Anti-DAT antibodies and compositions thereof

Abstract

The invention discloses an anti-DAT antibody which is formed by transcription and translation of a fragment gene, and the fragment gene comprises SEQ ID No: 2. The anti-DAT antibody provided by the invention can be prepared into a composition capable of crossing a blood brain barrier, and can achieve specific binding aiming at dopamine nerve cells, and achieve the excellent effects of reducing accumulation of protein synuclein in a striatum brain region of degeneration or lesion of the dopamine nerve cells and delaying the course of Parkinson's disease.

Description

TECHNICAL FIELD

[0001] An antibody, in particular an anti-DAT antibody and compositions thereof. BACKGROUND

[0002] Parkinson's disease (PD) is a common degenerative disease of the central nervous system (CNS) in the brain, the symptoms of which usually appear slowly over time, affecting the motor nervous system, and cannot be cured. Traditionally, the treatment method of administering a large amount of dopamine drug (L-DOPA) is used to delay disease progression, but as the nerve degenerates, loses, and is blocked by the blood-brain barrier, the treatment method of dopamine drug gradually fails. In addition, after the central nervous system in the brain is indiscriminately treated by a large amount of dopamine drug, it is difficult to avoid the influence of the drug on the physiological mechanism of the central nervous system in other brain areas. Therefore, developing other technologies to help slow down Parkinson's disease is an urgent goal in the related field. SUMMARY

[0003] In order to develop related technologies that can slow down Parkinson's disease, the present application provides an anti-DAT antibody for labeling dopamine transporters formed after transcription and translation of a fragment gene, wherein the fragment gene comprises SEQ ID No: 2.

[0004] The anti-DAT antibody provided by the present application can be further made into a composition according to the needs, comprising a target gene SEQ ID No: 3, any part of the target gene SEQ ID No: 3 is inserted into a fragment gene SEQ ID No: 2.

[0005] The anti-DAT antibody provided by the present application can be further made into a composition comprising an extracellular vesicle, wherein the anti-DAT antibody is combined with a transmembrane protein on the extracellular vesicle.

[0006] The extracellular vesicle is secreted by a cell transfected with a vector gene, and at least a part of the vector gene comprises SEQ ID No: 2 or SEQ ID No: 4.

[0007] The dopamine transporter antibody is formed in the extracellular loop between the third and fourth transmembrane domains of the transmembrane protein.

[0008] The transmembrane protein is CD63, and the cell is a HEK-293 cell

[0009] The extracellular vesicle contains a drug, and the drug includes but is not limited to a gene fragment (DNA, RNA), a protein sequence, a chemical agent, etc.

[0010] The targeted carrier provided by this invention can carry drugs across the blood-brain barrier and can specifically bind to dopamine neurons. It can also regulate the secretion of Parkinson's disease marker proteins and reduce the accumulation of protein synuclein in the striatum brain region where dopamine neurons are degenerated or diseased, thus producing excellent therapeutic effects in delaying the progression of Parkinson's disease. Attached Figure Description

[0011] Figure 1 This is a block diagram illustrating the steps of a preferred embodiment of the targeted carrier provided by the present invention;

[0012] Figure 2A and 2B The image shows the protein performance results of the first preferred embodiment of the targeting vector provided by the present invention.

[0013] Figure 3A A transmission electron microscope (TEM) image of a preferred embodiment of the targeting carrier provided by the present invention;

[0014] Figure 3B This is a diagram showing the results of the Western ink dot method in the first preferred embodiment of the targeted carrier provided by the present invention;

[0015] Figure 4A and 4B Electron microscopy (TEM) images of the first and second preferred embodiments of the targeted carrier provided by the present invention after loading the drug;

[0016] Figure 5A and 5B The flow cytometry results of the first and second preferred embodiments of the targeted carrier provided by the present invention after loading the drug are shown in the figure.

[0017] Figure 6A This is a simulation diagram of the blood-brain barrier cell culture model provided by the present invention;

[0018] Figure 6B This is a diagram of the immunofluorescence staining results in Experiment 1 provided by the present invention;

[0019] Figure 7A This is a graph showing the intracellular reactive oxygen species concentration detection in Experiment 2 provided by the present invention;

[0020] Figure 7B The result diagram of the Western ink dot method in Experiment 2 provided by this invention;

[0021] Figure 7C This is a graph showing the cell mortality rate results in Experiment 2 provided by the present invention;

[0022] Figure 8Distribution map of the targeting vector in vivo in the animal model of Parkinson's disease provided by this invention;

[0023] Figure 9 The image shows the results of a roller running test after the Parkinson's disease animal model provided by this invention was treated with a targeted vector in the first preferred embodiment.

[0024] Figures 10A to 10C The figure shows the results of open space trajectory analysis of the Parkinson's disease animal model provided by the present invention after treatment with the first preferred embodiment of the targeted vector;

[0025] Figures 11A to 11D The images show brain slices and blood analysis results of the Parkinson's disease animal model provided by this invention after treatment with a targeted vector in the first preferred embodiment.

[0026] Figure 12A and 12B The results of the roller running test and open space trajectory analysis were obtained by treating the Parkinson's disease animal model provided by the present invention with a targeted carrier in the second preferred embodiment.

[0027] Figure 12C The image shows the brain slice analysis results of the Parkinson's disease animal model provided by the present invention after treatment with a targeted vector in the second preferred embodiment.

[0028] Figure 13A and 13B The image shows the protein expression results of nerve cells (PD) differentiated from induced pluripotent stem cells (iPSCs) of Parkinson's disease patients provided by the present invention after treatment with a targeting vector in the first preferred embodiment.

[0029] Figure 14A This is a graph showing the concentration detection of intracellular and extracellular vesicle proteins in Experiment 7 provided by the present invention;

[0030] Figure 14B The result diagram of the Western ink dot method in Experiment 7 provided by this invention;

[0031] Figure 15A and 15B The results of the roller running test and open space trajectory analysis of the Parkinson's disease animal model provided by the present invention after treatment with the third preferred embodiment of the targeted vector are shown in the figure.

