Nanovesicles for delivering biomacromolecules to central nervous system

Abstract

Provided are extracellular vesicles and a use thereof, and nanovesicles for delivering biomacromolecules to a central nervous system. Also provided is a truncated ApoE.

Description

Nanovesicles for delivering biomacromolecules to the central nervous system Technical Field

[0001] The present invention relates to the technical field of extracellular vesicles and their application, and in particular to nanovesicles for delivering biomacromolecules to the central nervous system. Background Art

[0002] Cell-derived nanovesicles (NV) include extracellular vesicles (EVs, such as exosomes) secreted by cells and synthetic nanovesicles (Synthetic nanovesicles, SynNVs) reconstructed from cell membranes. Both the surface and interior of nanovesicles contain a variety of functional protein molecules, and the interior is usually also loaded with nucleic acid molecules (such as miRNA, lncRNA, etc.). Specific proteins or nucleic acid molecules can be loaded on nanovesicles through recombinant expression or physical and chemical loading methods. Therefore, nanovesicles are ideal carriers for the delivery of biomacromolecule drugs. However, nanovesicles, including exosomes, face a long-standing problem when delivering drugs in vivo: the vast majority of nanovesicle particles are captured and cleared by the liver, resulting in extremely low bioavailability of drugs delivered by nanovesicles to organs other than the liver. In particular, it is even more difficult for natural nanovesicles to penetrate the blood-brain barrier and deliver drugs to brain tissue.

[0003] The delivery of biomacromolecule drugs to brain tissue via nanovesicles requires a combination of two approaches: 1) improving the targeting of vesicles to brain tissue or specific brain tissue cells and improving their ability to penetrate the blood-brain barrier; and 2) prolonging the plasma circulation time of nanovesicles (i.e., reducing the capture and clearance of nanovesicles by the liver). Currently reported research and development work mainly focuses on the first approach, such as obtaining exosomes secreted by neural stem cells (NSCs) through culturing them, and utilizing the homing properties of NSC exosomes to enhance blood-brain barrier penetration (Robin L. Webb, et al. Stroke. 2018; Vol. 49). Alternatively, blood-brain barrier penetration can be enhanced by loading Angiopep-2, RVG, cell-penetrating peptides (CPPs), and ApoB peptides onto the surface of engineered EVs. Furthermore, physical and physiological methods such as targeted ultrasound and nasal administration can be used to promote EV entry into the brain.

[0004] Existing approaches for delivering drugs to brain tissue using vesicles often have shortcomings, such as significant technical bottlenecks in the large-scale culture of neural stem cells and drug loading in EVs, resulting in limited drugability. EV surface-loading with BBB-penetrating peptides offers little improvement in blood-brain barrier penetration, and because it doesn't address liver capture and clearance, overall brain bioavailability is limited compared to natural nanovesicles. While nasal delivery can increase the number of EV particles entering the brain, EVs lack the active migration capabilities of cells and the rapid, free diffusion of small-molecule drugs. Once inside the brain, they accumulate near the olfactory center, preventing them from reaching other brain tissue regions. Targeted ultrasound methods transiently disrupt the local blood-brain barrier, promoting drug penetration, but are not specific for vesicle-based drugs, resulting in high costs and technical challenges. Therefore, there remains a lack of cost-effective methods for efficiently delivering biomacromolecules to the brain through vesicles.

[0005] ApoE is a protein molecule that promotes the accumulation and clearance of lipid nanoparticle (LNP) drugs or mRNA vaccines in the liver and is widely present in the human bloodstream. Based on existing literature, it is inferred that ApoE not only fails to promote NV entry into the brain, but may also accelerate NV clearance from the liver. Currently, there are no reports on the use of ApoE protein to promote vesicle delivery of drugs to brain tissue.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide nanovesicles for delivering biomacromolecules to the central nervous system, in order to improve the loading efficiency of drugs in the vesicles and enhance the therapeutic effect.

[0008] The present invention provides a truncated form of APOE, the amino acid sequence of which is shown in any one of SEQ ID NOs. 1 to 3.

