CN115960835A

CN115960835A - Coronavirus envelope protein E-induced extracellular vesicle and preparation method and application thereof

Info

Publication number: CN115960835A
Application number: CN202111178724.1A
Authority: CN
Inventors: 高召兵; 夏冰清; 李双渠; 李佳
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2023-04-14
Also published as: WO2023061338A1

Abstract

The invention provides an extracellular vesicle induced by coronavirus envelope protein E and a preparation method and application thereof. Specifically, the invention provides an extracellular vesicle, wherein the extracellular vesicle is induced by host cells heterologously expressing coronavirus envelope protein E, and the particle size of the extracellular vesicle is 2-10 microns. The extracellular vesicles of the present invention have the ability to load biological substances such as small molecules, proteins, nucleic acids, and viruses, and can be used as a carrier for delivering substances for biological research and diagnostic treatment.

Description

Coronavirus envelope protein E induced extracellular vesicle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an extracellular vesicle induced by coronavirus envelope protein E and a preparation method and application thereof.

Background

Extracellular Vesicles (EVs) are spherical proteoliposomes with nanometer to micrometer size and double molecular layers, and the current Extracellular vesicles are mainly divided into three types, namely exosomes, microvesicles and apoptotic bodies, and the particle sizes of the Extracellular vesicles are respectively about: 0.04-0.15 μm,0.05-1 μm,0.5-2 μm.

The outer membrane vesicles have good cargo loading capacity, and the existing three types of outer membrane vesicles can load water-soluble proteins, mRNA (messenger ribonucleic acid), microRNA (ribonucleic acid) and other substances, and are widely applied as potential carriers of micromolecular medicaments or biomacromolecules. In addition, extracellular vesicles can carry intracellular contents and secrete extracellular contents, and can be used as disease markers according to the characteristics of carried goods, and are used for diagnosis of various diseases.

However, although extracellular vesicles have been studied as carriers, the current extracellular vesicle-related technology has various disadvantages, such as limited carrying capacity, complicated preparation process, difficulty in quality control, and the like.

Therefore, there is an urgent need in the art to develop new vehicles having excellent vehicle performance.

Disclosure of Invention

The invention aims to provide a novel vehicle with excellent carrying performance, a manufacturing method and application thereof.

In a first aspect of the invention, there is provided an extracellular vesicle preparation, comprising extracellular vesicles induced by heterologous expression of coronavirus envelope protein E by a host cell, wherein the extracellular vesicles have a particle size of 2-10 μm.

In another preferred embodiment, the extracellular vesicle brewing agent comprises isolated or purified extracellular vesicles.

In another preferred embodiment, the extracellular vesicles are giant extracellular vesicles.

In another preferred embodiment, the particle size of the extracellular vesicles is 2-8 microns.

In another preferred embodiment, the average particle size (D50) of the extracellular vesicles is 4.0 to 6.0 microns, preferably 4.2 to 5.8 microns, more preferably 4.3 to 5.6 microns.

In another preferred embodiment, the proportion of the extracellular vesicles of 3-5 microns in the extracellular vesicles is 50-90%, preferably 55-85%, more preferably 60-80%, most preferably 69-76%.

In another preferred embodiment, the host cell is selected from the group consisting of: somatic cells of mammals.

In another preferred embodiment, the mammal includes human and non-human mammals.

In another preferred embodiment, the host cell is an immortalized cell.

In another preferred embodiment, the somatic cell is selected from the group consisting of: kidney cells, cervical cells, lung cells, or a combination thereof.

In another preferred embodiment, the coronavirus envelope protein E comprises wild-type coronavirus envelope protein E and mutant coronavirus envelope protein E.

In another preferred embodiment, the coronavirus envelope protein E is an envelope protein E from a coronavirus capable of infecting a mammal.

In another preferred embodiment, the coronavirus comprises an alpha coronavirus or a beta coronavirus.

In another preferred embodiment, the coronavirus is a β genus coronavirus, preferably selected from the group consisting of: HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2.

In another preferred embodiment, particularly preferred coronavirus envelope proteins E are from SARS-CoV-2, SARS-CoV and HCoV-OC43, including coronavirus envelope proteins E from different virus strains.

In another preferred embodiment, the extracellular vesicles contain, within the vesicles, contents selected from the group consisting of: a small molecule compound, a protein, a nucleic acid, a virus, or a combination thereof.

In another preferred embodiment, the virus is not a coronavirus.

In another preferred embodiment, the virus is selected from the group consisting of: lentivirus, adenovirus, or a combination thereof.

In another preferred embodiment, the nucleic acid comprises: DNA, RNA, or a combination thereof.

In another preferred embodiment, the RNA comprises: mRNA, microRNA, lncRNA, or a combination thereof.

In another preferred embodiment, the protein comprises: a fluorescent protein, an antibody, an enzyme, or a combination thereof.

In another preferred embodiment, said extracellular vesicles are induced by heterologous expression of a plasmid containing the coronavirus E gene by the host cell.

In another preferred embodiment, the extracellular vesicles are spherical or substantially spherical.

In another preferred embodiment, the novel coronavirus envelope protein E is encoded by a coronavirus E gene.

In another preferred embodiment, the coronavirus E protein comprises: the E protein of coronavirus from different genera and different species, and the E protein is genetically engineered to form a mutein with the function of forming giant extracellular vesicles.

In another preferred embodiment, the mutein comprises: through all possible modifications such as amino acid mutation modification, truncation modification, addition modification, or selection of gene middle fragment segments and the like.

