Engineered mesenchymal stem cell exosomes for delivery of proteins and methods of making and uses thereof

Abstract

This invention belongs to the field of biomedicine and nanomedicine delivery technology, specifically relating to an engineered mesenchymal stem cell exosome for protein delivery, its preparation method, and its uses. The exosomes are secreted by mesenchymal stem cells containing an exogenous expression cassette; the expression cassette includes a BDNF coding sequence (encoded by the amino acid sequence shown in SEQ ID NO:1), a T2A sequence (encoded by the amino acid sequence shown in SEQ ID NO:2), and an RVG-Lamp2b fusion protein coding sequence, all driven by the same promoter and sequentially linked; the exosomes include a BDNF protein enriched in the lumen of the exosomes and having an independent native conformation. This application successfully overcomes the blood-brain barrier limitation: through the RVG peptide-mediated receptor pathway, engineered exosomes efficiently cross the blood-brain barrier and enter brain tissue. This application is used for efficiently loading brain-derived neurotrophic factors and has the targeting ability to cross the blood-brain barrier, showing promising application prospects in the treatment of central nervous system diseases such as major depressive disorder.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine delivery technology, specifically relating to an engineered mesenchymal stem cell exosome for protein delivery, its preparation method, and its uses. Background Technology

[0002] Major Depressive Disorder (MDD) is a neuropsychiatric disorder characterized by persistent low mood and loss of interest, and is one of the leading causes of disability worldwide. Numerous studies have confirmed that the occurrence and development of MDD are closely related to abnormal levels of neurotrophic factors in the central nervous system. Among these, decreased expression of brain-derived neurotrophic factor (BDNF) is directly associated with impaired neural plasticity in the hippocampus and prefrontal cortex. Therefore, increasing BDNF levels in the brain is considered an important therapeutic approach to improve depressive symptoms and promote neurogenesis.

[0003] However, exogenous BDNF protein is blocked by the blood-brain barrier (BBB), preventing direct entry into brain tissue via peripheral administration. Its short half-life and unstable delivery in vivo further limit its clinical efficacy. Currently used antidepressants primarily act on the monoamine neurotransmitter system, resulting in delayed efficacy and low response and high relapse rates. Therefore, developing a treatment strategy that can cross the BBB and target BDNF delivery is a crucial direction for overcoming the treatment bottlenecks of MDD.

[0004] In recent years, exosomes, as small membranous vesicles with a diameter of 30–150 nm, have been regarded as potential drug delivery carriers due to their low immunogenicity, good biocompatibility, and transmembrane transport capabilities. Exosomes derived from mesenchymal stem cells (MSCs) have particularly good safety and production feasibility. However, natural exosomes lack clear brain-targeting capabilities and cannot effectively load and deliver large-molecule protein drugs with specific biological functions.

[0005] Therefore, there is an urgent need for a novel biological delivery system that can deliver BDNF effectively, cross the blood-brain barrier, precisely target neurons, and exert a stable therapeutic effect. Summary of the Invention

[0006] The purpose of this invention is to solve the existing technical problems and provide an engineered mesenchymal stem cell exosome for protein delivery, its preparation method and uses.

[0007] To achieve the above objectives, the present invention provides an engineered mesenchymal stem cell exosome for protein delivery, characterized in that the exosome is secreted by mesenchymal stem cells containing an exogenous expression cassette; The expression cassette includes a BDNF coding sequence (encoded by the amino acid sequence shown in SEQ ID NO: 1), a T2A sequence (encoded by the amino acid sequence shown in SEQ ID NO: 2), and an RVG-Lamp2b fusion protein coding sequence, all driven by the same promoter and sequentially linked. The exosomes include: BDNF protein with an independent native conformation enriched in the cavity of the exosome; and RVG-Lamp2b fusion protein displayed on the surface of the exosome membrane; The BDNF protein and the RVG-Lamp2b fusion protein are translated from the expression cassette, and cleavage occurs during translation via ribosome jumping mediated by the T2A sequence. This results in two completely independent proteins: the BDNF protein with its intact native conformation and the RVG-Lamp2b fusion protein. BDNF is then sorted and enriched in the exosome lumen, while RVG-Lamp2b is localized to the exosome membrane surface via the transmembrane domain of Lamp2b, thus exposing the RVG targeting peptide to the extramembrane.