[0032] Figure 15C This is a protein surface analysis diagram of the brain region of the Parkinson's disease animal model provided by the present invention after treatment with a targeted vector in the third preferred embodiment.

[0033] Symbol explanation:

[0034] 10 Blood-brain barrier cell culture models

[0035] 11 First Training Area

[0036] 12 Second Cultivation Area

[0037] 13 perforations

[0038] Culture medium A

[0039] 20 endothelial cells

[0040] 30-week cells

[0041] 40 stellate cells

[0042] 50 Parkinson's disease model cell lines

[0043] Steps S1-S3 Detailed Implementation

[0044] The known pathological features of Parkinson's disease (PD) are the degeneration and death of dopamine neurons projected from the substantia nigra to the striatum. The expression level of the dopamine transporter (DAT) in the striatum can be used to assess the disease progression of Parkinson's disease.

[0045] Please refer to Figure 1 To produce a specific therapeutic effect, this invention genetically engineered a mother cell line so that the surface of an extracellular vesicle secreted by the mother cell line expresses a dopamine transporter antibody (hereinafter referred to as anti-DAT). A target extracellular vesicle (hereinafter referred to as DATEV vector) was designed so that the DATEV vector can specifically bind to dopamine neurons expressing dopamine transporter (DAT) through anti-DAT, thereby effectively promoting the phagocytic behavior of dopamine neurons and avoiding the binding reaction of normal cells to extracellular vesicles.

[0046] The dopamine transporter antibody (hereinafter referred to as anti-DAT) is preferably expressed on the membrane-penetrating proteins (such as CD63, CD81 and CD9) of the DATEV vector. In this embodiment, the dopamine transporter antibody is expressed on the CD63 membrane-penetrating protein of the DATEV vector.

[0047] In this embodiment, the fabrication steps of the DATEV carrier include:

[0048] Step S1: Constructing a dopamine transporter antibody plasmid: A gene fragment expressing a dopamine transporter antibody is prepared, and the nucleic acid sequence of the dopamine transporter antibody is inserted into a target gene of the membrane-penetrating protein using genetic engineering techniques, so that the gene fragment binds to the target gene and forms a vector gene. The present invention will subsequently use SEQ ID No:3 of the target gene of the membrane-penetrating protein CD63 expressed in the extracellular vesicle as an example for illustration.

[0049] The gene fragment contains either SEQ ID No:1 or SEQ ID No:2.

[0050] Specifically, this gene fragment is inserted into the gene sequence of the transmembrane protein in the extracellular loop between the third and fourth transmembrane domains.

[0051] In one embodiment, the fragment gene SEQ ID No:1 or SEQ ID No:2 is inserted into the target gene SEQ ID No:3 or SEQ ID No:5 to form the vector gene.

[0052] In one embodiment, the fragment gene SEQ ID No:2 is inserted into the target gene SEQ ID No:3 to form the vector gene SEQ ID No:4.

[0053] In one embodiment, the fragment gene SEQ ID No:1 is inserted into the target gene SEQ ID No:5 to form the vector gene SEQ ID No:6.

[0054] Next, the vector gene SEQ ID No:4 was recombined into a plasmid using gene recombination technology. In this embodiment, the plasmid was a pEXO plasmid (Addgene, Watertown, MA, USA).

[0055] Step S2: Transplant the vector gene into the mother cell line and culture: The plastid carrying the vector gene SEQ ID No:4 is inserted into the mother cell line using cell transfection technology to form a DAT cell line, which highly expresses the dopamine transporter antibody (anti-DAT). The cell transfection technology includes electroporation, cell extrusion, ultrasound, or chemical transfection. In this embodiment, liposome transfection (Lipofectamine 3000, L3000015, Invitrogen, Waltham, MA, USA) is used.

[0056] There are no restrictions on the choice of the mother cell line; it can be selected based on the cell characteristics of the line itself. For example, the Human Embryonic Kidney Cells 293 (HEK-293) cell line (hereinafter referred to as HEK-293 cell line) is highly efficient for cell transfection, and it grows rapidly and has a simple culture method, allowing for the rapid acquisition of a large number of cells and their secreted extracellular vesicles. Alternatively, mesenchymal stem cells (MSCs) are rich in growth factors and anti-inflammatory factors, and their secreted extracellular vesicles can simultaneously carry abundant growth factors and anti-inflammatory factors to achieve direct therapeutic effects.

[0057] 2×10 8 One DAT-293T cell was distributed in a culture dish. Benchtop Bioreactor (ESCO Aster, Singapore) was cultured with 500 mL of Dulbecco's modified Eagle's medium (DMEM), which contained exosome-depleted fetal bovine serum (exosome-depleted FBS, Gibco, Grand Island, NY, USA) and 1% antibiotics (penicillin / streptomycin / amphotericin B solution).

[0058] Step S3, collecting the DATEV vector: After culturing DAT-293T cells for 3 to 4 days, the culture medium of the DAT-293T cell line was collected and filtered through a 0.22 μm filter membrane. Then, it was concentrated and purified using a tangential flow filtration system with a molecular weight cutoff of 300 kDa (MAP.03-plus TFF System; Lefo Science). The supernatant obtained by elution by chromatography was then concentrated through a 30 kDa molecular weight cutoff membrane. Finally, the DATEV vector released into the culture medium was obtained and resuspended in phosphate-buffered saline (PBS).

[0059] The above steps were used to differentiate the DATEV vectors secreted by DAT-293T cell lines transfected with SEQ ID No:1 and SEQ ID No:2 of the gene fragment, respectively, using a first embodiment and a second embodiment. The DATEV vectors purified in the first and second embodiments exhibited an affinity of KD: 1.3 to 6.4 × 10⁻⁶. -9 Between M.

