CN110885789A - Preparation of engineered exosome of efficient controllable packaging endogenous nucleic acid and application thereof - Google Patents
Preparation of engineered exosome of efficient controllable packaging endogenous nucleic acid and application thereof Download PDFInfo
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Abstract
The invention relates to preparation of an engineered exosome of efficient controllable packaged endogenous nucleic acid and application thereof. The invention discloses an engineered exosome, comprising: 1) a nucleic acid molecule encoding a nucleic acid sequence of a binding protein linked to a nucleic acid drug, 2) a CIBN fusion protein fused to a membrane localization signal peptide, 3) a CRY2 fusion protein fused to a protein to which said nucleic acid sequence binds. The invention also provides a nucleic acid drug delivery system or kit comprising the exosome, methods of making the exosome and nucleic acid drug delivery system or kit, and uses of the engineered exosome or the nucleic acid drug delivery system to deliver a nucleic acid drug. The exosome has the advantages of endogenesis, stability, biocompatibility, nano size, capability of crossing blood brain barrier, designability of components and the like.
Description
Technical Field
The invention belongs to the field of drug delivery, and particularly relates to preparation and application of an engineered exosome for targeted delivery of nucleic acid drugs such as RNA drugs.
Background
Nucleic acid-based tumor therapy methods have been developed into clinical trials, mainly including siRNA, antisense oligonucleotide (ASO), RNA aptamer, synthetic mRNA and miRNA therapy, etc. Among them, miRNA-based therapy is also a potential cancer treatment. It is primarily through mimicking or inhibiting the function of the target miRNA. To date, some miRNA-targeted therapies have also reached the stage of clinical trials, such as the tumor suppressor miR-34 mimetic (NCT01829971) and miR-16 mimetic (NCT 02369198). In addition, various miRNA-based therapies such as oligonucleotides Against MiRNA (AMOs) and miRNA sponge (mirrnasponge) have been widely studied. In recent years, miRNA sponges have also shown great potential as a miRNA inhibitor. The miRNA sponge has repeated miRNA antisense sequences, can simultaneously target a plurality of miRNAs, and can continuously inhibit the functions of the miRNAs. Although miRNA-based therapeutic approaches have achieved many results, their delivery in vivo remains a challenge. Currently, the primary delivery system consists of viral delivery and non-viral delivery. Viral vectors can load endogenous RNA with high delivery efficiency, but safety issues require further investigation. Non-viral delivery includes lipid-based delivery, polymer-based delivery, and nanoparticle-based delivery. They have low immunotoxicity and good biocompatibility, but are unable to load endogenous RNA, and lack cargo protection, as well as low efficiency of endosomal escape. Therefore, there remains a need to develop a new delivery system that can efficiently load and deliver long RNAs into targeted cells for therapy.
Exosomes are cell-derived, membrane-bound extracellular vesicles, 30-150nm in size, produced in multivesicular bodies (MVBs), and secreted into the extracellular fluid by fusion of the MVBs with the cell membrane. Exosomes containing proteins, lipids and nucleic acids of blast origin play an important role in intercellular communication. Numerous studies have shown that actively functioning biomolecules can be exchanged between cells via exosomes and play a role in recipient cells. This unique property has led to the extensive interest of exosomes as natural drug delivery systems. Exosomes have been successfully used to date for the delivery of small molecules, short RNAs and proteins in vivo and in vitro. Compared to synthetic materials, exosome delivery systems have a number of advantages, such as their endogeneous, stability, biocompatibility, nano-size, ability to cross the blood-brain barrier, and ingredient designability. However, how to improve the capacity of exosomes to effectively load and deliver drugs to targets is still lacking, and the electroporation and transfection reagents mainly used at present have some problems, including low loading efficiency (macromolecules), influence on drug activity and the like. To overcome these challenges, a new design strategy is needed to improve the delivery efficiency of drugs.