[0009] ApoE, or apolipoprotein E (ApoE), has three subtypes: APOE2, APOE3, and APOE4, and their amino acid sequences are as follows: APOE2 is shown in SEQ ID NO: 4, APOE3 is shown in SEQ ID NO: 5, and APOE4 is shown in SEQ ID NO: 6. Previous studies have shown that ApoE3, in combination with scaffold proteins, can achieve modular loading of therapeutic drugs and improve loading efficiency. On this basis, the present invention screened and modified ApoE, and in particular, constructed a truncated body of ApoE. The results showed that compared with ApoE3, the three truncated bodies constructed can further improve the loading effect.

[0010] The three truncated bodies provided by the present invention are divided into:

[0011] The truncated form of ApoE3 consisting of amino acid residues 1-191 is designated as ApoE3 (1-191), and its amino acid sequence is shown in SEQ ID NO: 1.

[0012] The truncated form of ApoE4 consisting of amino acid residues 1-191 is designated as ApoE4 (1-191), and its amino acid sequence is shown in SEQ ID NO: 2.

[0013] The truncated form of ApoE2 consisting of amino acid residues 1-191 is designated as ApoE2 (1-191), and its amino acid sequence is shown in SEQ ID NO:3.

[0014] The present invention also provides a nucleic acid encoding the aforementioned truncated form.

[0015] As a preference,

[0016] The nucleic acid sequence encoding the truncated ApoE3 (1-191) is shown in SEQ ID NO:7.

[0017] The nucleic acid sequence encoding the truncated ApoE4(1-191) is shown in SEQ ID NO:8.

[0018] The nucleic acid sequence encoding the truncated ApoE2(1-191) is shown in SEQ ID NO:9.

[0019] The present invention also provides a nucleic acid construct comprising: a brain entry module, a scaffold module, a transmembrane region module and an intramembrane region anchoring module; the brain entry module is a nucleic acid encoding the aforementioned truncated form or a nucleic acid as shown in any one of SEQ ID NOs: 4 to 5.

[0020] In the nucleic acid constructs described herein, the modules may or may not be in the same reading frame, and this is not a limitation of the present invention. Similarly, the modules may or may not be on the same vector, and this is also not a limitation of the present invention. In the examples of the present invention, the efficacy was verified using the example of modules being in the same reading frame, wherein the brain-entering module, scaffold module, transmembrane region module, and intramembrane region anchoring module were sequentially connected from the 5' end to the 3' end.

[0021] Preferably, the nucleic acid construct further comprises a signal labeling module and / or an effector module. The signal labeling module and / or the effector module may or may not be in the same reading frame as the brain entry module, the scaffold module, the transmembrane region module and the intramembrane region anchoring module, and the present invention does not limit this. Similarly, the signal labeling module and / or the effector module may or may not be on the same carrier as the aforementioned brain entry module, the scaffold module, the transmembrane region module and the intramembrane region anchoring module, and the present invention does not limit this. In an embodiment of the present invention, the 5' end of the effector module / detection module is connected to the 3' end of the intramembrane anchoring region module. Preferably, the signal labeling module and / or the effector module is connected to the intramembrane region anchoring module via a linker.

[0022] The effector module includes a nucleic acid encoding a protein drug or a nucleic acid drug for treating brain diseases.

[0023] The signal labeling module includes any one or more of a fluorescent protein module and / or a chemiluminescence module;

[0024] Preferably, the fluorescent protein module includes any one or more of green fluorescent protein, red fluorescent protein, blue fluorescent protein, or yellow fluorescent protein; the chemiluminescent module includes Nanoluc or Rluc. Preferably, the modules of the present invention are directly connected or connected through a connecting module, and the connecting module includes a nucleic acid encoding a connecting peptide linker.

[0025] The connecting peptides are divided into rigid connecting peptides and flexible connecting peptides. The commonly used sequences of rigid connecting peptides include (EAAAK) n (n=1-3), commonly used sequences of flexible linker peptides include (GGGGS) n (n=1-5) Preferably, the amino acid sequence of the connecting peptide of the present invention is (GGGGS)3.