In another preferred embodiment, the coronavirus E-containing gene is inserted into an expression vector such as pcDNA5 or pcDNA3.1.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) A giant extracellular vesicle capsule according to the first aspect of the invention, and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: aerosol inhalant, eye drop, nasal drop, and injection.

In a third aspect of the present invention, there is provided a method for preparing a giant extracellular vesicle, the method comprising the steps of:

(a) Providing a genetically engineered host cell comprising an expression cassette for the expression of exogenous coronavirus envelope protein E;

(b) Culturing the genetically engineered host cell in a suitable culture system to induce the production of giant extracellular vesicles, wherein the particle size of the giant extracellular vesicles is 2-10 microns.

In another preferred embodiment, the expression cassette for expressing the exogenous coronavirus envelope protein E is located on a plasmid or on a chromosome of the host cell.

In another preferred embodiment, the expression cassette comprises a promoter operably linked to the coding sequence of coronavirus envelope protein E.

In another preferred embodiment, the promoter comprises a constitutive promoter, or an inducible promoter.

In another preferred embodiment, the method further comprises:

(c) Isolating said giant extracellular vesicles.

In another preferred embodiment, the separation comprises: and collecting separated supernatant into a centrifuge tube, and performing differential centrifugation to separate the giant extracellular vesicles.

In another preferred embodiment, the separation comprises:

(ii) subjecting the cells to a first centrifugation step (e.g., about 100 g. + -.20 g) such that the cells pellet and the giant extracellular vesicles remain in the supernatant;

(ii) precipitating the cell debris by secondary centrifugation (e.g., about 200g ± 50 g), while the giant extracellular vesicles remain in the supernatant;

subjecting the giant extracellular vesicles to a three-stage centrifugation (e.g., about 400 g. + -.50 g);

separating the giant extracellular vesicle precipitate from the supernatant to obtain the giant extracellular vesicle precipitate.

In another preferred example, the method further comprises: washing and re-suspending the giant extracellular vesicle precipitate; and optionally further removing impurities (e.g., by filtration through a filter of a certain cut-off size, e.g., by filtration through 1 μm and 10 μm filters of the resuspension solution)

In another preferred embodiment, the separation comprises: the exfoliated cells were removed by centrifugation at 100Xg for 10 min. The supernatant was transferred to a new 15ml centrifuge tube and centrifuged at 200Xg for 10min to remove disrupted cell debris and impurities. The supernatant was transferred again to a new 15ml centrifuge tube, centrifuged at 400Xg for 20min to collect the giant extracellular vesicles, removed and resuspended after 1 PBS wash. Finally, the resuspension was filtered through 1 μm and 10 μm filters to remove impurities.

In another preferred example, the method includes:

(d) Vero E6 cells or other host cells are transfected to express E gene expression plasmids;

and (3) performing an optimized separation method on the transfected cell supernatant to obtain giant extracellular vesicles, wherein the separation method comprises the steps of performing magnetic bead enrichment and density gradient centrifugation separation by using the vesicle specific marker, and performing flow fluorescence sorting by adding a fluorescence label.

In a fourth aspect of the present invention, there is provided a use of the giant extracellular vesicle preparation of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention, for preparing a medicament or a preparation for treating respiratory inflammation.

In a fifth aspect of the invention, there is provided a use of the giant extracellular vesicles of the invention as vehicles for loading and/or delivering chemical or biological substances such as small molecules, proteins, nucleic acids, and viruses.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows a flow diagram of the isolated extracellular vesicles of the invention and their shape and particle size characterization. Which comprises the following steps:

FIG. 1A shows a scheme for isolating SARS-CoV-2-E (2-E) induced production of extracellular vesicles by Vero E6 cells.

FIG. 1B shows scanning electron microscope images of SARS-CoV-2-E-induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells by differential centrifugation.

FIG. 1C shows confocal microscopy images of SARS-CoV-2-E-induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells by differential centrifugation.

FIG. 1D shows particle size statistics and distribution plots of SARS-CoV-2-E induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells by differential centrifugation.

FIG. 1E shows a colocalization map of SARS-CoV-2-E induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells by differential centrifugation with Golgi membrane markers and correlation analysis.

FIG. 2 shows the shape and particle size characterization of extracellular vesicles prepared using mutant SARS-CoV-2-E or different coronavirus E proteins. Which comprises the following steps:

FIG. 2A shows confocal microscopy images of two plasmids of mutant SARS-CoV-2-E and plasmids of different coronavirus E proteins induced extracellular vesicles.

FIG. 2B shows a particle size histogram of extracellular vesicles induced by plasmids of two mutant SARS-CoV-2-E and plasmids of different coronavirus E proteins.

FIG. 2C shows the particle size distribution of extracellular vesicles induced by two mutant SARS-CoV-2-E plasmids and different coronavirus E protein plasmids.

FIG. 3 shows confocal microscopy images of SARS-CoV-2-E (2-E) induced extracellular vesicles prepared from Vero E6 cells loaded with small molecules and secreted extracellularly, with the image scale being 5 microns.

FIG. 4 shows confocal microscope test results. Which comprises the following steps:

FIG. 4A shows confocal microscopy images of SARS-CoV-2-E-EGFP (2-E-eGFP) induced extracellular vesicles prepared from Vero E6 cells to load intracellular mCherry protein and secrete extracellular, with a5 micron image scale.