[0060] Figure 2AIn step 2B, the expression levels of CD63 and anti-DAT on the DATEV vector of the first embodiment were quantified using antibody-antigen technology, and compared with those of ordinary extracellular vesicles secreted by ordinary HEK-293 cells (hereinafter referred to as EV vectors) to confirm the difference in the expression of the transmembrane protein CD63 and anti-DAT protein between the two. The methods for culturing ordinary HEK-293 cells and collecting ordinary extracellular vesicles are the same as those for culturing DAT-293T cells and collecting DATEV vectors described above.

[0061] After collecting DATEV vectors and general extracellular vesicles, they were placed in reaction solutions containing fluorescent CD63 antibody and fluorescent DAT recombinant protein, respectively, and cultured for 6 to 12 hours. The reaction solutions were then replaced with a buffer solution (PBS-T containing 0.1% casein), and 50 μL (50 μL / well) was injected into each well of a detection disc. The proportion of DATEV vectors and general extracellular vesicles containing CD63 and / or anti-DAT on the surface was then calculated using an automated extracellular vesicle absolute quantification analyzer (ExoCounter, JVCKENWOOD Corporation, Yokosuka, Japan). The results showed that the expression of the transmembrane protein CD63 and anti-DAT protein in DATEV vectors obtained from the multi-DAT-293T cell line was superior to that in general extracellular vesicles obtained from the untransfected general HEK-293 cell line. In the second embodiment, the expression levels of CD63 and anti-DAT on the DATEV carrier were also as high as 94% and 91%, respectively (not shown in the figure).

[0062] The present invention also provides a third embodiment, which differs from the second embodiment in that the progenitor cell line is mesenchymal stem cell (MSC), and DAT-MSC cells are subsequently transfected using lentivirus technology. The DAT-MSC cells are cultured in 20 mL of fetal bovine serum (FBS) containing 8 μg / mL polybrene (PB), 50 mg / mL protamine sulfate (PS), and 1100 mg / mL synthesonic F108 (F108). Further, the mesenchymal stem cells (MSC) are inoculated with the lentivirus at a multiplicity of infection (MOI) greater than 2 (virus count / cell count).

[0063] Figure 3A As can be seen from the images taken by the transmission electron microscope (TEM), in the first, second and third embodiments, the DATEV vectors obtained by collection have the appearance of the dopamine transporter antibody (anti-DAT), and their overall size and shape are not affected compared with general EV vectors. Figure 3B In the study, the differences in protein expression levels between the DATEV and EV vectors were confirmed using Western blotting. It was clearly observed that the DATEV vector showed higher expression of membrane-penetrating proteins (especially CD63 and CD81) and anti-DAT protein compared to the EV vector. This confirms that the DATEV vector secreted by the DAT-293T cell line after gene transfection and anti-DAT protein expression, following the steps described above, exhibits anti-DAT protein expression.

[0064] The targeted carrier provided by this invention can be further loaded with a drug as needed, thereby creating a biocompatible medical preparation. The drug includes, but is not limited to, gene fragments (DNA, RNA), protein sequences, chemical agents, etc.

[0065] Curcumin possesses excellent medicinal properties, having been shown to aid in wound healing, fight cancer, combat COVID-19, and regulate the function of the human immune system. Numerous studies have also demonstrated that curcumin can effectively reduce the expression of neurodegenerative factors (phosphorylation of tau and β-amiloid precursor protein), showcasing its potential in the treatment of Parkinson's disease.

[0066] Brain-derived neurotrophic factor (BDNF) is one of the important active proteins in the brain, used to regulate the survival, growth, and remodeling of neurons. A considerable body of literature has indicated a strong correlation between insufficient BDNF levels and many neurodegenerative diseases, such as Alzheimer's and Parkinson's.

[0067] To verify that the targeting vector provided by this invention possesses targeting, loading, and release functions, this invention further encapsulates the DATEV vector with curcumin and messenger ribonucleic acid (mRNA)-BDNF-CY3 for expressing BDNF expression, forming a DATEV vector loaded with curcumin (hereinafter referred to as Cur@DATEV) and a DATEV vector loaded with mRNA-BDNF-CY3 (hereinafter referred to as BDNF@DATEV), to observe the effects of Cur@DATEV and BDNF@DATEV on the treatment of Parkinson's disease.

[0068] Please refer to Figure 4A as well as Figure 4B In the first embodiment, the DATEV vector and EV vector were coated with curcumin to form a curcumin-loaded DATEV vector (Cur@DATEV) and a curcumin-loaded EV vector (hereinafter referred to as Cur@EV). Subsequent cell and animal experiments were then conducted to confirm the specificity and functionality of the DATEV vector, as well as the effectiveness of curcumin in the treatment of Parkinson's disease. In the second embodiment, the DATEV vector was coated with mRNA-BDNF-CY3 to form a mRNA-BDNF-CY3-loaded DATEV vector (BDNF@DATEV). The EV vector loaded with mRNA-BDNF-CY3 was referred to as BDNF@EV in comparison, to confirm the specificity and functionality of the DATEV vector, as well as the effectiveness of BDNF downregulation in the treatment of Parkinson's disease.

[0069] In this embodiment, the DATEV vector and the EV vector are coated with curcumin and mRNA-BDNF-CY3 by ultrasound and electroporation, respectively.

[0070] The steps of ultrasonically encapsulating the curcumin in the DATEV and EV carriers include:

[0071] The DATEV and EV vectors were mixed with human serum albumin at weight ratios ranging from 1:0.1 to 1:2 to form a mixture. The mixture was then ultrasonically treated for 1-10 cycles at a 30-second on / off cycle, with the mixture cooled on ice for 2 minutes between cycles. The mixture was then filtered through a first-cutoff membrane with a molecular weight cutoff of 100 kDa. Curcumin was added to the filtered mixture at a ratio of 1:1:1 (μg) of human serum albumin, DATEV / EV vectors, and curcumin, and the mixture was subjected to another 6 ultrasonic cycles under the same conditions. Subsequently, the mixture was filtered twice through a second-cutoff membrane with a molecular weight cutoff of 30 kDa and resuspended in PBS buffer for subsequent experiments.