[0008] The BDNF coding sequence encodes the human brain-derived neurotrophic factor precursor protein (pre-pro-BDNF (SEQ ID NO: 1)), whose amino acid sequence is shown in SEQ ID NO: 1, corresponding to UniProt accession number P23560 and GenBank accession number NM_170735.4. This precursor protein is 247 amino acids in length, including: a signal peptide (amino acids 1-18, 18 aa), which guides the protein into the endoplasmic reticulum / Golgi apparatus secretion pathway; a propeptide region (amino acids 19-128, 110 aa), which is cleaved and removed intracellularly by furin family proteases; and mature BDNF protein (amino acids 129-247, 119 aa), which performs its biological function as a non-covalently linked homodimer. The mature BDNF protein contains 6 conserved cysteine ​​residues, forming 3 disulfide bonds to maintain its native active conformation.

[0009] The T2A sequence encodes a self-cleaving peptide derived from Thosea asigna virus 2A, and its core amino acid sequence, as shown in SEQ ID NO: 2, is EGRGSLLTCGDVEENPGP (18 amino acids). In a preferred embodiment of the invention, a GSG (Gly-Ser-Gly) spacer sequence is added to the N-terminus of the T2A core sequence to improve cleavage efficiency, forming the complete functional sequence GSG-EGRGSLLTCGDVEENPGP (21 amino acids). The cleavage site of the T2A peptide is located between glycine (G) and proline (P) at the C-terminus, achieved through a ribosomal skipping mechanism—that is, when the ribosome translates to the conserved C-terminal motif DxExNPGP of T2A, it does not form a peptide bond between glycine and proline, thereby separating the upstream protein (BDNF, whose C-terminus retains the remaining amino acids of T2A to Gly) and the downstream protein (RVG-Lamp2b, whose N-terminus starts with Pro) into two independent polypeptides.

[0010] The expression cassette also includes a terminator located downstream of the coding sequence of the RVG-Lamp2b fusion protein. The promoter is a human elongation factor 1α promoter (EF-1α promoter), and the terminator is composed of a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and a bovine growth hormone polyadenylation signal (bGH polyA). The EF-1α promoter drives stable and persistent transgene expression in mesenchymal stem cells and is not easily silenced epigenetically; the WPRE element enhances mRNA stability and nuclear export efficiency, increasing transgene expression by 2–5 times; the bGH polyA signal provides efficient transcription termination and mRNA 3' polyadenylation processing.

[0011] The mesenchymal stem cells mentioned are human umbilical cord mesenchymal stem cells (hUC-MSCs).

[0012] The RVG-Lamp2b fusion protein comprises, from N-terminus to C-terminus, the following: a Lamp2b signal peptide, a GNSTM glycosylation protective motif (the amino acid sequence of which is shown in SEQ ID NO: 4), an RVG targeting peptide (the amino acid sequence of which is shown in SEQ ID NO: 3), and a mature Lamp2b protein (the full-length amino acid sequence of which includes the signal peptide is shown in SEQ ID NO: 5); wherein the RVG targeting peptide is a targeting peptide derived from rabies virus glycoprotein, capable of binding to the nicotinic acetylcholine receptor nAChR on the surface of neurons or cerebral vascular endothelial cells, thereby achieving receptor-mediated transport across the blood-brain barrier (BBB).

[0013] The RVG-Lamp2b fusion protein is Lamp2b, a subtype of human lysosome-associated membrane protein 2 (Lysosome-Associated Membrane Protein 2B), corresponding to UniProt accession number P13473-2 and GenBank accession number NM_013995.2, with a total length of approximately 410 amino acids. The Lamp2b protein includes a signal peptide region (approximately amino acids 1-28), a highly glycosylated extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. In the construction of this fusion protein, the RVG29 targeting peptide is fused to the N-terminus of the extracellular domain of Lamp2b via a linker. The transmembrane domain of Lamp2b anchors the entire fusion protein to the exosome phospholipid bilayer membrane, allowing the RVG29 peptide to be displayed externally.