Disclosure of Invention
In some embodiments, the invention provides an engineered exosome comprising: 1) a nucleic acid molecule encoding a nucleic acid sequence of a binding protein linked to a nucleic acid drug, 2) a CIBN fusion protein fused to a membrane localization signal peptide, 3) a CRY2 fusion protein fused to a protein to which said nucleic acid sequence binds. In some embodiments, the membrane localization signal peptide can localize a protein, such as CIBN, fused thereto to the cell membrane. In some embodiments, the membrane localization signal peptide can further localize other components bound to the fusion protein, such as other fusion proteins, to the cell membrane. In some embodiments, the membrane localization signal peptide used in the present invention is not particularly limited as long as it can localize a polypeptide or protein linked thereto to a cell membrane and sort into exosomes. In this sense, the length of the membrane localization signal peptide is not particularly limited, and may include, for example, about 5 to 9 amino acid residues (peptide), 10 to 100 amino acid residues (polypeptide), or more than 100 amino acid residues (protein), such membrane localization signal peptides being widely known in the art. For example, in some embodiments, the membrane localization signal peptide of the present invention may be CAAX, PB, CD63, CD81, CD9, lamp2b, Palm, and the like. In some embodiments, the membrane localization signal peptide of the present invention is Palm.
Herein, unless otherwise specified or limited by context, the terms peptide, polypeptide, protein used are not particularly limited and may be substituted for each other where appropriate, for example, only that any one of the peptide, polypeptide or protein may include, for example, about 5 to 9 amino acid residues (peptide), 10 to 100 amino acid residues (polypeptide) or more than 100 amino acid residues (protein).
In some embodiments, the nucleic acid agents of the invention include DNA and RNA agents. In some embodiments, a nucleic acid drug refers to a nucleic acid, such as DNA or RNA, that is capable of delivering to a target cell, such as a disease cell, to function at the gene level. In some embodiments, the nucleic acid drug may modulate the expression of a gene, e.g., increase or inhibit the expression of a target gene. In some embodiments, DNA and RNA drugs include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA, ribozymes, antisense oligonucleotides, decoy oligonucleotides, peptide nucleic acids, Triplex Forming Oligonucleotides (TFO), genes, and the like. In some embodiments, the nucleic acid drug comprises an RNA binding molecule that binds to endogenous RNA. In some embodiments, endogenous RNAs include, for example, various types of RNAs capable of modulating gene expression, such as endogenous mirnas. In some embodiments, the nucleic acid drug may be a miRNA sponge that binds an endogenous miRNA. In some embodiments, the miRNA may be miRNA 21.
In some embodiments, the nucleic acid drug of the present invention may be a nucleic acid derived from a human, an animal, a plant, a bacterium, a virus, or the like, and in some embodiments, the nucleic acid drug of the present invention may also be a nucleic acid produced by chemical synthesis. In some embodiments, the nucleic acid may be any one of single-stranded, double-stranded, and triple-stranded, and the molecular weight thereof is not particularly limited.
In some embodiments, binding of a nucleic acid sequence of a binding protein (e.g., an aptamer or other nucleic acid sequence capable of binding to a protein) to the corresponding protein (also referred to herein as a nucleic acid binding protein) delivers a nucleic acid drug to the cell membrane and sorts into exosomes. In some embodiments, any suitable nucleic acid binding protein (e.g., aptamer binding protein) and corresponding nucleic acid sequence (e.g., aptamer) pair or protein (peptide) and specific RNA structural sequence that specifically binds to the protein (peptide) can be used. In some embodiments, the aptamer binding protein may be a phage protein MCP, and the corresponding said aptamer may be an aptamer that binds said phage protein MCP. In some embodiments, the protein may be an L7Ae protein, and the corresponding nucleic acid sequence is the RNA structural sequence C/Dbox (5'-CCA AGG GAU CAA UCG GUCUCU CGA GGG UCC GAG UCU AGA CCA GAU UGG UCU CUC UGG-3'). In some embodiments, nucleic acid sequences to which nucleic acid drugs are linked of the invention include aptamers, such as DNA aptamers and RNA aptamers. In some embodiments, the aptamer may be screened by SELEX, which is an oligonucleotide capable of specifically binding to a protein or other small molecule substance. In some embodiments, the aptamer binding protein and aptamer pair are not particularly limited, so long as the two are capable of interacting to deliver the nucleic acid drug to the cell membrane and sort into exosomes. In some embodiments, the aptamer delivers nucleic acid drug linked to it to a target site, e.g., to a cell membrane, by binding to an aptamer binding protein, such aptamers can be prepared by screening methods known in the art, or using any aptamer known in the art that specifically binds to an aptamer binding protein. In some embodiments, the aptamer of the invention is single-stranded and is about 20 to about 60 nucleotides in length. In some embodiments, the RNA aptamer may be MS 2.