[0072] The steps of electroporating the mRNA-BDNF-CY3 with the DATEV and EV vectors include:

[0073] Mix the DATEV / EV vector with mRNA-BDNF-CY3; then apply the mixture to an electroporation environment with a voltage of 100-250V, a pulse duration of 50-300μs, a pulse interval of 500-1500μs, and 4-6 cycles; then incubate in a cell culture incubator for 30-90 min, centrifuge, and resuspend in PBS buffer for subsequent experiments.

[0074] At Figure 4A Images taken using a transmission electron microscope (TEM) show that, compared to conventional EV carriers loaded with curcumin, the Cur@DATEV carrier coated with curcumin did not affect its overall size and shape compared to Cur@EVs. Similarly, Figure 4B In the study, there was no difference in size and shape between BDNF@DATEV and BDNF@EV, which proves that the DATEV carrier with the dopamine transporter antibody (anti-DAT) bound to its surface does not affect its ability to encapsulate drugs.

[0075] Figure 5A5B involves flow cytometry analysis. In the first and second embodiments, through the steps of coating curcumin and mRNA-BDNF-CY3 provided by this invention, the DATEV vector forms Cur@DATEV loaded with curcumin and BDNF@DATEV loaded with mRNA-BDNF-CY3. The flow cytometer identifies Cur@DATEV through the fluorescence reaction inherent in curcumin itself; and detects Cur@DATEV and BDNF@DATEV through the CY3 fluorescence reaction carried by mRNA-BDNF-CY3. The results are then calculated and converted by the system. Figure 5A as well as Figure 5B The results in quadrant Q2 show that, in the first embodiment, after the step of coating the DATEV vector with curcumin, approximately 88.3% of the vectors were successfully coated with curcumin (i.e., Cur@DATEV was formed); in the second embodiment, after the step of coating the DATEV vector with mRNA-BDNF-CY3, approximately 69.2% of the vectors were successfully coated with mRNA-BDNF-CY3 (i.e., BDNF@DATEV was formed).

[0076] Please refer to Figure 6A To demonstrate the effectiveness of the DATEV vector and / or Cur@DATEV in organisms, a blood-brain barrier cell culture model 10 is provided. The aim is to simulate the blood-brain barrier environment in organisms, ensuring that the drug carrier can cross the blood-brain barrier, enter the brain region, and specifically bind to a Parkinson's disease model cell line. The Parkinson's disease model cell line is differentiated from a human neuroblastoma cell line (hereinafter referred to as the SH-SY5Y cell line). Due to the DAT protein expression characteristic of the SH-SY5Y cell line, it has become a commonly used cell model in medical research related to Parkinson's disease.

[0077] SH-SY5Y cells were cultured in a 1:1 mixture of Minimum Essential Medium (MEM, Invitrogen) and Ham's F-12 Nutrient Mix (F-12, Thermo Fisher). This mixture was supplemented with 10% fetal bovine serum (FBS, US-sourced HyClone, GE), 1% sodium pyruvate (Thermo Fisher), and 1% GlutaMAX. TMSupplements and 1% penicillin-streptomycin (ThermoFisher) were added, and the cells were cultured at 37°C, 5% CO2, and under controlled humidity. When the cells reached a distribution that occupied 70–80% of the cell disc area, neural differentiation into the Parkinson's disease model cell line could be induced. The method for inducing neural differentiation was to first treat SH-SY5Y cells with 50 μM retinoic acid (RA) for two days, and then change the differentiation culture medium daily for the next 5 days. The differentiation culture medium was a mixed culture medium containing 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0078] The blood-brain barrier cell culture model 10 includes a first culture zone 11 and a second culture zone 12, which are connected by multiple perforations 13. The first culture zone 11 and the second culture zone 12 are filled with a culture medium A. Parkinson's disease model cell line 50 is cultured at the bottom of the first culture zone 11, while an endothelial cell 20, a pericyte cell 30, and an astrocyte 40 are sequentially arranged at the bottom of the second culture zone 12 adjacent to the multiple perforations 13. The resistance value of the blood-brain barrier cell culture model 10 is measured to be 2144 Ω·cm², effectively constructing the impedance environment of the blood-brain barrier. In subsequent experiments, EV vector, DATEV vector, Cur@DATEV or Cur@EV were sequentially administered to the second culture zone 12 of the blood-brain barrier cell culture model 10, and the Parkinson's disease model cell line 50 in the first culture zone 11 was used for experimental analysis to confirm the effectiveness and therapeutic effect of the drug carrier in crossing the second culture zone 12 to the first culture zone 11.

[0079] Experiment 1

[0080] First, it was confirmed that the DATEV vector could cross the impedance environment of the blood-brain barrier constructed in the blood-brain barrier cell culture model 10 and be phagocytosed by the Parkinson's disease model cell line 50. Please refer to [reference needed]. Figure 6B In this experiment, taking the DATEV vector of the first embodiment as an example, EV vector loaded with curcumin (Cur@EV) was introduced into the blood-brain barrier cell culture model 10 as the control group, and DATEV vector loaded with curcumin (Cur@DATEV) was introduced as the experimental group to compare the effects of the Parkinson's disease model cell line on phagocytosis of Cur@DATEV and Cur@EV.

[0081] The endocytosis efficiency of the Parkinson's disease model cell lines was determined after reacting with Cur@DATEV and Cur@293EV for 24 hours, respectively. The curcumin content in the Parkinson's disease model cell lines was detected using the fluorescent properties of curcumin itself to compare the phagocytic effects of Cur@DATEV and Cur@EV on the Parkinson's disease model cell lines. Simultaneously, immunofluorescence staining and extracellular vesicle protein labeling techniques (Protein EV Labeling Kit (Red), ExoGlow) were used. TM The study, conducted by System Biosciences (Palo Alto, CA, USA), allows for the identification of extracellular vesicle proteins of Cur@DATEV and Cur@EV in the Parkinson's disease model cell line via red fluorescence to confirm whether Cur@DATEV and Cur@EV enter the Parkinson's disease model cell line via endocytosis; F-actin can be identified via purple fluorescence to confirm the cytoskeleton; the cell nucleus is identified via blue fluorescence; and curcumin can be identified via green fluorescence to confirm whether it enters the Parkinson's disease model cell line via Cur@DATEV and / or Cur@EV.