FIG. 4B shows confocal microscopy images of SARS-CoV-2-E-mCherry (2-E-mCherry) prepared from Vero E6 cells inducing extracellular vesicles to load intracellular EGFP protein and secrete extracellular EGFP protein.

FIG. 5 shows the results of the nucleic acid gel, which shows a verification graph of the DNA loadable into SARS-CoV-2-E-induced extracellular vesicles prepared from Vero E6 cells. Lane 1: transfecting a no-load carrier by using Vero E6 cells; lane 2 and lane 3: vero E6 cells transfect SARS-CoV-2-E and mCherry plasmid together and then separate the extracellular vesicle; lane 4: vero E6 cells were transfected with mCherry.

FIG. 6 shows that the extracellular vesicles of the invention can be loaded with virus. Which comprises the following steps:

FIG. 6A shows confocal microscopy images of SARS-CoV-2-E-induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells loaded with lentivirus on a5 micron scale.

FIG. 6B shows the results of the nucleic acid gel, which shows the confirmation that SARS-CoV-2-E-induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells can be loaded with lentivirus.

FIG. 6C shows the results of Western immunoblotting showing the confirmation that SARS-CoV-2-E-induced extracellular vesicles (2-E-EVs) prepared from Vero E6 cells can be loaded with lentivirus.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly developed an oversized Extracellular Vesicle (EV). The giant extracellular vesicles are formed by inducing envelope protein E of coronavirus, and the envelope protein E of different types of coronavirus and the E protein obtained by genetic engineering can be induced to produce the giant extracellular vesicles. The extracellular vesicles of the invention are useful as vectors for efficient loading of a variety of different cargo, such as small molecules, proteins, nucleic acids, and viruses. The present invention has been completed on the basis of this finding.

Specifically, the invention proves through experiments that the envelope protein E expression of coronavirus can induce host cells to produce a large amount of spherical Extracellular Vesicles (EV) with the average particle size of about 5 microns. Extracellular vesicles prepared from the E protein expression system can be loaded with proteins, small compounds, DNA, and viruses and secreted extracellularly. The extracellular vesicles generated by the expression induction of the envelope protein E have high generation efficiency and simple preparation; in addition, the average particle size of the Extracellular Vesicles (EV) is far larger than that of other extracellular vesicles, the loading efficiency is high, the cargo loading is wide, and the EV can be used as a novel carrier for biological research and therapeutic diagnosis. The present invention provides a potentially efficient biological delivery system.

Term(s) for

As used herein, "formulation of the invention" refers to a formulation containing giant extracellular vesicles of the invention.

As used herein, the term "comprising" or variations thereof, such as "comprises" or "comprising," etc., are understood to imply the inclusion of stated elements or components but not the exclusion of any other elements or components.

As used herein, the term "novel coronavirus," or "SARS-CoV-2," are used interchangeably, the 2019 novel coronavirus being the 7 th coronavirus known to infect humans and causing new coronary pneumonia (COVID-19), one of the serious infectious diseases threatening global human health.

Coronavirus and envelope protein E

Coronaviruses (CoV) belong to the family of the nested virus order (Nidovirales) Coronaviridae (Coronaviridae), a positive-stranded enveloped RNA virus, a subfamily of which comprises the four genera α, β, δ and γ.

Among the coronaviruses currently known to infect humans, HCoV-229E and HCoV-NL63 belong to the genus alphacoronavirus, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are each the genus betacoronavirus. SARS-CoV-2 is also known as 2019-nCov.

The coronavirus envelope protein E is the smallest structural protein in coronavirus, is about 75-84 amino acids long, and consists of three parts, namely hydrophilic amino terminal (NTD), hydrophobic transmembrane domain (TMD) and long and hydrophilic Carboxyl Terminal (CTD), wherein the transmembrane structural region is the main ion transport pore region. The homology of E protein of different kinds of coronaviruses is 40%, wherein, the homology of SARS-CoV E and SARS-CoV-2E is as high as 95%, and there is a certain homology between HCoV-OC43 and HCoV-HKU1, HCoV-229E and HCoV-NL 63. Most of the coronavirus E protein enters the cells and can be positioned on endoplasmic reticulum, golgi apparatus and membrane structures between the endoplasmic reticulum and the Golgi apparatus of host cells, and oligomerization and assembly are carried out on a plasma membrane to form an ion channel so as to play a role.

Preferably, in the present invention, the coronavirus envelope protein E is an envelope protein E from a coronavirus that can infect a mammal, for example from an alphacoronavirus or a betacoronavirus. More preferably, from the genus beta coronavirus, such as HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2.

Particularly preferred coronavirus envelope proteins E are from SARS-CoV-2, SARS-CoV and HCoV-OC43, including coronavirus envelope proteins E from different virus strains.

In the present invention, the envelope protein E of coronavirus includes wild type and mutant type. Representative examples include, but are not limited to: coronavirus envelope protein E having G10A, N64H, or a combination thereof.

Representative mutant novel coronavirus envelope proteins E include (but are not limited to): SARS-CoV-2-E ^G10A (abbreviated as 2-E) ^G10A )、SARS-CoV-22-E ^N64H (abbreviated as 2-E) ^N64H )。

Giant extracellular vesicle

As used herein, the terms "extracellular vesicles of the invention", "EVs of the invention", "giant extracellular vesicles of the invention", "envosome" and "Envesome" are used interchangeably and refer to giant extracellular vesicles induced by heterologous expression of coronavirus envelope protein E by a host cell.