In some embodiments, engineered exosomes of the present invention may be modified in order to target disease cells. In some embodiments, the engineered exosomes of the present invention further comprise a disease-targeting cell aptamer on its surface. As described above, the aptamer may specifically bind to a protein or other small molecule substance. In some embodiments, the invention comprises modifying the exosomes to add nucleic acid aptamers that target disease cells on their surface. In some embodiments, the aptamer may be an aptamer that targets a protein specifically expressed on a disease cell, such as a tumor cell. In some embodiments, the aptamer may be an aptamer that targets a protein that is overexpressed on disease cells, such as tumor cells. For example, in some embodiments, the protein overexpressed on tumor cells may be interleukin 3(IL3), nucleolin, integrin, Prostate Specific Membrane Antigen (PSMA), Epidermal Growth Factor Receptor (EGFR), and the like. In some embodiments, the disease cell-targeting nucleic acid aptamer targets a receptor protein on the surface of a disease cell, particularly a surface protein specific to a disease cell. As is widely known in the art, many disease cells, such as cancer cells, express a specific protein, thereby enabling the specific targeting of drugs, such as nucleic acid drugs, to the disease cells by molecules, such as aptamers, that specifically bind to the protein. Such disease cell surface-specifically expressed proteins are widely known in the art.
In some embodiments, engineered exosomes of the present invention may be modified in order to target disease cells. For example, in some embodiments, a protein or peptide targeting a disease cell may be further included on the surface of the engineered exosomes of the present invention, or the protein or peptide targeting a disease cell may be sorted onto the exosome membrane during exosome formation. Proteins or peptides may interact with each other to form specific binding, as known to those skilled in the art. In some embodiments, the invention includes modifying the exosomes to include proteins or peptides that target disease cells. In some embodiments, the protein or peptide may be a protein or peptide that targets a protein specifically expressed on a disease cell, such as a tumor cell. In some embodiments, the protein or peptide may be a protein or peptide that targets a protein that is overexpressed on disease cells, such as tumor cells. For example, in some embodiments, the protein overexpressed on tumor cells may be interleukin 3(IL3), nucleolin, integrin, Prostate Specific Membrane Antigen (PSMA), Epidermal Growth Factor Receptor (EGFR), and the like. In some embodiments, the protein or peptide targeting the disease cell targets a receptor protein on the surface of the disease cell, particularly a surface protein specific to the disease cell. As is widely known in the art, many disease cells, such as cancer cells, express a specific protein, thereby enabling specific targeting of drugs, such as nucleic acid drugs, to the disease cells by proteins or peptides that specifically bind to the protein. Such disease cell surface-specifically expressed proteins are widely known in the art.
In some embodiments, the disease cells include cancer cells, such as epithelial cancers, including lung cancer, breast cancer, esophageal cancer, gastric cancer, cervical cancer, colon cancer, and the like; sarcomas, including rhabdomyosarcoma, liposarcoma, osteosarcoma, and the like; leukemias, including Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), hyperproliferative acute leukemia, adult T-lymphocytic leukemia, plasma cell leukemia, mast cell leukemia, eosinophilic leukemia, basophilic leukemia, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), myelodysplastic syndrome (MDS), and the like; lymphomas, including non-hodgkin lymphoma (NHL) and Hodgkin Lymphoma (HL), and the like; myeloma, and the like. In some embodiments, the disease cell can be a leukemia cell.
In some embodiments, the nucleic acid aptamer targeted to the disease cell may be a DNA aptamer or an RNA aptamer that specifically targets the disease cell. In some embodiments, the aptamer of the invention is single-stranded and is about 20 to about 60 nucleotides in length. In some embodiments, the aptamer is a DNA aptamer to nucleolin that is overexpressed on leukemia cells. In some embodiments, the aptamer may be modified for introduction into an exosome membrane. For example, in some embodiments, it may be immobilized to the exosome membrane by modifying the interaction of cholesterol at its terminus with the exosome membrane.