[0082] Depend on Figure 6B The results showed that in the group treated with Cur@DATEV, the fluorescence of the extracellular vesicle protein (red fluorescence) and the fluorescence reaction of curcumin (green fluorescence) in the Parkinson's disease model cell line were significantly higher than those in the group treated with Cur@EV. This confirms that the DATEV vector should have the specificity to label DAT protein and can enter the Parkinson's disease model cell line through cytophagy.

[0083] Experiment 2

[0084] Similarly, using the DATEV vector from the first embodiment as an example, it was confirmed that Cur@DATEV can cross the impedance environment of the blood-brain barrier constructed in the blood-brain barrier cell culture model 10 and be phagocytosed by the Parkinson's disease model cell line 50. Further confirmation was made regarding whether Cur@DATEV can release curcumin within the Parkinson's disease model cell line 50 and achieve therapeutic effectiveness. The following were provided in the blood-brain barrier cell culture model 10:

[0085] The group that did not undergo any additive treatment served as the primary control group (PD);

[0086] The group treated with the added EV vector served as the second control group (EV);

[0087] The groups treated with the DATEV vector were used as the third control group (DATEV).

[0088] The group treated with added curcumin served as the fourth control group (Cur);

[0089] The group treated with EV vectors loaded with curcumin served as the fifth control group (Cur@EV); and

[0090] The experimental group (Cur@DATEV) was treated with DATEV vector loaded with curcumin to confirm whether curcumin was released after the Parkinson's disease model cell line engulfed Cur@DATEV and the effectiveness of the treatment.

[0091] After 48 hours of reaction in each group, the response mechanisms of the Parkinson's disease model cell line 50 in each group were examined, including: mortality rate, intracellular reactive oxygen species (ROS) concentration, and protein expression of Parkinson's disease-related factors. Alpha-synuclein (α-syn) is a marker protein commonly found in dopaminergic neurons of Parkinson's patients, while Parkin protein, DJ-1 protein, and TH protein are marker proteins expressed by dopaminergic neurons after damage to achieve cell repair.

[0092] In this experiment, the intracellular reactive oxygen species (ROS) concentration of the Parkinson's disease model cell line 50 was determined by flow cytometry analysis. Figure 7A The test results showed that the intracellular reactive oxygen species concentration of the Parkinson's disease model cell line 50 in the fourth control group, the fifth control group, and the experimental group was significantly lower than that in the first to third control groups.

[0093] Figure 7B By detecting alpha-synuclein (α-syn), Parkin, DJ-1, and TH proteins in the Parkinson's disease model cell line 50 in the above groups using Western blotting, it was observed that the expression of alpha-synuclein was reduced after Cur@DATEV treatment, while Parkin, DJ-1, and TH proteins showed high expression. This indicates that the Parkinson's disease model cell line exhibited enhanced cell repair capabilities after Cur@DATEV treatment.

[0094] Please refer to the following: Figure 7CThe Parkinson's disease model cell line 50 was considered as degenerated / pathological dopamine neurons, exhibiting low cell repair capacity and a high mortality rate. In this experiment, the number of remaining cells in each group was standardized on days 1, 3, and 7 after treatment, using the number of remaining cells in the first control group as a baseline. Starting from day 3, the number of remaining cells in the fourth control group, fifth control group, and experimental group was significantly higher than that in the first to third control groups. More notably, on day 7, the number of remaining cells in the experimental group was significantly higher than that in the fourth and fifth control groups, confirming that after treatment with Cur@DATEV, the DATEV vector can release curcumin intracellularly and enhance the effectiveness of curcumin treatment (compared to the results of the fourth and fifth control groups).

[0095] The present invention then constructs an animal model of Parkinson's disease to test the behavioral changes of the animal model after treatment with Cur@DATEV and BDNF@DATEV. Rats were trained to run on a roller, and their motor values ​​were measured in a healthy state. The behavioral performance of the healthy rats was also assessed using an open field test. Next, an injection of oxydopamine (6-hydroxydopamine, 6-OHDA) was performed into the left dorsal striatum of the rats to destroy dopamine neurons in the dorsal striatum, thus creating an animal model of Parkinson's disease that simulates the symptoms of the disease. The unit volume of each injection of the hydroxydopamine (6-hydroxydopamine, 6-OHDA) agent is 3.2 μL, and each unit volume of the hydroxydopamine (6-hydroxydopamine, 6-OHDA) agent contains 11 μg of hydroxydopamine (6-hydroxydopamine, 6-OHDA) powder.

[0096] While the specificity of DATEV delivery in an animal model of Parkinson's disease has not been confirmed, this invention uses the first embodiment as an example. In this animal model, Cur@DATEV and Cur@EV were administered intravenously. Prior to intravenous injection, Cur@DATEV and Cur@EV were reacted with a lipophilic fluorescent dye (XenoLight DiR, PerkinElmer) to impart fluorescence. Twenty-four hours after injection, the absorption of Cur@DATEV and Cur@EV in various organs of the Parkinson's disease animal model was detected using an In Vivo Imaging System (IVIS Imaging System) with fluorescence. Figure 8 The fluorescence values ​​measured in various organs of this Parkinson's disease animal model were standardized based on the Cur@EV group. It can be seen that in the Cur@DATEV group, a clearly measurable fluorescence response was observed in the brain. This not only demonstrates the specificity of DATEV but also proves that DATEV has the ability to cross the blood-brain barrier. Furthermore, it was found that DATEV can remain in the brain for up to 7 days (results not shown).