The giant Extracellular Vesicles (EV) of the invention have a size much larger than known extracellular vesicles. Typically, the extracellular vesicles of the invention have a particle size of 2-10 microns, preferably 2-8 microns.

Typically, the extracellular vesicles of the invention have an average particle size (D50) of 4.0 to 6.0 microns, preferably 4.2 to 5.8 microns, more preferably 4.3 to 5.6 microns.

In addition, the extracellular vesicles of the invention are characterized by uniform size. In the extracellular vesicles, the proportion of the 3-5 μm extracellular vesicles is very high (. Gtoreq.50%), for example 50-90%, preferably 55-85%, more preferably 60-80%, most preferably 69-76%.

In the present invention, the host cell that can be used to produce the giant extracellular vesicles is a somatic cell, preferably a mammalian somatic cell. The mammals include human and non-human mammals.

Preferably, the host cell is an immortalized cell. Representative such somatic cells include (but are not limited to): kidney cells, cervical cells, lung cells, or a combination thereof.

In the present invention, the envelope protein E of coronavirus which can be used includes wild-type and mutant envelope proteins E of coronavirus. Preferably, the coronavirus envelope protein E is an envelope protein E from a coronavirus capable of infecting a mammal.

In another preferred embodiment, the extracellular vesicles may contain additional desirable contents within their vesicles, such as pharmaceutical substances or fluorescent proteins or enzymes. Representative inclusions include (but are not limited to): small molecule compounds, proteins, nucleic acids, viruses, or combinations thereof.

Preparation method

The invention also provides a preparation method of the giant extracellular vesicles.

Typically, the preparation method of the present invention comprises:

(a) Transfecting host cells (such as Vero E6 cells or other host cells) to express E gene expression plasmids;

(b) Carrying out differential centrifugal separation on the transfected cell supernatant to obtain vesicles;

application of giant extracellular vesicles of the invention

The invention also provides the use of the giant extracellular vesicles as vehicles, particularly for delivery of various substances such as small molecules, proteins, nucleic acids, and viruses.

Preferably, the present invention provides a pharmaceutical composition comprising (a) a preparation of giant extracellular vesicles of the present invention, and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a cell-free and cell debris-free pharmaceutical composition.

Preferably, in the pharmaceutical composition, the extracellular vesicles in the giant extracellular vesicle preparation further comprise components for therapeutic or prophylactic or diagnostic purposes, such as small molecules, proteins, nucleic acids and viruses with therapeutic effects, immunogens with prophylactic effects, etc., or molecules for diagnostic purposes.

In another preferred embodiment, the pharmaceutical composition of the present invention is a vaccine composition.

The invention also provides a composition for non-medical use (e.g. scientific research composition) comprising (a) a preparation of giant extracellular vesicles according to the invention, and (b) a suitable carrier (e.g. water, buffer, etc.). Preferably, the giant extracellular vesicles of the invention contain a detectable label, e.g., a co-expressed fluorescent protein, such as green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc., or an exogenous fluorescent molecule, etc.

Typically, the novel extracellular vesicles of the present invention are applicable for loading different kinds of cargo and can be used for drug delivery, biological research, diagnostic therapy.

The main advantages of the invention include:

(1) The giant extracellular vesicles of the invention have oversized size, the particle size of the extracellular vesicles is intensively distributed between 2-10 microns, and the particle size is larger and uniform.

(2) The giant extracellular vesicle membrane component of the invention is mostly composed of an intracellular Golgi apparatus membrane, and the envelope protein E is expressed on the membrane surface of the extracellular vesicle.

(3) The preparation method of the giant extracellular vesicles is simple and convenient, and the giant extracellular vesicles can be efficiently prepared by adopting methods such as differential centrifugation.

(4) The giant extracellular vesicles of the invention can encapsulate and deliver various substances such as small molecules, proteins, nucleic acids, viruses, and the like.

(5) The giant extracellular vesicles of the invention can be used for targeted delivery of different substances.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Methods and materials

The experimental procedures used in the examples are conventional unless otherwise specified.

Materials, reagents and the like used in examples are commercially available unless otherwise specified.

Transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher; cellTracker Green CMFDA (5-Chloromethylfluoroscein Diacetate) viable cell tracer CMFDA,2 × Hieff PCR Master Mix (with Dye), agarose, yeaRed nucleic acid Dye purchased from san Ding Corp in Nei; the primer mCherry-F/R is ordered from Huada Gene; d-lysine (P6407) was purchased from Sigma; the SARS-COV-2 virus strain is from the Guangdong province disease prevention and control center (China); fetal bovine serum (total bovine serum) was purchased from Gibco, USA.

Centrifuge 5702 low speed centrifuges were purchased from Eppendorf corporation; veriti 96well Thermal Cycler purchased from Thermo Fisher; EPS 600 electrophoresis apparatus was purchased from Tanon corporation; laika ultra-high inverted laser confocal microscope is purchased from laika corporation.

Sequence of

SARS-CoV-2-E amino acid sequence (SEQ ID No: 1)

MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV

Example 1 preparation, isolation and identification of novel coronavirus envelope protein E (-E) -induced extracellular vesicles

In this example, after expressing the SARS-CoV-2-E plasmid by host cells, extracellular vesicles were separated from the supernatant by differential centrifugation and examined under a microscope. The method comprises the following steps:

step one, preparing and separating SARS-CoV-2-E induced extracellular vesicle

Vero E6 cells were purchased from ATCC, SARS-CoV-2-E gene sequence was synthesized by Huada Gene Co., ltd and constructed on pcDNA5 vector, SARS-CoV-2-E gene number YP _009724392.1.