In some embodiments, the invention provides a nucleic acid drug delivery system comprising the engineered exosomes described above, comprising a nucleic acid drug, the engineered exosomes encapsulating the nucleic acid drug, an aptamer targeting a disease cell, and the disease cell.
In some embodiments, the aptamer targeting a disease cell and the disease cell are as described above. In some embodiments, the aptamer targeting the disease cell is a DNA aptamer to nucleolin overexpressed on a leukemia cell, which is a leukemia cell.
In some embodiments, the invention provides kits comprising the engineered exosomes described above, further comprising instructions for using the engineered exosomes of the invention.
In some embodiments, the present invention provides a method of making an engineered exosome or a nucleic acid drug delivery system or kit comprising the same, the method comprising:
1) construction of vectors encoding for transfection of cells
i) A CIBN fusion protein fused to a membrane localization signal peptide, and
ii) a CRY2 fusion protein fused to the bacteriophage protein MCP, and
iii) an aptamer binding to the bacteriophage protein MCP linked to a nucleic acid drug,
2) constructing a stable transgenic cell line using the constructed vector, and
3) collecting and obtaining the engineered exosome from the stable transfer cell line
In some embodiments, the vector used in the methods of the invention is a viral vector. In some embodiments, the vector used in the methods of the invention is a retroviral vector. In some embodiments, the vector used in the methods of the invention is a lentiviral vector.
In some embodiments, the transfected cells used in the methods of the invention are not particularly limited, and may be any cells known in the art suitable for transfection, such as 293T cells. In some embodiments, transfection may be performed using an appropriate transfection agent. In some embodiments, prior to harvesting exosomes, the methods of the invention comprise culturing transfected cells under intermittent irradiation with an inducer, such as blue light. In some embodiments, exosomes may be collected using any suitable method, for example, methods of collecting engineered exosomes, including ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, magnetic bead immunization, PEG-base precipitation, or using commercially available exosome extraction kits. In some embodiments, the methods of the invention use ultracentrifugation to collect engineered exosomes.
In some embodiments, the methods of the invention further comprise the step of modifying the engineered exosomes with aptamers that target a disease, such as tumor cells. In some embodiments, the targeted cell in the methods of the invention is a leukemia cell. In some embodiments, the nucleic acid aptamer is a DNA aptamer to nucleolin that is overexpressed on leukemia cells.
The invention also provides the use of a nucleic acid drug delivery system comprising an engineered exosome in the preparation of a nucleic acid drug delivery system or kit for delivering a nucleic acid drug into cells of a disease patient, for example a tumour patient.
In some embodiments, the invention provides engineered exosomes capable of large enrichment for cellular endogenous mRNA. In some embodiments, the present invention provides a novel vehicle for drug delivery. In some embodiments, the invention provides a use of an engineered exosome in targeted therapy of leukemia.
In some embodiments, the delivery system of nucleic acid drug provided by the present invention and its application can include nucleic acid drug, engineered exosome encapsulating nucleic acid drug, aptamer targeting tumor cells and leukemia cells.
In some embodiments, exosomes may be obtained by:
first, 3 elements required for the formation of engineered exosomes, palm-CINB, CRY2-MCP and mir21 sponge-MS 2, were constructed into the lentiviral vector PLVX, and then transfected into 293T cells, respectively. palm is a membrane localization sequence, CIBN and CRY2 are two reversible blue-light-inducible interacting proteins, and MCP is a phage protein. After the membrane-CINB was localized to the cell membrane by the membrane, CRY2-MCP was recruited to the cell membrane by the interaction of CIBN and CRY2 under blue light. Subsequently, mir21 sponge-MS 2 expressed mRNA enriched mir21 sponge-MS 2 in the membrane by interaction of the MS2 region with MCP proteins, MS2 being an RNA aptamer to MCP proteins. mir21 sponge is an inhibitor of mir21 (sequence 5 'uagcuuaucagacugauguuga 3'). After the lentivirus has formed in the cells, the lentivirus is collected from the cell culture supernatant. The 293T cells were infected with the collected lentiviruses separately and then screened for stable transgenic cell lines required for the formation of engineered exosomes by resistance.