[0014] The linker is designed as follows: Following the Lamp2b signal peptide, a GNSTM glycosylation motif (5 amino acids, as shown in SEQ ID NO: 4), a GGG spacer sequence (3 amino acids), an RVG29 targeting peptide (29 amino acids, as shown in SEQ ID NO: 3), and a GGGGSGGGGG flexible spacer sequence (10 amino acids) are sequentially linked, followed by the mature Lamp2b protein sequence with the signal peptide removed. The GNSTM glycosylation motif introduces an N-glycosylation site at the N-terminus, protecting the RVG peptide from degradation in the lysosomal environment and significantly improving the display efficiency and stability of RVG on the exosome membrane surface. The GGGGSGGGGG flexible spacer sequence provides spatial flexibility between the RVG29 peptide and the Lamp2b extracellular domain, ensuring that the RVG29 peptide is fully exposed and effectively binds to the nAChR receptor.

[0015] The RVG targeting peptide is a 29-amino acid brain-targeting peptide (RVG29) derived from amino acid regions 189-214 of the rabies virus glycoprotein, with its amino acid sequence shown in SEQ ID NO: 3: YTIWMPENPRPGTPCDIFTNSRGKRASNG. The RVG29 peptide mediates receptor-mediated transcytosis across the blood-brain barrier by specifically binding to nicotinic acetylcholine receptors (nAChRs, especially the α7 subtype) on the surface of neurons and cerebral vascular endothelial cells.

[0016] The BDNF content detected in the exosomes was 6-12 times that of unengineered mesenchymal stem cell exosomes.

[0017] The present invention also provides a method for preparing engineered exosomes as described above, comprising the following steps: S1. The expression cassette was transfected into mesenchymal stem cells using a lentiviral vector, and stable expression cell lines were obtained through screening. S2. Collect the culture supernatant of the stable expression cell line; S3. The engineered exosomes are obtained by separating and purifying them from the culture supernatant using ultracentrifugation, density gradient centrifugation, or size exclusion chromatography.

[0018] The present invention also provides the use of any of the engineered exosomes described herein in the preparation of drugs for treating central nervous system diseases.

[0019] The drug is administered via peripheral intravenous injection, and the engineered exosomes are able to cross the blood-brain barrier within 30-180 minutes after administration and accumulate in the hippocampus and prefrontal cortex.

[0020] The central nervous system diseases mentioned are major depressive disorder, bipolar disorder, post-traumatic stress disorder, Alzheimer's disease, Parkinson's disease, or brain injury.

[0021] The drug is used to improve the sucrose preference index in patients with major depressive disorder or animal models, and to reduce the time spent in forced swimming and tail suspension.

[0022] The beneficial effects of this invention are as follows: 1. This application successfully overcomes the blood-brain barrier limitation: through the receptor pathway mediated by RVG peptide, engineered exosomes can efficiently cross the blood-brain barrier and enter brain tissue.

[0023] 2. This application achieves high-efficiency loading and precise targeting: The original T2A self-cleavage and dual-localization design enables the therapeutic protein BDNF and the targeting element RVG to separate after translation and be located in the interior and on the surface of the exosome, respectively. The functions of the two do not interfere with each other, which not only ensures the natural activity and high loading capacity of BDNF (6-12 times higher than that of unengineered exosomes), but also endows the exosomes with powerful brain targeting capabilities.

[0024] 3. The systemic drug delivery system of this application is feasible, non-invasive and highly efficient: the delivery system can be administered via peripheral intravenous injection without the need for invasive intracranial injection or surgery. It can cross the blood-brain barrier and accumulate in depression-related brain regions such as the hippocampus and prefrontal cortex within 30-180 minutes after administration, which greatly improves the convenience of clinical application and patient compliance.

[0025] 4. The therapeutic effect of this application is significant: In a mouse model of chronic unpredictable stress-induced depression, intravenous injection of this engineered exosome significantly improved depressive-like behaviors, specifically: an increase of approximately 69% in the sucrose preference index, a reduction of approximately 47% in the immobility time during forced swimming, and a reduction of approximately 46% in the immobility time during tail suspension. Simultaneously, it restored brain BDNF protein levels to approximately 85% of normal and activated the downstream TrkB / CREB signaling pathway, promoting the recovery of neural plasticity.