The first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. After 24h, transfect SARS-CoV-2-E-mCherry plasmid 4000 ng/well, and replace fresh culture medium 8h after transfection. And culturing for 14-16h after liquid replacement, collecting cell supernatant, and performing extracellular vesicle separation. Centrifuging for the first time at 100g for 10min, discarding the precipitate after centrifugation, and collecting the supernatant; centrifuging again for 200g,10min, discarding the precipitate and collecting the supernatant; finally, 400g was centrifuged for 20min, and the pellet was collected by discarding the supernatant and collected after washing one time with PBS containing 2% ribonuclease A (RnaseA) (FIG. 1A).

Step two, characterization of post-vesicle shape and particle size of SARS-CoV-2-E-induced extracellular vesicles

The SARS-CoV-2-E-induced extracellular vesicles prepared by the method described in step one of example 1 were analyzed for shape and size.

The post-vesicle shape induced by SARS-CoV-2-E was observed using a Scanning Electron Microscope (SEM). The sample preparation method of the scanning electron microscope is as follows: fixing the separated extracellular vesicles on a slide coated with poly-D-lysine; placing the extracellular vesicles on the cover glass in 2.5% glutaraldehyde at 4 ℃ for fixing for 16 hours; after glutaraldehyde fixation, the coverslips were washed 3 times with 0.1% PBS for 10 minutes each, then fixed with 1% osmic acid at room temperature for 1h; washing the cover glass with ethanol with different concentrations for 10 minutes each time, wherein the ethanol concentration is 30%, 50%, 70%, 80%, 90%, 95% and 100%; then, critical point drying and gold spraying treatment are carried out for 15 minutes. Finally, observation was performed using a FEI Quanta 250 scanning electron microscope (cellular analysis platform of the central innovation of molecular cell science, china academy of sciences).

The results are shown in FIG. 1B, the extracellular vesicles are spherical, 2-8 microns in size, and smooth on the surface.

Dripping the separated extracellular vesicles into a glass bottom eight-link plate, observing by a Leica ultrahigh inverted laser confocal microscope, and counting the particle size.

As a result, as shown in FIGS. 1C-D, extracellular vesicles prepared from Vero E6 cells were spheronized and, after statistical analysis of particle size, had a size of 2 to 8 microns and an average particle size of 4.3 microns, with extracellular vesicles at 3-5 microns accounting for 72% of the post-isolation.

Step three, membrane characteristic characterization after separation of SARS-CoV-2-E induced extracellular vesicles

Vero E6 cells were purchased from ATCC, and SARS-CoV-2-E gene sequence fusion expressed red tag protein mCherry (SARS-CoV-2-E-mCherry), synthesized by Huada Gene Co, and constructed on pcDNA5 vector. The Golgi membrane specific marker plasmid is 1-82Aa of beta-1, 4-Galactosyltransferase 1 (B4 GALT 1), and fusion expresses YFP tag protein (Golgi-YFP).

The first day, 3X 10 ⁴ Vero E6 cells per well were plated on glass dishes for culture. After 24h, 3000 ng/well of SARS-CoV-2-E-mCherry plasmid and 1000 ng/well of Golgi marker Golgi-YFP plasmid were co-transfected, and fresh medium was replaced 8h after transfection. After the solution was changed, the cell supernatant was collected by culturing for 14 to 16 hours, and after extracellular vacuoles were separated, observation was performed using a come card ultra-high inverted laser confocal microscope (fig. 1A).

As a result, as shown in fig. 1E, the extracellular vesicles prepared from Vero E6 cells were spheroidized, the membrane surface exhibited red fluorescence and green fluorescence expression, the membrane components of visible extracellular vesicles were mostly composed of golgi membranes, and envelope protein E could be co-localized to the extracellular vesicle membranes with high correlation of co-localization, R2=0.89, p < -0.0001.

EXAMPLE 2 engineering of the novel coronavirus envelope protein E (SARS-CoV-2-E) Gene and the extracellular vesicle characteristics induced by other coronavirus envelope proteins

In this example, the SARS-CoV-2-E mutant plasmid (2-E) was expressed by host cells ^G10A Or 2-E ^N64H ) Or other coronavirus envelope protein E (SARS-CoV-E or HCoV-OC 43-E), and adopting fluorescence co-reactionThe extracellular vesicle generation morphology was observed with a focusing microscope, and extracellular vesicles were separated from the supernatant by differential centrifugation and subjected to particle size statistics. The method comprises the following steps:

step one, SARS-CoV-2-E mutant plasmid (2-E) ^G10A Or 2-E ^N64H ) And other coronavirus envelope protein E (SARS-CoV-E or HCoV-OC 43-E) plasmid.