The selected stable transformed cell line was cultured in a large amount, and the culture supernatant was collected. Exosomes in the supernatant were then obtained by differential centrifugation at different steps.
And after the exosomes are collected, performing targeted modification on the exosomes. The targeted modification is to modify an aptamer capable of recognizing leukemia cells on the surface of an exosome. The aptamer is a DNA aptamer to nucleolin that is overexpressed on leukemia cells. The DNA aptamer is immobilized to an exosome membrane by modifying the interaction of cholesterol at its terminus with the exosome membrane.
And (3) incubating the modified exosome with a leukemia cell K562 cell line, and observing the apoptosis condition of the leukemia cell and the expression change of the mir21 target protein PTEN.
Compared with the prior art, the invention has the following advantages and effects:
the invention firstly provides a method for enriching cell endogenous nucleic acid into exosome, and secondly provides a specific application result for the feasibility of the exosome as a nucleic acid drug delivery vector. The results show that the engineered exosome synthesized by the invention can enrich specific nucleic acid expressed in cells and can enhance the capacity of the exosome to target disease cells through targeted modification. Secondly, after the exosome carrying the nucleic acid drug acts on the leukemia cell, compared with a control group, the apoptosis of the leukemia cell is obviously increased, and the expression of the target protein PTEN of the mirna acting on the corresponding nucleic acid drug is also increased. Therefore, the exosome of the present invention can be used as a drug carrier to successfully deliver an active nucleic acid drug to a target cell.
Drawings
FIG. 1 is a graph demonstrating enrichment of RNA in exosomes by RT-PCR. a: and (3) carrying out agarose gel electrophoresis on the qualitative analysis of the PCR result of the BFP-miRNA-21 sponge-MS 2 in the exosome. Lane 1: marker two lane 2: BFP-miRNA-21 sponge-MS 2; lanes: miRNA-21 sponge-MS 2. b: semi-quantitative analysis of mir21 sponge in exosomes by Q-PCR. On: exosomes were collected under blue light stimulation in 293T cell culture, off: exosomes were collected in 293T cell culture without blue light stimulation.
FIG. 2 is a flow cytometer analyzing the apoptosis of leukemia cells after the AS 1411-RNA-exosomes target the leukemia cells and the exosomes convey BFP-miRNA 21 sponge-MS 2 to the leukemia cells K562.
Figure 3 is a plasmid map of BFP-miRNA 21 sponge-MS 2.
FIG. 4 is a plasmid map of CRY2 PHR-mCHERRY-MCP.
FIG. 5 is a Pahn-EGFP-CIBN plasmid map.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, but the scope of the present invention is not limited thereto.
The relevant nucleic acids used in the present invention were synthesized by bioengineering (Shanghai) Co., Ltd. The miRNA-21 sponge and MS2 sequences are derived from CMV-d2EGFP-21(addge, 21972) and pSL-MS2-6X (addge, 27118) plasmids. The BFP, EGFP, mCherry, CIBN and CRY2 sequences were synthesized by bioengineering (Shanghai) Inc. Transfection reagent: lipofectamine 3000(L3000015) was purchased from ThermoFisher. Anti-actin (mouse, abconal, AC004), anti-CD 63 (mouse, abcam, MX-49.129.5). PTEN (rabbit, abclonal, a 11193). RNA extraction kit (TOYOBO, SCQ-101) and reverse transcription kit (TOYOBO, FSQ-201). qPCR kit: (Transgen Biotech, AQ 141-03). Hoechst 33342(invitrogen, H3570). Alexa Fluor 647-conjugated Annexin V and PI kit (Yeasen, shanghai).