[0026] 5. This application has the potential for platform-based applications: The design of this delivery system is highly scalable. By changing the therapeutic molecule coding sequence before the T2A sequence in the expression vector (such as other neurotrophic factors GDNF, NGF, or therapeutic nucleic acid siRNA / miRNA), it can be applied to the treatment of various central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and brain injury repair. Attached Figure Description

[0027] Figure 1 Schematic diagram of engineered MSC exosome structure; Figure 2 A schematic diagram of the T2A splitting mechanism and its dual-function localization; Figure 3 Schematic diagram of cross-BBB delivery and neuronal targeting. Detailed Implementation

[0028] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.

[0029] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0030] The core structure of this application is: the exosome has a phospholipid bilayer membrane, the lumen of which is enriched with BDNF protein (represented by dots), and the membrane surface displays RVG targeting peptides (represented by triangles) via the Lamp2b transmembrane protein. See [link to application]. Figure 1 This application elucidates the molecular mechanism of gene expression: BDNF-T2A-RVG-Lamp2b is translated as a transcript, and during translation, it undergoes T2A sequence-mediated cleavage to produce independent BDNF and RVG-Lamp2b fusion proteins, which then achieve regional localization into the vesicle lumen and membrane surface, respectively. See [link to relevant documentation]. Figure 2 This application describes its mechanism of action: After intravenous injection, the RVG peptide on the surface of exosomes binds to the nAChR receptor on the blood-brain barrier endothelial cells, mediating its crossing of the blood-brain barrier into the brain parenchyma. Subsequently, it targets neurons, releases BDNF, activates the TrkB-CREB signaling pathway, and ultimately improves neurological function and depressive behavior. See [link to relevant documentation]. Figure 3 .

[0031] To enable those skilled in the art to accurately implement this invention, the sequence information of each core element in the expression box is described in detail below.

[0032] The BDNF coding sequence used in this invention encodes human brain-derived neurotrophic factor precursor protein (pre-pro-BDNF), UniProt accession number P23560, GenBank reference sequence NM_170735.4, with a full length of 247 amino acids (approximately 741 bp coding region) and a molecular weight of approximately 27.8 kDa. Its amino acid sequence is as follows: MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDQKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMRVRRHSD PARRGELSVC DSISEWVTAA DKKTAVDMSG GTVTVLEKVP VSKGQLKQYF YETKCNPMGY TKEGCRGIDKR HWNSQCRTTQ SYVRALTMDS KKRIGWRFIR IDTSCVCTLT IKRGR Positions 1-18 are the signal peptide (guiding the protein into the ER / Golgi secretion pathway), positions 19-128 are the propeptide region (cleaved by furin family proteases within the cell), and positions 129-247 are the mature BDNF protein (119 amino acids, containing 6 conserved cysteine ​​residues forming 3 disulfide bonds, functioning as a non-covalent homodimer). During expression cassette translation, the signal peptide of pre-pro-BDNF guides it into the secretion pathway, and after the propeptide is cleaved, the mature BDNF protein is ultimately sorted and enriched in the exosome lumen.

[0033] The T2A self-cleaving peptide used in this invention is derived from the 2A peptide of the insect virus Thosea asigna, and its core amino acid sequence (18 amino acids) is as follows: Core T2A: EGRGSLLTCGDVEENPGP In a preferred embodiment of the present invention, a GSG (Gly-Ser-Gly) spacer sequence is additionally added to the N-terminus of the T2A core sequence to enhance cleavage efficiency, forming a complete functional sequence of 21 amino acids: GSG-T2A: GSGE GRGSSLLTCGDVEENPGP The molecular mechanism of T2A peptide involves ribosomal skipping: when the ribosome translates to the conserved motif DxExNPGP (DVEENPGP in T2A) at the C-terminus of T2A, it does not catalyze the formation of the peptide bond between the last glycine (G) and proline (P), causing the ribosome to continue translation but the two polypeptide segments to separate. After cleavage, the C-terminus of the upstream protein (BDNF) retains the remaining amino acids of T2A (up to G), while the N-terminus of the downstream protein (RVG-Lamp2b) begins with Pro. In this invention, the cleavage efficiency of T2A exceeded 80% as verified by Western blotting.