The SARS-CoV-2-E mutant gene is synthesized by using specially designed primer and wild SARS-CoV-2-E nucleotide sequence as template, and is constructed on PcDNA3.1-mCherry carrier, and the SARS-CoV-2-E gene number is YP _009724392.1. The mutation sites are respectively 2-E ^G10A And 2-E ^N64H The primers are as follows:

2-E ^G10A -F:taagcaaagccttctctgtcgatgcaattatcaattatcgc(SEQ ID No:2),2-E ^G10A -R:attcgtttcggaagagacagctacgttaatagttaatagcg(SEQ ID No:3)；

2-E ^N64H -F:aaaaatgcaaatgagagcacaatttgtagacttaagaagatctcaagg(SEQ ID No:4),

2-E ^N64H -R:tttttacgtttactctcgtgttaaacatctgaattcttctagagttcc(SEQ ID No:5)。

the SARS-CoV-E gene sequence is synthesized by Huada Gene company and constructed on pcDNA3.1-mCherry vector, the SARS-CoV-E gene number is NC-004718.3; the HCoV-OC43-E gene sequence is synthesized by Huada Gene company and is constructed on a pcDNA3.1-mCherry vector, and the HCoV-OC43-E gene number is NC _006213.1.

Step two, extracellular vesicle preparation and morphological characterization

The first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. After 24h, the SARS-CoV-2-E mutant plasmid (2-E) was transfected separately ^G10A Or 2-E ^N64H ) Or other coronavirus envelope protein E (SARS-CoV-E or HCoV-OC 43-E) plasmid 4000 ng/well, and fresh medium was replaced 8h after transfection. After the medium was changed, the cells were cultured for 14 to 16 hours to collect cell supernatants, and extracellular vesicles were isolated by the method of example 1.

The generation form of extracellular vacuoles was observed using a Leica ultra-high inverted laser confocal microscope. As a result, as shown in fig. 2A, round and spherical extracellular vesicles were secreted on the surface of the maternal cells.

As a result, as shown in FIGS. 2B to C, the extracellular vesicles prepared from Vero E6 cells were spherical, and after statistical analysis of the particle size, 2-E was found ^G10A The resulting extracellular vesicles had an average particle size of 4.89 microns, with extracellular vesicles at 3-5 microns accounting for 69% after isolation; discovery 2-E ^N64H The resulting extracellular vesicles had an average particle size of 5.01 microns, with extracellular vesicles at 3-5 microns accounting for 73% after isolation; the average size of the extracellular vesicles produced by SARS-CoV-E was found to be 4.97 microns, with extracellular vesicles between 3 and 5 microns accounting for 76% of the total size after isolation; HCoV-OC43-E was found to produce extracellular vesicles with an average size of 5.54 microns, with extracellular vesicles at 3-5 microns accounting for 59% of the total size after separation.

EXAMPLE 3 novel coronavirus envelope protein E (SARS-CoV-2-E) -induced extracellular vesicles can be loaded with small molecules

In this example, viable cells were stained with fluorescent small molecule CMFDA, and host cells simultaneously expressed SARS-CoV-2-E, verifying the ability of the novel coronavirus envelope protein E (SARS-CoV-2-E) induced extracellular vesicles to be loaded with small molecules. The method comprises the following steps:

the first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. The next day, staining was performed using a fluorescent small molecule CMFDA (CMFDA is an ester fluorescent small molecule, and lipophilic groups in CMFDA can be hydrolyzed by intracytoplasmic nonspecific esterase to generate green fluorescent 5-chloromethyl fluorescein (molecular weight about 464.85), which has a charge and cannot freely penetrate cell membrane and can be well retained in the cell); diluting the CMFDA stock solution to a working solution concentration of 1. Mu.M using serum-free medium DMEM, discarding the supernatant after transfection, adding 1 ml of 1. Mu.M CMFDA dye, and 5% CO at 37% ₂ Incubating for 30min in a cell incubator; after staining, removing staining solution, adding fresh complete culture medium (DMEM +10% FBS), continuing to culture for 14-16h, and then transfecting the SARS-CoV-2-E-mCherry plasmid 4000 ng/hole; and (4) replacing the culture medium with fresh culture medium 8h after transfection, culturing for 14-16h, and observing by using a come card ultrahigh inverted laser confocal microscope.

Results

As shown in fig. 3, in the CMFDA staining group, the cells were green, and the cell surface was smooth and free of extracellular vesicles; in the CMFDA staining and SARS-CoV-2-E-mCherry group, the cells were red, a large number of extracellular vesicles were produced, the extracellular vesicles were red and green fluorescence was visible in the vesicles.

This demonstrates that CMFDA small molecules can be loaded by SARS-CoV-2-E induced extracellular vesicles. Based on these results, it was confirmed that SARS-CoV-2-E induced extracellular vesicles can be used to deliver small molecules.

Example 4 novel coronavirus envelope protein E (SARS-CoV-2-E) -induced extracellular vesicle loadable protein

In this example, the green fluorescent protein EGFP and the red fluorescent protein mCherry were used as tool proteins, and the two tool protein plasmids were transfected into host cells together with SARS-CoV-2-E plasmids, respectively, to express 3 foreign proteins (i.e., SARS-CoV-2-E, EGFP and mCherry) in the host cells, and the ability of extracellular vesicles to carry proteins was verified by microscopic examination. The method comprises the following steps:

the first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. After 24h, transfect SARS-CoV-2-E-mCherry plasmid 2000ng (SARS-CoV-2-E and mCherry are expressed in the form of fusion protein)/hole and EGFP (synthesized by Huada Gene company and constructed on pcDNA3.1 vector) plasmid 2000 ng/hole together, and change fresh culture medium after 8 h; or transfect SARS-CoV-2-E-EGFP plasmid 2000ng (SARS-CoV-2-E and EGFP express in the form of fusion protein)/hole and mCherry (synthesized by Huada Gene company and constructed on pcDNA3.1 vector) plasmid 2000 ng/hole together, transfect 8 hours, then change fresh culture medium, change liquid, culture for 14-16 hours, use Leica's card super-inverted laser confocal microscope to observe.