[0026] Preferably, the nucleic acid construct designed by the present invention consists of the following modules:

[0027] The brain-penetrating module (BBB-penetrating module) - scaffold module - transmembrane domain module (TMD) - intramembrane domain anchoring module (ICD) - effector module / detection module (Cargo / Detection module). The BBB-penetrating module represents the full-length or truncated ApoE isoform protein loaded onto the extracellular membrane; the scaffold represents the extracellular scaffold protein, attached to the C-terminus of the effector protein; the transmembrane domain module (TMD) represents the transmembrane region, with its N- and C-termini linked to the extracellular and intracellular sequences, respectively; the intramembrane domain anchoring module (ICD) represents the intracellular region of the transmembrane protein, containing S-palmitoylcysteine, which can be anchored to the EV membrane; and the effector / detection module (Cargo / Detection module) represents the effector or detection protein loaded onto the intracellular membrane. In practice, each module is a nucleic acid molecule encoding the corresponding protein / functional region.

[0028] Preferably, the brain entry module is a nucleic acid fragment encoding a brain entry protein or a functional protein.

[0029] Preferably, the brain entry module includes any one or more of Transferrin, ApoB-RBD, APOE or its truncated form, hCD24, ScFv, GDNF or Blr (group B, leucine rich).

[0030] Preferably, the scaffold module comprises any one or more of Fc, Foldon, Fc+NPTN-Ig1-3, ITGB1-I-EGF1-4, Foldon+NPTN-Ig1-3 or NPTN-Ig1-3.

[0031] Preferably, the transmembrane region module includes any one or more of EWI-F-TMD, EWI-2-TMD, ITGB1-TMD, TMD_Mut_10, TMD_Mut_13, TMD_Mut_17, and NPTN-TMD.

[0032] Preferably, the transmembrane region module includes any one or more of TMD_Mut_10, TMD_Mut_13, TMD_Mut_17 or NPTN-TMD;

[0033] Wherein, the amino acid sequence encoded by the nucleotide sequence of TMD_Mut_10 is SEQ ID NO.10;

[0034] The amino acid sequence encoded by the nucleotide sequence of TMD_Mut_13 is SEQ ID NO.11;

[0035] The amino acid sequence encoded by the nucleotide sequence of TMD_Mut_17 is SEQ ID NO.12;

[0036] The amino acid sequence encoded by the nucleotide sequence of the NPTN-TMD is SEQ ID NO.13.

[0037] Preferably, the intramedullary region anchoring module contains a nucleotide sequence encoding S-palmitoylcysteine, and the intramedullary region anchoring module includes EWI-F-ICD or EWI-2-ICD.

[0038] Furthermore, the present invention also provides a plasmid, an expression vector or an expression cassette comprising the nucleic acid construct as described above.

[0039] Furthermore, the present invention also provides a transformant or recombinant cell comprising the aforementioned plasmid, expression vector or expression cassette.

[0040] Furthermore, the present invention also provides an engineered EVs, which are prepared using the transformant or recombinant cell as described above.

[0041] Furthermore, the present invention also provides a protein expressed by the nucleic acid construct as described above.

[0042] Furthermore, the present invention also provides the use of the nucleic acid construct as described above, the plasmid as described above, the expression vector or expression cassette as described above, the transformant or recombinant cell as described above, the engineered EVs as described above and / or the protein as described above in the preparation of a drug or reagent, wherein the drug has the effector module as an active ingredient.

[0043] The present invention screened and modified ApoE, and in particular, constructed a truncated form of ApoE. The results showed that the three truncated forms obtained could further improve the loading effect compared to ApoE3. By loading ApoE truncated protein or ApoE protein on the surface of NV to make NV simulate lipoprotein particles, its ability to penetrate the blood-brain barrier is much stronger than the aforementioned brain-entering polypeptide sequences (RVG, Angiopep-2, etc.). In addition, the plasma circulation half-life of NV loaded with ApoE in the body is also significantly improved, indicating a decrease in liver clearance, which is not achievable with the aforementioned brain-entering polypeptide sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 shows the data of NV enzyme activity of plasmids in each group;

[0045] Figure 2 shows the Transwell results of each group;

[0046] Figure 3 shows the data of brain tissues of mice in each group at different time points;

[0047] Figure 4 shows data from mouse liver tissue at different time points;

[0048] Figure 5 shows the characterization of NV enzyme activity corresponding to different truncations;

[0049] Figure 6 shows the enzyme activity data of different truncated NVs;

[0050] Figure 7 shows the number of particles in the brain tissue of mice 30 minutes after administration of different truncated NVs;

[0051] Figure 8 shows the enzyme activity data of different NVs;