AS1411：5’GGTGGTGGTGGTTGTGGTGGTGGTGG3’
miRNA21：5’uagcuuaucagacugauguuga3’
MS2：5’ACATGAGGATCACCCATGT3’
miR-21 sponge: 5 'TCAACATCAGGACATAAGCTA 3'
The preparation and application of the engineered exosome related in the embodiment mainly comprise the following steps:
(1) establishment of Stable cell lines
The plasmids required for the preparation of engineered exosomes were first constructed, including three plasmids, Palm-EGFP-CIBN (sequence shown below), CRY 2-mChery-MCP (sequence shown below) and BFP-6X miRNA21 sponge-6X MS2 (derived from CMV-d2EGFP-21(addge, 21972 and pSL-MS2-6X (addge, 27118) plasmids). EGFP, mChery and BFP were used only to screen for stable transduction lines and to localize fusion proteins.first, the amino acid fragments of the different proteins were ligated together.palm is a 16 amino acid peptide fragment whose sequence was ligated to the base sequence of CIBN by primer design (bioengineered (Shanghai) Co., CRY2 (bioengineered (Shanghai) Co., and ligated by the ligation site of MCP endonucleases, 21 sea and MS2 were also ligated by the ligation site of endonucleases after ligation of the ligation fragments, and then ligated to the different DNA-pXpXpXpXl vectors PLVX vectors have different fluorescent proteins on them.
Sequence of CRY2-mCherry-MCP (where bold part is CRY2 sequence, italics part is mCherry sequence, underlined part is MCP sequence, linker sequence between CRY2 sequence and mCherry sequence and between mCherry sequence and MCP sequence):
sequence of palm-EGFP-CIBN (where bold part is the palm sequence, italic part is the EGFP sequence, underlined part is the CIBN sequence, linker sequence between the palm sequence and the EGFP sequence and between the EGFP sequence and the CIBN sequence):
after the plasmids are constructed, the constructed plasmids are respectively transfected into 293T cells to form lentiviruses packaged with target plasmids. After transfection for 48h, cell culture supernatant was collected, then cell debris in the supernatant was removed by centrifugation at 1000g, and lentivirus in the supernatant was collected and stored at-80 ℃ for further use. In order to form cells stably expressing the above plasmids, 293T cells were infected with the collected viruses together, and after 48 hours, stably transformed cells into which the three plasmids had been incorporated were selected by fluorescence using a flow cytometer from the infected cells.
(2) Collection of exosomes
The exosome is collected by adopting a differential centrifugation method. The method comprises the following specific steps:
1. and (3) carrying out amplification culture on the obtained stably transformed cells, changing the culture medium into a culture medium containing 10% of exosome serum-removed after the cells grow to 60-70% of confluence, and collecting exosomes from cell culture supernatant after continuous culture for 48 h. To generate exosomes enriched in miRNA21 sponge, cells were cultured under interval irradiation of blue light during 48h of exosome-deprived serum culture. The blue light is provided by a self-assembled blue light lamp. The irradiation intensity of the blue light can be determined according to specific experimental results, and the irradiation time is 60s at intervals of 60 s.
2. The cell culture broth was collected and centrifuged at 500g at 4 ℃ for 5 minutes to remove the cells remaining in the culture broth. Then, the cells were centrifuged at 2000g for 20 minutes at 4 ℃ to remove cell debris remaining in the culture solution.
3. The supernatant obtained in step 1 was centrifuged again at 10000g at 4 ℃ for 30 minutes to remove large vesicles.
4. The supernatant was collected and then centrifuged at 100,000g for 90 minutes at 4 ℃ to obtain an exosome pellet. Discarding the supernatant, washing the precipitate with PBS, and centrifuging at 100,000g for 90 min at 4 deg.C to obtain relatively pure exosome.
(3) Verification of exosome-packaged miRNA21 sponge
To demonstrate that the engineered exosomes designed by the present invention can enrich miRNA21 sponge, the exosomes collected when the mother cells are cultured under blue light irradiation are used as a positive group, and the exosomes collected when the mother cells are cultured under dark conditions are used as a negative group. Then, RNA in the exosomes was extracted, and the amount of miRNA21 sponge in the exosomes was relatively quantified by qPCR.