[0097] Experiment 3

[0098] Having confirmed that Cur@DATEV can cross the blood-brain barrier in this Parkinson's disease animal model, this experiment further confirms, using the first embodiment, whether Cur@DATEV can release curcumin within the Parkinson's disease animal model and alter the Parkinson's disease symptoms, thereby slowing disease progression or even producing a therapeutic effect. The Parkinson's disease animal model was treated with intravenous injection every 7 days from the day of surgery (week 0) to week 5, and was divided into the following groups:

[0099] The group that underwent sham surgery in the dorsal striatum brain region and did not receive any drug treatment was the healthy control group.

[0100] The animal model of Parkinson's disease that was not treated with any drugs served as the first control group (PD);

[0101] The Parkinson's disease animal model treated with EV vector served as a second control group (EV);

[0102] The Parkinson's disease animal model treated with the DATEV vector served as the third control group (DATEV).

[0103] The Parkinson's disease animal model treated with curcumin served as the fourth control group (Cur);

[0104] The Parkinson's disease animal model treated with an EV vector loaded with curcumin served as the fifth control group (Cur@EV). In both the fourth and fifth control groups (Cur and Cur@EV), 1 mg of curcumin per 100 g of mouse weight was administered.

[0105] The Parkinson's disease animal model treated with the DATEV vector loaded with curcumin was used as the experimental group (Cur@DATEV) to confirm whether curcumin was released in the brain of the Parkinson's disease animal model after Cur@DATEV treatment and the effectiveness of the treatment.

[0106] Please refer to Figure 9 Each group began the rotarod test at week 0 (W0), and the test was conducted at weeks 2 (W2), 4 (W4), 6 (W6), and 8 (W8) to observe the performance of each group's motor values, which included the duration of time spent on the rotarod. The results showed that, compared with the healthy control group, the motor values ​​of the Parkinson's disease animal models in the first to fifth control groups and the experimental group were significantly reduced.

[0107] Furthermore, compared with the second (EV), third (DATEV), fourth (Cur), and fifth control group (Cur@EV), only in the experimental group (Cur@DATEV) did the motor function of the Parkinson's disease animal model significantly increase with the number of Cur@DATEV treatments and weeks. Notably, after the final intravenous injection treatment in week 5, the motor function of the Parkinson's disease animal model in the experimental group (Cur@DATEV) not only did not decline in weeks 6 to 8, but even showed a trend of further improvement.

[0108] Please refer to Figures 10A to 10CEach group underwent open field trajectory analysis at week 0, and at weeks 2, 4, 6, and 8 to observe the performance of motor values ​​in each group. The motor values ​​included speed, total distance traveled, and total rest time. Results corresponding to the rotarod test were obtained. In the group treated with curcumin-DATEV-encapsulated material (Cur@DATEV), the motor values ​​(speed, total distance traveled, and total rest time) of the Parkinson's disease animal model significantly improved with increasing weeks and the number of Cur@DATEV treatments. This indicates that treatment with curcumin-DATEV-encapsulated material not only alleviated the symptoms of Parkinson's disease (decreased motor ability) but also potentially restored the behavioral performance of the Parkinson's disease animal model.

[0109] Experiment 4

[0110] Experiment 3 demonstrated that treatment with the curcumin-coated DATEV vector could restore the behavioral performance of the Parkinson's disease animal model. Further, this experiment involved biochemical analysis of brain slices and blood samples from each of the aforementioned groups of Parkinson's disease animal models.

[0111] In this study, striatum brain slices were obtained from each group of animal models of Parkinson's disease, and antibody antigen reactions were used to detect α-synuclein (α-syn), a marker of Parkinson's disease, TH protein, a marker of cell repair, and DAT protein, a marker of dopamine neurons.

[0112] It is possible to Figure 11A The results showed that in the experimental group of the Parkinson's disease animal model treated with Cur@DATEV, the dorsal striatum brain region not only had significantly less accumulation of α-synuclein (α-syn) than the first to fifth control groups, but also significantly higher levels of TH and DAT proteins. Apart from this, there was no significant difference in the expression of α-syn, TH, and DAT proteins between the experimental group (Cur@DATEV) and the healthy control group (Health).

[0113] After obtaining blood from each of the Parkinson's disease animal models in each group, biochemical analysis was performed on a protein microarray using antigen labeling technology to observe the expression of proteins related to nerve regeneration and inflammatory response in the blood of each Parkinson's disease animal model after treatment.

[0114] The proteins related to nerve regeneration include BDNF, NGF, VEGF, IL-10, and CNTF; the proteins related to inflammatory responses include INF-γ, IL-1β, IL-6, and TNF-α. The values ​​of the aforementioned items obtained by detection are standardized and analyzed and plotted based on the values ​​obtained from the healthy control group (Health).

[0115] Figure 11B The results show the protein analysis results related to nerve regeneration in this Parkinson's disease animal model in each group. The results indicate that, compared to the first to fifth control groups, the experimental groups showed an increasing trend in the expression of proteins related to nerve regeneration, especially BDNF, IL-10, and CNTF. Notably, the expression of the nerve regeneration-related protein BDNF in the experimental groups was even significantly higher than that in the healthy control group.

[0116] Figure 11C This section presents the results of protein analysis related to inflammation in each group of the Parkinson's disease animal model. The results show that, compared to the first to fifth control groups, the experimental groups exhibited a decreasing trend in the expression of inflammation-related proteins, particularly INF-γ, IL-1β, IL-6, and TNF-α. Notably, the expression of TNF-α in the experimental groups was even significantly lower than that in the healthy control group.

[0117] Figure 11D The study compared the expression of neural stem cell-related proteins in brain slices from the dorsal striatum of a Parkinson's disease animal model between the first control group (PD) and the experimental group (Cur@DATEV) using immunofluorescence staining. These neural stem cell-related proteins included Nestin and Ki67. The results showed that the expression of neural stem cell-related proteins in the experimental group (Cur@DATEV) was significantly higher than that in the first control group (PD). This indicates that the curcumin-encapsulated DATEV vector can maintain the activity of neural stem cells in the dorsal striatum brain region, thereby achieving the effect of dopamine-induced neuronal regeneration and differentiation.