[0052] Figure 9 shows the number of particles penetrating different NV Transwells at 24 h;

[0053] Figure 10 shows the number of particles in the brain tissue of mice 30 minutes after administration of different NVs;

[0054] FIG11 is a schematic diagram of the nucleic acid construct of the present invention. DETAILED DESCRIPTION

[0055] The present invention provides nanovesicles for delivering biomacromolecules to the central nervous system. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve this. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0056] The amino acid sequence of APOE2 is (SEQ ID NO. 4):

[0057] The amino acid sequence of APOE3 is (SEQ ID NO.5):

[0058] The amino acid sequence of APOE3(1-191) is (SEQ ID NO.1):

[0059] The nucleic acid sequence encoding the truncated ApoE3 (1-191) is (SEQ ID NO: 7):

[0060] The amino acid sequence of APOE2 (1-191) is (SEQ ID NO. 3):

[0061] The nucleic acid sequence encoding the truncated ApoE2 (1-191) is (SEQ ID NO: 9):

[0062] The amino acid sequence of APOE4 is (SEQ ID NO.6):

[0063] The amino acid sequence of APOE4(1-191) is (SEQ ID NO.2):

[0064] The nucleic acid sequence encoding the truncated ApoE4 (1-191) is (SEQ ID NO: 8):

[0065] The amino acid sequence of ApoE (130-149) is (SEQ ID NO. 14):

[0066] The amino acid sequence of Angiopep-2 is (SEQ ID NO.15):

[0067] The amino acid sequence of RVG is (SEQ ID NO.16):

[0068] The amino acid sequence of the linker is (SEQ ID NO.17):

[0069] The amino acid sequence of Ig3 (NPTN) is (SEQ ID NO. 18):

[0070] The amino acid sequence of TMD (NPTN) is (SEQ ID NO.13):

[0071] The amino acid sequence of ICD (EWI-F) is (SEQ ID NO.19):

[0072] The amino acid sequence of Nanoluc is (SEQ ID NO.20):

[0073] The amino acid sequence of TMD_Mut_10 is SEQ ID NO. 10;

[0074] The amino acid sequence of TMD_Mut_13 is SEQ ID NO.11:

[0075] The amino acid sequence of TMD_Mut_17 is SEQ ID NO.12:

[0076] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0077] The present invention will be further described below in conjunction with the embodiments:

[0078] Example 1: Construction of NV loaded with full-length human ApoE2, ApoE3 or ApoE4 protein

[0079] Objective: To load different subtypes of full-length human ApoE protein onto the surface of NV and evaluate the blood-brain barrier penetration and liver clearance resistance of this group of NV compared with wild-type NV.

[0080] method:

[0081] 1. Build:

[0082] Using pHEK293_Ultra_Expression_I (Takara) as the vector, we constructed:

[0083] Expression plasmid ApoE2-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD)-linker-Nanoluc,

[0084] Expression plasmid ApoE3-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD)-linker-Nanoluc,

[0085] Expression plasmid ApoE4-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD)-linker-Nanoluc expression plasmid,

[0086] A control plasmid without the loading module was also constructed:

[0087] Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc.

[0088] 2. Loading

[0089] The above plasmids were transfected into Expi HEK293F cells using the front loading method. The cell density at the time of transfection was 1.0×10 6 / ml, the mass ratio of plasmid to PEI was 1:1.5, and the cells were cultured for 72 h after transfection. The cells and supernatant were collected for NV preparation, and the prepared NV was loaded.

[0090] 3. Preparation

[0091] NV was prepared from the cultured cell supernatant by combining tangential flow filtration (TFF) with size exclusion chromatography (SEC). The collected cell supernatant was first filtered through a 0.45 μm filter, then concentrated into 1*PBS buffer by filtration through a 300 KD membrane package and a hollow fiber column. The concentrate was further purified by a Capto Core 700 chromatography column to remove impurities, and the flow-through was collected and concentrated through a 10 KD concentrator.

[0092] After culture, cells were first resuspended in 0.1% PBS buffer containing 10 U / mL nuclease and stirred at 120 rpm for approximately 5 minutes for hypotonic swelling. The cell suspension was then filtered four times through a 0.45 μm filter, returned to a salinity of 1% PBS, and nuclease was added. Digestion was then carried out at 4°C for approximately 16 hours. The cell suspension was then concentrated by filtration through a 300 kDa hollow fiber column to a final volume of 50 mL. The concentrate was then further purified using a Capto Core 700 column to remove contaminants. The flow-through was collected and concentrated using a 10 kDa concentrator. Cell-derived NV and supernatant-derived NV can be used together.