(4) Targeted modification of exosomes
After the exosomes are collected, the aptamers are modified on the surface of the exosomes so as to enhance the targeting capacity of leukemia cells. The modified aptamer is AS1411 (shown in sequence below), and can specifically recognize nucleolin protein. The aptamer was synthesized by the company (bioengineering (Shanghai) Co., Ltd.), and was modified with cholesterol at the 5' end. The combination of the aptamer and the exosome is completed through the interaction of cholesterol and an exosome membrane, and the specific steps are as follows: mu.l of 1. mu.g/ml exosomes were incubated with 5. mu.l of 10. mu.M aptamer overnight at 4 ℃ and then unbound aptamer was removed by ultracentrifugation. Sequence of AS 1411: GGTGGTGGTGGTTGTGGTGGTGGTGG
(5) Targeted study of exosomes
After obtaining the modified engineered exosomes, they were added to K562 cells and incubated for 1 h. The cells were then removed and centrifuged at 1000g for 10min to remove excess exosomes. And washing and resuspending the centrifuged cells by PBS, then dripping the cells onto a glass slide, and observing the uptake of the cells to exosomes by using a structured light illumination microscope. AS a control, engineered exosomes not modified by aptamer AS1411 were also incubated with cells, and then uptake of exosomes by K562 cells was observed under a confocal microscope.
(6) Validation of exosome results for delivery of functional RNA
In order to further verify that the obtained engineered exosome can package miRNA21 sponge and can be successfully delivered to target cells to play a function, the exosome after targeted modification is incubated with K562 cells for 48 hours, then the cells are collected, and the apoptosis condition of the cells is analyzed by a flow cytometry technology. Meanwhile, the whole protein of the cell is extracted, and the expression of the PTEN protein is analyzed through western blotting. In this, exosomes, which were not treated and as such did not package miRNA21 sponge, were used as controls.
Claims (10)
1. An engineered exosome, comprising:
1) a nucleic acid molecule encoding a nucleic acid sequence of a binding protein linked to a nucleic acid drug,
2) a CIBN fusion protein fused with a membrane localization signal peptide,
3) a CRY2 fusion protein fused to a protein to which said nucleic acid sequence binds.
2. The engineered exosome of claim 1, wherein the nucleic acid drug comprises DNA and RNA drugs, e.g. the nucleic acid drug comprises an RNA binding molecule that binds endogenous RNA, e.g. the endogenous RNA comprises an endogenous miRNA, e.g. the nucleic acid drug may be a miRNA sponge that binds endogenous miRNA.
3. The engineered exosome of claim 1 or 2, wherein the membrane localization signal peptide comprises CAAX, PB, CD63, CD81, CD9, lamp2b and Palm.
4. The engineered exosome according to any one of claims 1-3, wherein the nucleic acid sequence is an aptamer, including DNA aptamers and RNA aptamers.
5. The engineered exosome according to any one of claims 1-4, wherein the engineered exosome further comprises a modification to target disease cells, such as tumor cells, the modification comprising a peptide or aptamer to target disease cells, such as tumor cells, e.g. to a protein specifically expressed on disease cells, such as tumor cells, e.g. to a protein overexpressed on disease cells, such as tumor cells.
6. A nucleic acid drug delivery system or kit comprising the engineered exosome of any one of claims 1-6.
7. A method of making the engineered exosome of any one of claims 1-5 or the nucleic acid drug delivery system or kit of claim 6, the method comprising:
1) constructing a vector for transfecting a cell, the vector encoding i) a CIBN fusion protein fused to a membrane localization signal peptide, and ii) a CRY2 fusion protein fused to a bacteriophage protein MCP, and iii) a nucleic acid aptamer binding to the bacteriophage protein MCP, linked to a nucleic acid drug,
2) constructing a stable transgenic cell line using the constructed vector, and
3) collecting the engineered exosome from the stable transfer cell line.
8. The method of claim 7, wherein the vector is a viral vector, such as a retroviral vector, such as a lentiviral vector.
9. The method according to claim 7 or 8, wherein the method comprises the step of modifying the engineered exosomes with aptamers targeting disease, such as tumor cells.
10. Use of the engineered exosome of any one of claims 1-5 or the nucleic acid drug delivery system of claim 6 in the preparation of a drug delivery system or kit for delivering a nucleic acid drug into a disease, such as a tumor patient.
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