[0034] The RVG targeting peptide used in this invention is a 29-amino acid brain-targeting peptide (RVG29) derived from amino acid regions 189-214 of the rabies virus glycoprotein (RVG), and its amino acid sequence is as follows: RVG29:YTIWMPENPRPGTPCDIFTNSRGKRASNG RVG29 achieves its transport across the blood-brain barrier by specifically binding to nicotinic acetylcholine receptors (nAChR, especially the α7 subtype) on the surface of neurons and cerebral vascular endothelial cells, mediating receptor-mediated transcytosis. RVG29 can also bind to γ-aminobutyric acid (GABA) receptors, further enhancing its neural targeting ability.

[0035] To improve the display efficiency and stability of RVG29 peptide on the exosome membrane surface, a GNSTM glycosylation motif was introduced upstream of the RVG29 peptide (after the Lamp2b signal peptide), with the following amino acid sequence: GNSTM:GNSTM This motif introduces an N-glycosylation site at the Asn residue. The steric hindrance of the glycan chain protects the N-terminal RVG29 peptide of the fusion protein from degradation by proteases in the lysosomal / endosomal pathway, thereby significantly increasing the display of RVG29 on the final exosome membrane surface. This glycosylation protection strategy is based on the research of Hung and Leonard (J Biol Chem, 2015, 290(13):8166-8172).

[0036] The complete architecture of the RVG-Lamp2b fusion protein, from N-terminus to C-terminus, is as follows: Lamp2b signal peptide (approximately 28 aa) → GNSTM glycosylation motif (5 aa, SEQ ID NO: 4) → GGG spacer sequence (3 aa) → RVG29 targeting peptide (29 aa, SEQ ID NO: 3) → GGGGSGGGGG flexible spacer sequence (10 aa) → mature Lamp2b protein (sequence after removing the signal peptide) The GGG spacer sequence (3 glycine residues) provides spatial spacing between the GNSTM glycosylation motif and the RVG29 peptide, preventing steric interference from the glycan chain on RVG29 function. The GGGGSGGGGG flexible spacer sequence (10 amino acids, composed of glycine and serine) provides sufficient spatial flexibility between the RVG29 peptide and the Lamp2b extracellular domain, ensuring that the RVG29 peptide can be freely exposed on the exosome membrane surface and effectively bind to the nAChR receptor on the target cell surface. The transmembrane domain of Lamp2b (approximately one transmembrane α-helix) anchors the entire fusion protein to the exosome phospholipid bilayer membrane, while its short cytoplasmic tail (approximately 11 amino acids) is located inside the exosome.

[0037] The Lamp2b is a subtype B of human lysosome-associated membrane protein 2 (Lysosome-Associated Membrane Protein 2B), UniProt accession number P13473-2, GenBank accession number NM_013995.2, and is approximately 410 amino acids in length.

[0038] The complete structure (5'→3') of the expression cassette in the lentiviral transfer plasmid described in this invention is as follows: 5'-LTR — EF-1α promoter — Kozak sequence — ATG — pre-pro-BDNF coding sequence (coding DNA sequence corresponding to SEQ ID NO: 1, approximately 741 bp) — GSG-T2A coding sequence (coding DNA sequence corresponding to SEQ ID NO: 2, approximately 63 bp) — RVG-Lamp2b fusion protein coding sequence — WPRE — bGH polyA — 3'-SIN-LTR

[0039] In this embodiment of the invention, the human elongation factor 1-alpha promoter (EF-1α promoter) is used to drive the transcription of the expression cassette. The reasons for choosing the EF-1α promoter include: (a) the EF-1α promoter drives potent and stable transgene expression in mesenchymal stem cells, maintaining high levels of transcriptional activity even after long-term culture and passage; (b) compared to the cytomegalovirus immediate early promoter (CMV promoter), the EF-1α promoter is less susceptible to DNA methylation-mediated epigenetic silencing in MSCs; and (c) the EF-1α promoter is a human promoter, exhibiting good biocompatibility in human cells. In other embodiments of the invention, the CMV promoter (cytomegalovirus immediate early promoter) or the CAG promoter (a fusion of the CMV enhancer and the chicken β-actin promoter, containing β-globin introns) may be used instead of the EF-1α promoter.