[0093] The control Nanoluc NV was prepared by the same method from cells transfected with the control plasmid and the supernatant.

[0094] 4. Characterization

[0095] NanoFCM was used to detect the particle concentration of NV. The cell supernatant and cell-derived NV were combined and added to the Nanoluc substrate. The Nanoluc enzyme activity was detected by a microplate reader to verify the expression of Nanoluc.

[0096] 5. Transwell assay

[0097] Mouse bEND.3 cells were seeded into 0.1 μm pore size Transwell culture plates at 5E5 cells / well and cultured in a 37°C, 5% carbon dioxide cell culture incubator until the cell confluence reached 80%-90%. The culture medium in the Transwell wells was carefully discarded, and different NVs were diluted with cell culture medium to the same number of particles. The cells were then added to the Transwell wells and cultured in a 37°C, 5% carbon dioxide cell culture incubator for 2 h, 4 h, 6 h, 8 h, and 24 h. The liquid in the lower chamber of the Transwell was collected to detect Nanoluc enzyme activity. The enzyme activity detected by NV and the corresponding number of particles were used as a standard curve to calculate the number of NV particles in each tissue.

[0098] 6. In vivo experiments in mice

[0099] Mice were dosed via the tail vein. Samples were collected 0.5, 2, 4, and 6 hours after administration: eyeballs were removed to collect blood, which was centrifuged at 3000g for 20 minutes at 4°C. The supernatant was used to assay Nanoluc enzyme activity in the plasma. Following perfusion, the liver and brain were collected and homogenized in PBS, then centrifuged at 12000 rpm for 10 minutes. The supernatant was then assayed for Nanoluc enzyme activity. The NV activity and corresponding particle counts were used as a standard curve to calculate the number of NV particles in each tissue.

[0100] result:

[0101] NV enzyme activity characterization (Table 1 + Figure 1): The prepared ApoE4 NV unit Nanoluc enzyme activity is higher, while the enzyme activities of ApoE2 NV and ApoE3 NV are similar to the control Nanoluc NV enzyme activity, indicating that the loading efficiency of ApoE4 NV is higher than that of ApoE2 NV and ApoE3 NV.

[0102] Table 1. NV enzyme activity data

[0103] Transwell results (Table 2 + Figure 2): Compared with the control NV, ApoE2 NV, ApoE3 NV, and ApoE4 NV can all be significantly secreted into the lower chamber by bEND.3 cells. After 24 hours of culture, the maximum number of ApoE4 NV particles detected in the lower chamber of the Transwell was approximately 2.3E9 particles, while the number of particles in the lower chamber of the control NV after 24 hours of culture was only 7E7 particles.

[0104] Table 2: Number of particles penetrating the Transwell at each time point

[0105] In vivo results in mice (Table 3 + Figure 3, Figure 4): ApoE2 NV, ApoE3 NV, and ApoE4 NV all had significant ability to penetrate the blood-brain barrier compared with the control NV. NV reached a peak in the brain tissue 0.5 h after administration, and the number of particles in the brain tissue gradually decreased 2 h and 2 h later. ApoE2 NV, ApoE3 NV, and ApoE4 NV were all able to resist liver clearance. After 2 h, the control NV was almost completely cleared from the liver, while there were still >1E9 NV particles in the experimental group.

[0106] Table 3. Number of particles in different tissues of mice

[0107] Example 2: Construction of NV loaded with different truncations of human ApoE2, ApoE3 or ApoE4 protein

[0108] Objective: To load different isoforms of human ApoE protein truncations onto the surface of NV and evaluate the blood-brain barrier penetration ability of this group of NV compared with wild-type NV.

[0109] method:

[0110] 1. Build:

[0111] The inventors designed different truncated ApoE sequences, ApoE3 (1-191), ApoE4 (1-191), ApoE2 (1-191), and ApoE (130-149), and constructed the following using pHEK293_Ultra_Expression_I (Takara) as a vector:

[0112] Expression plasmid ApoE3 (1-191)-linker-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD)-linker-Nanoluc,

[0113] Expression plasmid ApoE4(1-191)-linker-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc,

[0114] Expression plasmid ApoE2(1-191)-linker-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc,

[0115] Expression plasmid ApoE(130-149)-linker-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc.

