure can deliver the RNA segment to the brain to realize the treatment of the glioblastoma.
Further, the RNA fragment comprises 1, two or more specific RNA sequences of medical significance, which are siRNA, shRNA or miRNA of medical significance.
In order to prove that the viral vector has the effects of in vivo enrichment and self-assembly, the invention randomly adopts 2 siRNAs, 2 shRNAs and 2 miRNAs, and is named as siRNA1, siRNA2, shRNA1, shRNA2, miRNA1 and miRNA2, and the enrichment and self-assembly effects of the viral vector are verified through experiments under the condition that the viral vector contains the RNAs independently or the viral vector contains any of the RNAs, and are shown in figures 3-6 specifically. 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 kinds of RNA sequences in 1) above;
3) The group 3 of RNA fragments comprising any 3 RNA sequences in 1) above;
4) The above 1), and 2 sets of RNA fragments comprising 2 additional RNA sequences.
Further, the viral vector 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 a host, the targeting structure is positioned on the surface of the complex structure, and the complex structure is capable of delivering the RNA segment into the brain of a target tissue through the targeting structure.
In order to prove that the viral vectors have the effect of in vivo enrichment and self-assembly, the invention randomly provides a group of experimental data that the viral vectors only contain a targeting peptide tag or a targeting protein tag, and verifies the enrichment and self-assembly effect of the viral vectors through experiments, as shown in fig. 16-17.
For the explanation of the above viral vectors, RNA fragments, targeting tags, etc. in this example, reference can be made to example 1, which is not described herein again.
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, flavors, adjuvants, desiccants, adsorptive 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 drug provided by the embodiment takes the virus as a carrier and the virus carrier as a mature injectant, the safety and reliability of the drug are fully verified, and the drug potency is very good. The RNA sequence which finally exerts the effect is wrapped 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 viral vectors is much cheaper than that of exosomes or substances such as proteins and polypeptides, and is economical. The drugs provided herein are 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 prematurely degraded, but also can maintain the exosome inThe stability in circulation is favorable for 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 use of the RNA delivery system for glioblastoma treatment in conjunction with the following two assays.
In this experiment, EGFR siRNA-carrying lines (AAV-CMV-RVG-siR) were carried out using liver high affinity AAV-type 5 adeno-associated virus E ) And EGFR siRNA, TNC siRNA line (AAV-CMV-RVG-SiR) E+T ) Tail vein injection 100 μ L titer 10 12 V.g/ml of AAV solution into mice. The AAV system expression in vivo was monitored by a small animal living body, and after 3 weeks, stable expression of the AAV system in vivo, particularly in the liver, was observed.
Then selecting mice, injecting glioblastoma cells (U-87 MG-Luc cells) into the mice, and injecting PBS buffer solution/AAV-CMV-scrR/AAV-CMV-RVG-siR into the mice every two days from day 7 to day 21 E /AAV-CMV-RVG-siR E+T (5 mg/kg) to form PBS group/AAV-scrR group/AAV-CMV-RVG-siR E group/AAV-CMV-RVG-siR E+T And (4) grouping.
Survival analysis was performed on each group of mice, and the survival rates of each group of mice after 20 days, 40 days, 60 days, and 80 days after the treatment were counted, and the results are shown in FIG. 1A, in which AAV-CMV-RVG-SiR can be seen E+T The survival time of the group mice is longest, AAV-CMV-RVG-siR E And then the combination is carried out.
The tumor evaluation of each group of mice was performed by BLI in vivo imaging test on day 7, 14, 28, and 35, and the result is shown in FIG. 1B, in which AAV-CMV-RVG-siR can be seen E+T The inhibition effect of the glioblastoma in the group of mice is the most remarkable.
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 (19)
1. An RNA delivery system for treating glioblastoma comprising a viral vector carrying RNA fragments capable of treating glioblastoma, said viral vector being capable of being enriched in the host's organ tissues and forming spontaneously and endogenously in said host's organ tissues a complex structure containing said RNA fragments capable of treating glioblastoma, said complex structure being capable of delivering said RNA fragments to the brain and effecting the treatment of glioblastoma.
2. The RNA delivery system of claim 1, wherein the viral vector is an adenovirus-associated virus.
3. The RNA delivery system of claim 2, wherein the adeno-associated virus is adeno-associated virus type 5, adeno-associated virus type 8, or adeno-associated virus type 9.
4. The RNA delivery system for treating glioblastoma according to claim 1, wherein said RNA segment comprises 1, two or more specific RNA sequences of medical interest, said RNA sequences being siRNA, shRNA or miRNA of medical interest.
5. The RNA delivery system of claim 1, wherein the viral vector 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 a surface of the complex structure, and wherein the complex structure is capable of delivering the RNA fragment into a brain of a target tissue via the targeting structure.
6. The RNA delivery system according to claim 5, wherein the viral vector comprises any one or a combination of the following lines: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each viral vector comprises at least one RNA segment and one targeting label, wherein the RNA segment and the targeting label are positioned in the same line or different lines.
7. The RNA delivery system for treating glioblastoma of claim 6, wherein said viral vector further includes flanking sequences, including a 5 'flanking sequence and a 3' flanking sequence, and loop sequences, that enable the lines to fold into the correct structure and to be expressed;
the virus vector comprises any one line or combination of several lines: 5 '-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3 'flanking sequence, 5' -promoter-targeting label-5 'flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence.
8. The RNA delivery system of claim 7, wherein the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence having greater than 80% homology thereto;
the loop sequence is gttttggccactgactgac or a sequence with homology of more than 80%;
the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with homology of more than 80 percent;
the compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted.
9. The RNA delivery system according to claim 6, wherein, in the case where at least two lines are present in the viral vector, adjacent lines are connected to each other by a sequence consisting of the 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.
10. The RNA delivery system of claim 9, wherein in the presence of at least two lines in the viral vector, adjacent lines are connected by sequence 4 or a sequence having greater than 80% homology to sequence 4;
wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
11. The RNA delivery system of claim 1, wherein the organ tissue is liver and the complex structure is exosome.
12. The RNA delivery system for the treatment of glioblastoma according to claim 5, wherein said targeting tag is selected from targeting peptides or targeting proteins with targeting functions.
13. The RNA delivery system for treating glioblastoma of claim 12, wherein said targeting peptides include RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, 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.
14. The RNA delivery system for treating glioblastoma of claim 5, wherein the RNA sequence is 15 to 25 nucleotides in length.
15. The RNA delivery system according to claim 14, wherein 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 molecule encoding the above-mentioned RNA.
16. The RNA delivery system for treating glioblastoma according to claim 15,
the siRNA of the EGFR gene comprises UGUUGCUUCUCUUAAUUCCU, AAAUGAUCUUCAAAAGUGCCC, UCUUUAAGAAGGAAAGAUCAU, AAUAUUCGUAGCAUUUAUGGA, UAAAAAUCCUCACAUAUACUU, 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 the TNC gene comprises UAUGAAAUGUAAAAAAAGGGA, AAUCAUAUCCUUAAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAAAUCCUUAGUUUUCAU, AGAAGUAAAAAACUAUUGCGA, other sequences which can inhibit the expression of the TNC gene and sequences with homology of more than 80 percent with the sequences.
17. The RNA delivery system for treating glioblastoma according to claim 1, wherein said delivery system is a delivery system for use in mammals, including humans.
18. Use of an RNA delivery system according to any one of claims 1 to 17 for the treatment of glioblastoma in medicine.
19. The use of claim 18, wherein the medicament is administered orally, by inhalation, subcutaneously, intramuscularly or intravenously.
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