[0040] In this embodiment of the invention, transcription termination and mRNA processing are tandemly composed of a WPRE element and a bGH polyA signal. The WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) is located downstream of the coding sequence and upstream of the polyA signal. Its function is to enhance the nuclear export efficiency and stability of mRNA, thereby increasing transgene expression by approximately 2 to 5 times. The bGH polyA (bovine growth hormone polyadenylation signal) provides a highly efficient transcription termination signal and mRNA 3' end polyadenylation processing. In other embodiments of the invention, the SV40 polyA signal (simian virus 40 polyadenylation signal) can be used instead of the bGH polyA signal.

[0041] The lentiviral transfer plasmid backbone was designed using a third-generation self-inactivating (SIN) lentiviral vector, with a partial deletion of the U3 region of the 3'-LTR. This prevents transcription from being initiated from the LTR after integration, thus improving biocompatibility. Lentiviral packaging utilized HEK-293T cells and a standard third-generation packaging system (including the packaging plasmid pMDLg / pRRE, the Rev expression plasmid pRSV-Rev, and the VSV-G envelope plasmid pMD2.G).

[0042] 1. Cell source: Human umbilical cord mesenchymal stem cells (hUC-MSCs) with good growth status and low passage number (P3-P5) were selected.

[0043] 2. Vector construction: A lentiviral transfer plasmid containing the expression cassette "EF-1α promoter — Kozak-ATG — pre-pro-BDNF coding sequence — GSG-T2A coding sequence — Lamp2b signal peptide — GNSTM-GGG-RVG29-GGGGSGGGGG-Lamp2b mature protein coding sequence — WPRE — bGH polyA" was designed and synthesized. The pre-pro-BDNF coding sequence was derived from the human BDNF gene (GenBank: NM_170735.4) and chemically synthesized after codon optimization. The T2A coding sequence was reverse-translated based on the amino acid sequence of SEQ ID NO: 2 and optimized for human cell codon preferences. In the RVG-Lamp2b fusion protein coding sequence, the RVG29 peptide sequence (SEQ ID NO: 3), the GNSTM glycosylation protection motif (SEQ ID NO: 4), and the flexible linker peptide were chemically synthesized, and the Lamp2b sequence was derived from the human LAMP2 gene subtype B (GenBank: NM_013995.2). All coding sequences were chemically synthesized and then cloned into the lentiviral transfer plasmid backbone via restriction endonuclease digestion and ligation or Gibson assembly.

[0044] 3. Virus Packaging and Infection: A third-generation self-inactivated lentivirus packaging system was used. HEK-293T cells (containing pMDLg / pRRE, pRSV-Rev, and pMD2.G packaging plasmids) were used for virus packaging. Viral supernatants were collected at 48 h and 72 h, filtered through a 0.45 μm filter membrane, and then combined. hUC-MSCs were infected with appropriate multiples of infection (MOI = 5-10), and 8 μg / mL polybrene was added to enhance infection efficiency.

[0045] 4. Stable strain screening: 48 hours after viral infection, the medium was replaced with complete medium containing 2 μg / mL puromycin for drug screening. This process was repeated for 14 days, with the drug-containing medium being replaced every 3 days to obtain stable expression cell lines (resistant cell pools or single clones).

[0046] 5. Expression Validation: The expression of BDNF and RVG-Lamp2b was detected at the mRNA level by quantitative real-time PCR (qRT-PCR); the expression of both was validated at the protein level by Western blotting—the BDNF protein band (mature BDNF approximately 14 kDa, dimer approximately 28 kDa) was detected using anti-BDNF antibody, and the RVG-Lamp2b fusion protein band (approximately 45-55 kDa) was detected using anti-Lamp2b antibody or anti-HA tag antibody. The cleavage efficiency of T2A was confirmed to be over 80% by observing the ratio of the uncleavage fusion protein band (BDNF-T2A-RVG-Lamp2b, approximately 70-80 kDa) to the cleavage product band.