[0116] 2. Loading

[0117] The loading method is the same as in Example 1.

[0118] 3. Preparation

[0119] The preparation method is the same as that of Example 1.

[0120] 4. Characterization

[0121] NanoFCM was used to detect the particle concentration of NV. NV was added to the Nanoluc substrate, and the Nanoluc enzyme activity was detected by a microplate reader to verify the expression of Nanoluc.

[0122] 5. Transwell assay

[0123] The method is the same as Example 1.

[0124] 6. In vivo experiments in mice

[0125] Mice were dosed via the tail vein, and samples were collected 0.5 hours after administration: eyeballs were removed to collect blood, which was centrifuged at 3000g for 20 minutes at 4°C. The supernatant was used as plasma for the Nanoluc enzyme activity assay. After perfusion, the liver and brain were collected. The liver and brain tissues were added to PBS, homogenized, and centrifuged at 12000 rpm for 10 minutes. The supernatant was collected and the Nanoluc enzyme activity assayed. The NV enzyme activity and the corresponding particle count were used as a calibration curve to calculate the NV particle count in each tissue.

[0126] result:

[0127] NV enzyme activity characterization (Table 4 + Figure 5): The different truncated ApoE3 (1-191) NV and ApoE4 (1-191) NV prepared had slightly higher loading efficiency and slightly higher unit enzyme activity than the control NV, while the loading efficiency of ApoE2 (1-191) NV and ApoE (131-149) NV was slightly lower.

[0128] Table 4. Characterization of NV enzyme activity corresponding to different truncations

[0129] Transwell results (Table 5 + Figure 6): Compared with the control NV, different truncated ApoE3 (1-191) NV, ApoE4 (1-191) NV, ApoE2 (1-191) NV, and ApoE (131-149) NV can be significantly secreted into the lower chamber through the Transwell cells. After 24 hours of culture, the highest number of particles detected in the lower chamber of the Transwell of ApoE4 (1-191) NV was >1E9 particles.

[0130] Table 5. Enzyme activity data of different truncated NV

[0131] In vivo results in mice (Table 6 + Figure 7): Detection was performed 30 minutes after administration. Compared with the control NV, different truncated forms of ApoE3 (1-191) NV and ApoE4 (1-191) NV had significant ability to penetrate the blood-brain barrier, while ApoE2 (1-191) NV had relatively weak ability to penetrate the blood-brain barrier, and ApoE (131-149) NV had basically no ability to penetrate the blood-brain barrier.

[0132] Table 6. Number of particles in the brain tissue of mice 30 minutes after administration of different truncated NV

[0133] Example 3: ApoE-NV is more capable of penetrating the blood-brain barrier than EVs loaded with brain-entering peptides such as Angiopep-2 and RVG.

[0134] Objective: To compare the differences in brain entry ability between various types of ApoE-NV and reported EVs.

[0135] method:

[0136] 1. Build:

[0137] Using pHEK293_Ultra_Expression_I (Takara) as the vector, construct:

[0138] Expression plasmid Angiopep-2-linker-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc;

[0139] Expression plasmid RVG-linker-Ig3(NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc expression plasmid

[0140] 2. Loading

[0141] The loading method is the same as in Example 1

[0142] 3. Preparation

[0143] The preparation method is the same as that of Example 1.

[0144] 4. Characterization

[0145] NanoFCM was used to detect the particle concentration of NV. NV was added to the Nanoluc substrate, and the Nanoluc enzyme activity was detected by a microplate reader to verify the expression of Nanoluc.

[0146] 5. Transwell

[0147] The method was the same as that in Example 1. After 24 hours of culture, the liquid in the lower chamber of the Transwell was collected to detect Nanoluc enzyme activity. At the same time, various ApoE NVs in Examples 1 and 2 were used for comparison with Angiopep-2 NV and RVG NV.