Results

As shown in FIG. 4A, in the SARS-CoV-2-E-EGFP/mCherry cotransfer group, a large number of extracellular vesicles were produced, and mCherry red fluorescence was observed in the green extracellular vesicles.

As shown in FIG. 4B, in the SARS-CoV-2-E-mCherry/EGFP cotransfer group, a large number of extracellular vesicles were produced, and EGFP green fluorescence was observed in the red extracellular vesicles.

This indicates that foreign proteins such as mCherry red protein and EGFP green protein, once expressed in cells, can be loaded into the extracellular vesicles induced by SARS-CoV-2-E. Based on these results, it was confirmed that SARS-CoV-2-E-induced extracellular vesicles can be used for protein delivery.

eGFP sequence (SEQ ID No: 6):

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa

mCherry sequence (SEQ ID No: 7):

atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaa

example 5 novel coronavirus envelope protein E (SARS-CoV-2-E) -induced extracellular vesicles loadable with DNA

In this example, mCherry plasmid was used as a tool DNA, and mCherry plasmid and SARS-CoV-2-E plasmid were co-transfected into host cells, and after extracellular vesicles were separated, DNA was extracted, and the ability of extracellular vesicles to carry DNA was verified by PCR. The method comprises the following steps:

step one, preparing an extracellular vesicle. The first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. After 24h, 2000 ng/well of SARS-CoV-2-E plasmid and 2000 ng/well of mCherry plasmid were co-transfected into Vero E6 cells, after 8h transfection, fresh medium was replaced, and after medium replacement, SARS-CoV-2-E induced extracellular vesicles were isolated by differential centrifugation as described in example 1 for 14-16 h.

And step two, extracting DNA. The collected extracellular vesicles and cell samples were taken and 37.5. Mu.l of each lysate (25mM NaOH,0.2mM EDTA, ddH, etc.) ₂ O dissolution), cracking for 25min at 95 ℃ in a metal bath; after lysis, 40. Mu.l of a neutralization solution (40 mM Tris-HCl, ddH) was added ₂ O lysis, PH = 5.5), and the supernatant was collected as a template for subsequent PCR.

And step three, PCR amplification. Two different pairs of mCherry-specific primers were used for PCR with the Hieff PCR Master Mix, as follows:

primer information is shown in the following table

PCR Using Veriti 96well Thermal Cycler according to the following Table PCR program

Step four, agarose electrophoresis detection. Prepare 2% agarose gel, load 10. Mu.l of PCR product per well, electrophorese for 20min at 170V voltage, and finally take pictures of bands.

Results

As shown in FIG. 5, no mcherry-specific band was seen in the transfection-only plasmid vector group. A specific band for mCherry was seen in the group of cells co-transfected with SARS-CoV-2-E and mCherry, as well as in isolated extracellular vesicles. In the positive control group, only the mcherry-transfected group showed a mcherry-specific band.

The above results indicate that SARS-CoV-2-E induced extracellular vesicles can be used for DNA delivery.

EXAMPLE 6 novel coronavirus envelope protein E (SARS-CoV-2-E) -induced extracellular vesicles loadable with the virus

In this example, a green fluorescent protein lentivirus was used as a tool virus, a host cell was infected with the virus, and then a SARS-CoV-2-E-mCherry plasmid was transfected, and the ability of the extracellular vesicle to carry the virus was verified by microscopic examination, western blotting, and nucleic acid gel. The method comprises the following steps:

the first day, 3X 10 ⁴ Vero E6 cells per well were plated in 6-well plates for culture. After 24h, the infectious titer was 6.72X 10 ⁸ TU/ml lentivirus (pLentipLentipLenti-CBh 3FLAG-luc 2-tCMV-mNeonGreen-F2A-Puro), changing a fresh culture medium after infecting for 24 hours, simultaneously transfecting a SARS-CoV-2-E-mCherry plasmid 4000 ng/hole, changing the fresh culture medium after transfecting for 8 hours, and culturing for 14-16 hours after changing the culture solution to observe by using a Leica super-inverted laser confocal microscope; in addition, SARS-CoV-2-E-induced extracellular vesicles were isolated by differential centrifugation as described in example 1. Collecting extracellular vesicles, and extractingTaking protein and nucleic acid in the vesicle.

As a result, the

As shown in fig. 6A, in the group infected with lentivirus only, cells exhibited green fluorescence of meneongreen, with cells intact; in the transfection SARS-CoV-2-E-mCherry plasmid group, the cells are red, a large number of extracellular vesicles are generated, and the vesicles are red; when the SARS-CoV-2-E-mCherry plasmid group is transfected at the same time of lentivirus infection, a large number of extracellular vesicles are generated, and green virus fluorescence can be seen in red vesicles.

As shown in fig. 6B, nucleic acids of the isolated extracellular vesicles were extracted and detected using lentivirus-specific primers, and it was seen that virus-specific bands were present in the vesicle group after virus infection.

As shown in FIG. 6C, when the expression of the protein in the isolated extracellular vesicles was detected using antibodies against mCherry and Flag, it was found that mCherry was expressed in the extracellular vesicles transfected with only the SARS-CoV-2-E-mCherry plasmid group, and that both mCherry expression and the virus-borne Flag expression were expressed in the extracellular vesicles transfected with the SARS-CoV-2-E-mCherry plasmid group at the same time as the infection with lentivirus.