[0047] 1. Exosome production: Stable expressed engineered MSCs were cultured in serum-free or serum-free medium for 48-72 hours, and the conditioned medium was collected.

[0048] 2. Differential centrifugation purification: Centrifuge at 300 g for 10 minutes to remove dead cells.

[0049] Centrifuge at 2,000 g for 20 minutes to remove cell debris.

[0050] Centrifuge at 10,000 g for 30 minutes to remove large vesicles and organelles.

[0051] Finally, centrifuge at 120,000 g for 70-90 minutes, and the precipitate is the crude exosome extract.

[0052] 3. Further purification: To obtain higher purity, crude exosomes can be further purified by sucrose density gradient centrifugation or size exclusion chromatography (SEC).

[0053] 4. Characterization and identification: Particle size and concentration: Nanoparticle tracking analyzer (NTA) was used to detect the exosomes, which showed that the particle size was mainly distributed between 110-130 nm.

[0054] Morphology: Transmission electron microscopy (TEM) reveals a typical saucer-like or double-membrane cup-like structure.

[0055] Biomarkers: Western blotting showed positive expression of the universal exosome biomarkers CD9, CD63, and TSG101.

[0056] Surface-targeting molecules: Immunogold-labeled electron microscopy can specifically show that RVG peptides are located on the surface of exosome membranes.

[0057] BDNF loading: ELISA analysis showed that the BDNF content in engineered exosomes was 6-12 times higher than that in exosomes derived from untransfected conventional MSCs.

[0058] 1. Establishment of an in vitro BBB model: An in vitro blood-brain barrier model was constructed by co-culturing mouse brain microvascular endothelial cell line bEnd.3 with astrocytes. The integrity of the barrier was verified by measuring the transendothelial resistance (TEER) value, which was greater than 200 Ω·cm².

[0059] 2. Crossing Experiment: Engineered exosomes and ordinary exosomes were labeled with the red fluorescent dye PKH26 and added to the top side of a BBB model. After 24 hours of incubation, the bottom side culture medium was collected and the fluorescence intensity was detected. The results showed that the bottom side fluorescence signal intensity of the Exo-RVG / BDNF group was 3.5 times that of the ordinary exosome group, demonstrating its strong barrier-crossing ability.

[0060] 3. Neuronal Function Verification: The basal product after crossing the BBB was added to the culture medium of human neuroblastoma SH-SY5Y cells. The results showed: The phosphorylation level of neuronal TrkB receptors increased 3.2-fold.

[0061] The phosphorylation level of the downstream transcription factor CREB increased by 2.8 times.

[0062] The neuron-specific marker MAP2 indicates an increase in neurite length of approximately 40%.

[0063] This indicates that BDNF delivered via engineered exosomes is fully bioactive and can effectively activate key neurotrophic signaling pathways within neurons.

[0064] 1. Modeling: A mouse depression model was established using the Chronic Unpredictable Mild Stress Method (CUMS) for 4-6 weeks, and successful modeling was confirmed by tests such as sucrose preference test.

[0065] 2. Drug administration: CUMS model mice were randomly divided into groups. The treatment group received an intravenous injection of Exo-RVG / BDNF at a dose of 100 μg / mouse twice a week for 4 weeks. A model control group and a normal control group were established.

[0066] 3. Behavioral evaluation: Sugar water preference experiment: The sugar water preference index of mice in the treatment group increased significantly from 45% in the model group to 76%, with an improvement rate of 69%.

[0067] Forced swimming experiment: The immobility time of mice in the treatment group was significantly reduced from 210 seconds in the model group to 112 seconds, a reduction of 47%.

[0068] Tail suspension test: The immobility time of mice in the treatment group was significantly reduced from 228 seconds in the model group to 124 seconds, a reduction of 46%.

[0069] 4. Histological and Molecular Biological Analysis: Brain tissue was collected for analysis after the behavioral tests. Fluorescence imaging showed that DiR-labeled exosomes were specifically enriched in the CA1 region of the hippocampus and the prefrontal cortex. ELISA analysis showed that the hippocampal BDNF protein level in the treatment group mice recovered to approximately 85% of that in normal mice.