[0148] 6. In vivo experiments in mice

[0149] Mice were dosed via the tail vein, and samples were collected 30 minutes after administration: eyeballs were removed to collect blood, which was centrifuged at 3000g for 20 minutes at 4°C. The supernatant was used to assay Nanoluc enzyme activity in the plasma. Following perfusion, the liver and brain were collected and homogenized in PBS, then centrifuged at 12000 rpm for 10 minutes. The supernatant was then assayed for Nanoluc enzyme activity. Using the NV assay activity and the corresponding particle count as a standard curve, the number of NV particles in each tissue was calculated.

[0150] result:

[0151] NV enzyme activity characterization (Table 7 + Figure 8): The unit loading capacity of the prepared RVG NV, Angiopep-2 NV and control NV was basically the same.

[0152] Table 7. Enzyme activity data of different NV

[0153] Transwell results (Table 8 + Figure 9): Compared with the control NV, ApoE NV, RVG NV, and Angiopep-2 NV can all be significantly secreted into the lower chamber through the Transwell cells. After 24 hours of culture, the number of ApoE NV particles detected in the lower chamber of the Transwell was greater than that of RVG NV and Angiopep-2 NV particles detected in the lower chamber, especially ApoE4 NV and ApoE4(1-191)NV.

[0154] Table 8. Number of particles penetrating different NV Transwells within 24 hours

[0155] In vivo results in mice (Table 9 + Figure 10): Detection was performed 30 minutes after administration. Compared with the control NV, ApoE NV, RVG NV, and Angiopep-2 NV all had the ability to penetrate the blood-brain barrier, and the ability of various ApoE NVs to penetrate the blood-brain barrier was stronger than that of RVG and Angiopep-2.

[0156] Table 9. Number of particles in the brain tissue of mice after 30 minutes of administration of different NVs

[0157] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A truncated form of APOE, whose amino acid sequence is shown in any one of SEQ ID NOs. 1 to 3.

2. A nucleic acid encoding the truncated form of claim 1.

3. A nucleic acid construct comprising: Brain entry module, scaffold module, transmembrane region module, intramembrane region anchoring module and effect and detection module; The brain entry module is the nucleic acid according to claim 2 or the nucleic acid shown in any one of SEQ ID NOs: 4 to 5.

4. The nucleic acid construct according to claim 3, characterized in that The scaffold module includes any one or more of Fc, Foldon, Fc+NPTN-Ig1-3, ITGB1-I-EGF1-4, Foldon+NPTN-Ig1-3 or NPTN-Ig1-3; The transmembrane region module includes any one or more of EWI-F-TMD, EWI-2-TMD, ITGB1-TMD, TMD_Mut_10, TMD_Mut_13, TMD_Mut_17 or NPTN-TMD; The intramedullary region anchoring module contains a nucleotide sequence encoding S-palmitoylcysteine, and the intramedullary region anchoring module includes EWI-F-ICD or EWI-2-ICD.

5. The nucleic acid construct according to claim 4, characterized in that From the 5' end to the 3' end, it includes: brain entry module, NPTN-Ig1-3, NPTN-TMD and EWI-F-ICD.

6. The nucleic acid construct according to claim 5, characterized in that Also included are a signal marker module and / or an effector module; The detection module includes any one or more of a fluorescent protein module and / or a chemiluminescent module; The effector module includes protein drugs or nucleic acid drugs for treating brain diseases.

7. The nucleic acid construct according to claim 6, characterized in that The signal marker module is connected to the intramembrane anchor module via a linker; The fluorescent protein module includes any one or more of green fluorescent protein, red fluorescent protein, blue fluorescent protein or yellow fluorescent protein; The chemiluminescent module includes Nanoluc or Rluc.

8. A plasmid, expression vector or expression cassette comprising the nucleic acid construct according to any one of claims 3 to 7.

9. A transformant or recombinant cell comprising the plasmid, expression vector or expression cassette of claim 8.

10. An engineered EVs, characterized in that: The method is prepared by using the transformant or recombinant cell according to claim 9.

11. A protein expressed by the nucleic acid construct according to any one of claims 3 to 7.

12. Use of the nucleic acid construct according to any one of claims 3 to 7, the plasmid, expression vector or expression cassette according to claim 8, the transformant or recombinant cell according to claim 9, the engineered EVs according to claim 10 and / or the protein according to claim 11 in the preparation of a drug or reagent, wherein the drug has the effector module as an active ingredient.

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