The above results indicate that the confirmation of SARS-CoV-2-E induced extracellular vesicles can be used to deliver viruses.

Discussion of the related Art

Extracellular Vesicles (EVs) are bilayer globular proteoliposomes of nanometer to micrometer size, and the current Extracellular vesicles are mainly divided into three types, namely Exosomes (Exosomes), microvesicles (microviscles) and Apoptotic bodies (Apoptotic bodies), and have particle sizes respectively of about: 0.04-0.15 μm,0.05-1 μm,0.5-2 μm.

The outer membrane vesicles have good cargo loading capacity, and the three existing outer membrane vesicles (exosomes, microvesicles and apoptotic bodies) can be loaded with water-soluble proteins, mRNA (messenger ribonucleic acid), microRNA (ribonucleic acid) and other substances.

The surface of the extracellular vesicle carries a large number of protein and carbohydrate macromolecules, and the molecular characteristics of the surface determine the interaction between the extracellular vesicle and a target cell thereof, so that the extracellular vesicle becomes an important organism for cell-cell communication. The current multifunctional modified tumor vaccine vector utilizes the characteristic to increase the capability of taking specific antigens in a targeted manner by DC cells, thereby effectively promoting the processing and presentation of the antigens and enhancing the specific immune response induced by the antigens.

In addition, the outer surface of the outer-cell membrane vesicle is surrounded by a phospholipid bilayer, and the outer-cell membrane vesicle has the characteristics of good biocompatibility and the like, so that the outer-cell membrane vesicle can more easily penetrate various physiological barriers and can be used as a high-efficiency drug delivery system.

In contrast, the traditional drug carrier has large particle size and toxicity, is complex to synthesize, is easy to be phagocytized by macrophages and the like, and cannot effectively reach tumor parts; the surface of the traditional medicine carrier is easy to combine with serum protein with negative charge due to the existence of permanent positive charge, so that the traditional medicine carrier has poor targeting property and cannot circulate in vivo for a long time. When the extracellular vesicles are used as drug carriers, the extracellular vesicles have certain targeting property and greatly reduce side effects according to the surface characteristics of the extracellular vesicles, and donor cells can be selected according to recipient cells, so that efficient and durable delivery of goods is achieved.

The invention provides an ultra-large type extracellular membrane vesicle, the average particle size of which is about 5 microns and is obviously larger than the average particle size of the three types of the existing extracellular vesicles. The average size of the extracellular vesicles of the invention is about 4 times the size and about 64 times the volume of the largest apoptotic bodies. This suggests that there is a further advantage in terms of the loading of the individual extracellular vesicles of the invention.

The experimental result of the invention shows that the wild type and mutant SARS-CoV-2-E or similar coronavirus envelope protein E can efficiently induce to generate extracellular vesicles, and the extracellular vesicles have the capacity of loading biological substances such as small molecules, proteins, nucleic acids and viruses, and can be used as carriers for delivering substances and used for biological research and diagnosis and treatment.

In conclusion, the extracellular vesicles of the invention are different from the reported extracellular vesicles in morphology and preparation mode, and are novel extracellular vesicles, so the inventor names the extracellular vesicles as "Envesome", the naming is derived from taking a prefix of Envelope protein English Envelope and taking an English suffix of Exosome.

The novel extracellular vesicles have the advantages of convenience in preparation, high yield, high efficiency, diversified loading capacity, large loading capacity and the like, and have a great application prospect in the field of delivery of biological samples (or certain chemical substances).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai pharmaceutical research institute of Chinese academy of sciences

<120> coronavirus envelope protein E-induced extracellular vesicle and preparation method and application thereof

<130> P2021-2597

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<170> PatentIn version 3.5

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Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

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aaaaatgcaa atgagagcac aatttgtaga cttaagaaga tctcaagg 48

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ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

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Claims

1. An extracellular vesicle preparation, wherein said extracellular vesicle preparation comprises extracellular vesicles induced by heterologous expression of coronavirus envelope protein E by a host cell, and wherein said extracellular vesicles have a particle size of 2-10 μm.

2. The formulation of claim 1, wherein the proportion of the extracellular vesicles of 3-5 microns is 50-90%, preferably 55-85%, more preferably 60-80%, most preferably 69-76%.

3. The formulation of claim 1, wherein the host cell is selected from the group consisting of: somatic cells of mammals.

4. The formulation of claim 1, wherein the coronavirus envelope protein E comprises wild-type and mutant coronavirus envelope proteins E.

5. The formulation of claim 1, wherein the coronavirus envelope protein E is an envelope protein E from a coronavirus that infects a mammal;

preferably, the coronavirus comprises an alpha coronavirus or a beta coronavirus.

6. The formulation of claim 1, wherein the extracellular vesicles contain inclusions within the vesicles selected from the group consisting of: small molecule compounds, proteins, nucleic acids, viruses, or combinations thereof.

7. A pharmaceutical composition, comprising: (a) The giant extracellular vesicle formulation of claim 1, and (b) a pharmaceutically acceptable carrier.

8. A method for preparing giant extracellular vesicles, comprising the steps of:

9. The method of claim 8, wherein the method further comprises:

(c) Isolating said giant extracellular vesicles.

10. Use of the giant extracellular vesicle formulation of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 for the preparation of a medicament or formulation for the treatment of respiratory inflammation.