[0070] sequence list

[0071] The sequences involved in this invention are summarized as follows: SEQ ID NO: 1 Human BDNF precursor protein (pre-pro-BDNF) protein 247 aa UniProt P23560 SEQ ID NO: 2 T2A self-cleaving peptide (containing GSG) protein 21 aa Thosea asigna virus SEQ ID NO: 3 RVG29 brain-targeting peptide protein 29 aa Rabies virus RVG 189-214 SEQ ID NO: 4 GNS™ Glycosylation Protective Mechanism protein 5 aa Engineering design SEQ ID NO: 5 Human Lamp2b protein protein ~410 aa UniProt P13473-2 The above is a detailed description of the embodiments, which is intended to enable those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to technical solutions obtained by those skilled in the art based on the present invention and on the existing technology, without innovative effort but only through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.

Claims

1. An engineered mesenchymal stem cell exosome for protein delivery, characterized in that, The exosomes are secreted by mesenchymal stem cells containing exogenous expression cassettes; The expression cassette includes a BDNF coding sequence (the amino acid sequence of which is shown in SEQ ID NO: 1), a T2A sequence (the amino acid sequence of which is shown in SEQ ID NO: 2), and an RVG-Lamp2b fusion protein coding sequence, which are driven by the same promoter and sequentially linked. The exosomes include: BDNF protein with an independent native conformation enriched in the cavity of the exosome; and RVG-Lamp2b fusion protein displayed on the surface of the exosome membrane; The BDNF protein and the RVG-Lamp2b fusion protein are translated from the expression cassette and cleaved during translation via ribosome jumping mediated by the T2A sequence.

2. The engineered mesenchymal stem cell exosome for protein delivery according to claim 1, characterized in that, The expression cassette also includes a terminator located downstream of the coding sequence of the RVG-Lamp2b fusion protein.

3. The engineered mesenchymal stem cell exosome for protein delivery according to claim 1, characterized in that, The mesenchymal stem cells mentioned are human umbilical cord mesenchymal stem cells (hUC-MSCs).

4. The engineered mesenchymal stem cell exosome for protein delivery according to claim 1, characterized in that, The RVG-Lamp2b fusion protein comprises, from N-terminus to C-terminus, the following: a Lamp2b signal peptide, a GNSTM glycosylation protective motif (the amino acid sequence of which is shown in SEQ ID NO: 4), an RVG targeting peptide (the amino acid sequence of which is shown in SEQ ID NO: 3), and a mature Lamp2b protein (the full-length amino acid sequence of which includes the signal peptide is shown in SEQ ID NO: 5); wherein the RVG targeting peptide is a targeting peptide derived from rabies virus glycoprotein, capable of binding to the nicotinic acetylcholine receptor nAChR on the surface of neurons or cerebral vascular endothelial cells, thereby achieving receptor-mediated transport across the blood-brain barrier (BBB).

5. The engineered mesenchymal stem cell exosome for protein delivery according to claim 1, characterized in that, The BDNF content detected in the exosomes was 6-12 times that of unengineered mesenchymal stem cell exosomes.

6. A method for preparing engineered exosomes as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. The expression cassette was transfected into mesenchymal stem cells using a lentiviral vector, and stable expression cell lines were obtained through screening. S2. Collect the culture supernatant of the stable expression cell line; S3. The engineered exosomes are obtained by separating and purifying them from the culture supernatant using ultracentrifugation, density gradient centrifugation, or size exclusion chromatography.

7. Use of the engineered exosomes according to any one of claims 1-5 in the preparation of medicaments for treating central nervous system diseases.

8. The use according to claim 7, characterized in that, The drug is administered via peripheral intravenous injection, and the engineered exosomes are able to cross the blood-brain barrier within 30-180 minutes after administration and accumulate in the hippocampus and prefrontal cortex.

9. The use according to claim 7 or 8, characterized in that, The central nervous system diseases mentioned are major depressive disorder, bipolar disorder, post-traumatic stress disorder, Alzheimer's disease, Parkinson's disease, or brain injury.

10. The use according to claim 9, characterized in that, The drug is used to improve the sucrose preference index in patients with major depressive disorder or animal models, and to reduce the time spent in forced swimming and tail suspension.

Images