[0118] Based on the above results, it can be comprehensively evaluated that after treatment with curcumin-coated DATEV carrier, curcumin can not only cross the blood-brain barrier via DATEV to reach the dorsal striatum brain region, thereby reducing the secretion and accumulation of synuclein proteins and slowing down the production of inflammatory responses, but also promote the decline of the expression of inflammatory response-related proteins and the increase of the expression of neural regeneration-related proteins. At the same time, it can maintain and sustain the activity of neural stem cells, achieve the effect of dopamine-induced neural cell regeneration and differentiation, and effectively improve the motor ability of the rat model of Parkinson's disease.

[0119] Experiment 5

[0120] This experiment used the DATEV vector from the second embodiment as an example to confirm whether BDNF@DATEV could release mRNA-BDNF-CY3 within the Parkinson's disease model cell line 50 and effectively downregulate BDNF expression. The Parkinson's disease animal model was treated with intravenous injection every 7 days from the day of surgery (week 0) to week 5, and was divided into the following groups:

[0121] The group that underwent sham surgery in the dorsal striatum brain region and did not receive any drug treatment was the healthy control group.

[0122] The animal model of Parkinson's disease that was not treated with any drugs served as the first control group (PD);

[0123] An animal model of Parkinson's disease treated with an EV vector loaded with mRNA-BDNF-CY3 served as a second control group (BDNF@EV); and

[0124] The experimental group (BDNF@DATEV) was treated with the DATEV vector loaded with mRNA-BDNF-CY3 to confirm whether mRNA-BDNF-CY3 was released after the Parkinson's disease model cell line engulfed BDNF@DATEV and the effectiveness of the treatment.

[0125] Please refer to Figure 12A and 12B The above-mentioned groups of Parkinson's disease animal models began at week 0 (W0), and underwent the rotarod test and open field trajectory analysis test at weeks 2 (W2), 4 (W4), 6 (W6), 8 (W8), and 12 (W12) to observe the performance of each group's motor values, which included the duration of time spent on the rotarod and the total distance traveled.

[0126] Results from both the roller running test and the open field trajectory analysis experiment revealed that the motor values ​​of the Parkinson's disease animal models in the first control group, the second control group, and the experimental group were significantly lower than those in the healthy control group. However, it is noteworthy that the motor values ​​of the Parkinson's disease animal models in the experimental group gradually increased over time, especially at week 12. The duration of the roller running test in the experimental group increased by more than 60% compared to week 0, and the total distance traveled in the open field trajectory analysis experiment increased by more than 32% compared to week 0. This indicates that the BDNF@DATEV treatment in the experimental group not only alleviated the symptoms of Parkinson's disease (decreased motor ability) but also had a restorative effect on the behavioral performance of the Parkinson's disease animal models.

[0127] Figure 12C Immunofluorescence staining was used to compare the expression of neural stem cell-related proteins in brain slices from the dorsal striatum of the Parkinson's disease animal models in each group. F-actin was identified using red fluorescence to confirm the cytoskeleton; the cell nucleus was identified using blue fluorescence; and green fluorescence was used to identify alpha-synuclein (α-syn) accumulation. The figures clearly show that the experimental group treated with BDNF@DATEV showed a significant improvement in α-syn accumulation compared to the second control group (BDNF@EV). This result is consistent with the related experimental results from the first embodiment.

[0128] Experiment Six

[0129] Furthermore, to demonstrate that the DATEV vector can produce the same excellent effects on human cells, Figures 13A to 1 3 provides an experimental method for differentiating induced pluripotent stem cells (iPSCs) from Parkinson's disease patients into neural cells (PDs). Figure 13A In this study, Western blotting was used to compare the cells with normal cells (Health). The high expression of α-synin in differentiated neural cells (PD) confirmed that the neural cells differentiated from induced pluripotent stem cells (iPSCs) of Parkinson's disease patients were consistent with the protein physiological indicators of Parkinson's disease patients.

[0130] Next, the differentiated neurons were treated with Cur@DATEV, and the expression of synuclein (α-syn), Parkin, and DJ-1 proteins was observed on day 10. Figure 13B It was found that, consistent with the results of the previous experiments, the synuclein expression was reduced in the group treated with Cur@DATEV, while the expression of Parkin protein and DJ-1 protein showed an increasing trend.

[0131] Please refer to Figures 14A to 14B To demonstrate that DATEV can exhibit different functions depending on the cellular characteristics of the parent cell line, Parkinson's disease model cell line 50 was cultured in blood-brain barrier cell culture model 10 to verify the therapeutic efficacy of the third embodiment.

[0132] Experiment 7

[0133] Provided separately in the blood-brain barrier cell culture model 10:

[0134] The group that did not undergo any additive treatment served as the primary control group (PD);

[0135] A second control group (EV) was added, consisting of samples of the general EV vector collected from these 293 cells.

[0136] A group treated with a general EV vector collected via this mesenchymal stem cell was added as a third control group (MSCEV);

[0137] The group treated with DATEV vector collected via DAT-293 cells was added as the fourth control group (293-DATEV);

[0138] DATEV vector collected via DAT-MSC cells was added as the experimental group (MSC-DATEV). The endocytosis efficiency of the Parkinson's disease model cell line was confirmed after reacting with each of the above groups for 24 hours, and the therapeutic effect was confirmed after 48 hours of reaction when the Parkinson's disease model cell line phagocytosed DATEV collected via DAT-MSC cells.

[0139] Using immunofluorescence staining and extracellular vesicle protein labeling technology (Protein EV Labeling Kit (Red), ExoGlow) TMThe study, conducted by System Biosciences (Palo Alto, CA, USA), enabled the fluorescent labeling of these exosomal proteins of MSCEV and DATEV in a Parkinson's disease model cell line, and allowed for flow cytometry analysis to determine the efficiency of MSCEV or DATEV uptake in the Parkinson's disease model cell line. Figure 14B Results from the third control group (MSCEV) and the experimental group (MSC-DATEV) showed that the Parkinson's disease model cell line 50 could efficiently take up extracellular vesicles secreted by MSC cells. In particular, the proportion of intake of extracellular vesicles in the experimental group (MSC-DATEV) was as high as 95%.

[0140] Experiment 8

[0141] Further animal experiments confirmed that the DATEV vector obtained from DAT-MSC cells could directly exert physiological effects on this Parkinson's disease animal model. The Parkinson's disease animal model was treated intravenously every 7 days from week 0 (the day of surgery) to week 5, and was divided into the following groups:

[0142] The group that underwent sham surgery in the dorsal striatum brain region and did not receive any drug treatment was the healthy control group.

[0143] The animal model of Parkinson's disease that was not treated with any drugs served as the first control group (PD);

[0144] The group treated with conventional EV vectors collected via these mesenchymal stem cells served as the second control group (MSCEV);

[0145] DATEV vectors collected via DAT-MSC cells were administered as the experimental group (MSC-DATEV) to confirm the physiological mechanisms of the Parkinson's disease animal model after MSCEV treatment, as well as the protein expression of Parkinson's disease-related factors α-syn, neurogenesis-related protein IL-10, and inflammatory response-related protein NF-γ in the dorsal striatum brain region.

[0146] Please refer to Figure 15A and 15BThe above-mentioned groups of Parkinson's disease animal models began with open field trajectory analysis experiments at week 0 (W0), week 2 (W2), week 4 (W4), week 6 (W6), week 8 (W8), and week 12 (W12) to observe the performance of each group's motor values, which included the duration of time spent on the roller and the total distance traveled.

[0147] Results from both the roller running test and the open field trajectory analysis experiment revealed that the motor values ​​of the Parkinson's disease animal models in the first control group, the second control group, and the experimental group were significantly lower than those in the healthy control group. However, it is noteworthy that the motor values ​​of the Parkinson's disease animal models in the experimental group gradually increased over time, especially at week 12. The duration of the roller running test in the experimental group increased by more than 48% compared to week 0, and the total distance traveled in the open field trajectory analysis experiment increased by more than 17% compared to week 0. This indicates that the experimental group treated with MSCEV effectively alleviated the symptoms of Parkinson's disease (decreased motor ability) and even produced a restorative effect on the behavioral performance of the Parkinson's disease animal models.

[0148] Depend on Figure 15B The results showed that, over time, the expression of α-syn, a Parkinson's disease-related factor, decreased in the dorsal striatum brain region of the experimental group of the Parkinson's disease animal model, while the expression of IL-10, a protein related to nerve regeneration, and NF-γ, a protein related to inflammation, tended to increase. This confirmed that extracellular vesicles secreted by DAT-MSC cells have the effect of directly regulating physiological mechanisms.

[0149] The DATEV carrier provided by this invention can carry drugs across the blood-brain barrier and can specifically bind to dopamine nerve cells, thereby achieving excellent therapeutic effects in regulating the secretion of Parkinson's disease marker proteins and delaying the progression of Parkinson's disease.

[0150] Further loading curcumin into the DATEV vector can effectively reduce the accumulation of synuclein and the expression of inflammation-related proteins (INF-γ, IL-1β, IL-6, TNF-α), enhance the expression of Parkin protein, DJ-1 protein, TH protein, and neuroregeneration-related proteins (BDNF, NGF, VEGF, IL-10, CNTF), and maintain the activity of neural stem cells (nesin and Ki67 protein) in the dorsal striatum. The DATEV vector can also load BDNF mRNA, thereby delaying the symptoms of Parkinson's disease by downregulating BDNF expression and reducing the accumulation of synuclein. It can even achieve the effects of dopamine neuron regeneration and differentiation, showing great potential for the treatment of Parkinson's disease.

Claims

1. An anti-DAT antibody, characterized in that, The dopamine transporter is formed by transcription and translation of a gene fragment containing SEQ ID No:

2.

2. A composition, characterized in that, It contains a target gene SEQ ID No:3, and a fragment of gene SEQ ID No:2 is inserted into any part of the target gene SEQ ID No:

3.

3. A composition, characterized in that, It comprises an extracellular vesicle containing an antibody bound to a transmembrane protein, the extracellular vesicle being secreted by a cell transfected with a vector gene, and the vector gene at least partially containing SEQ ID No:

2.

4. The composition according to claim 3, characterized in that, The dopamine transporter antibody forms in the extracellular loop between the third and fourth transmembrane domains of this transmembrane protein.

5. A composition, characterized in that, It comprises an extracellular vesicle wherein a transmembrane protein is bound to the anti-DAT antibody as provided in claim 1.

6. A composition, characterized in that, It contains an extracellular vesicle in which an anti-DAT antibody is bound to a transmembrane protein. The extracellular vesicle is secreted by a cell transfected with a vector gene containing SEQ ID No:

4.

7. The composition according to any one of claims 3 to 6, characterized in that, The membrane-penetrating protein is CD63, and the cell type is HEK-293.

8. The composition according to any one of claims 3 to 6, characterized in that, The membrane-penetrating protein is CD63, and the cell is a mesenchymal stem cell.

9. The composition according to any one of claims 3 to 6, characterized in that, The extracellular vesicle is loaded with a drug, which may include gene fragments (DNA, RNA), protein sequences, or chemical agents.

10. The composition according to any one of claims 3 to 6, characterized in that, The extracellular vesicles are coated with messenger ribonucleic acid for expressing BDNF.

11. Use of the composition comprising any one of claims 2 to 9 for reducing the accumulation of synuclein protein in the striatum brain region of dopamine-affected neurons that are degenerating or diseased.

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