CN114934070B

CN114934070B - Mesenchymal stem cells and anti-inflammatory application thereof

Info

Publication number: CN114934070B
Application number: CN202210058414.4A
Authority: CN
Inventors: 秦大江; 徐洪杰; 岑晓彤; 吴迪
Original assignee: Bioisland Laboratory
Current assignee: Bioisland Laboratory
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2023-05-02
Anticipated expiration: 2042-01-18
Also published as: CN114934070A

Abstract

The present invention provides methods of treating inflammatory diseases using recombinant mesenchymal stem cells comprising lentiviral vectors over-expressing a plurality of cytokines. The invention also relates to a host cell containing the lentiviral vector, and application of the lentiviral vector and the host cell containing the lentiviral vector in preparing a medicament for treating inflammatory diseases.

Description

Mesenchymal stem cells and anti-inflammatory application thereof

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a viral expression vector and application thereof in cell therapy.

Background

Clinically many pathological conditions, such as diabetes, and also conditions like aging or sub-health, cause inflammation in the human body. The mesenchymal stem cells have wide sources, low immunogenicity and no obvious adverse reaction due to the excellent anti-inflammatory and repair promoting properties, are widely applied to the treatment of various inflammations, and achieve encouraging results. However, the mesenchymal stem cell types currently studied are almost entirely unmodified cells. Improving the specific performance of mesenchymal stem cells aiming at different pathological states is a key research direction of future stem cell patent medicine.

Lentiviral vectors are a class of exogenous gene-carrying viral vectors developed by engineering elements of lentivirus itself, such as LTR, gag, pol, env, etc., based on lentiviral genome. Depending on its origin, there are mainly the following types: HIV-type 1 (human immunodeficiency virus type 1) vector systems, HIV-type 2 (human immunodeficiency virus type) vector systems, simian immunodeficiency virus (simian immunodeficiency virus, SIV), feline immunodeficiency virus (felines immunodeficiency virus, FIV), and the like, with HIV-type 1 vector systems being most widely and deeply studied.

Interleukins are a very important family of cytokines whose functions involve many processes such as maturation, activation, proliferation, regulation of immune cells, and are involved in a variety of physiological and pathological responses in the body. Among the interleukin families, there are mainly 1L-1, 1L-6 and IL-8 pro-inflammatory cytokines, and anti-inflammatory factors mainly include IL-4, IL-10 and IL-13.

For inflammatory diseases, there is a need for cell therapy with significantly improved anti-inflammatory effects.

Disclosure of Invention

The present invention provides the use of recombinant stem cells for the treatment of inflammatory diseases. The recombinant stem cells, particularly recombinant Mesenchymal Stem Cells (MSCs), are capable of expressing IL-10, IL-13 and IL-4 simultaneously.

In a first aspect, the invention provides the use of an expression vector carrying coding sequences for the expression of IL-10, IL-13 and IL-4, a host cell comprising said expression vector, or a cell pharmaceutical composition comprising said expression vector and a pharmaceutically acceptable excipient or carrier, in the manufacture of a medicament for the treatment of an inflammatory disease.

In some embodiments, the expression vector expresses IL-10, IL-13 and IL-4 simultaneously.

In some embodiments, the expression vector is a lentiviral vector.

Preferably, the lentiviral vector comprises a vector plasmid comprising a 5'LTR comprising a psisequence, a 3' LTR, a gene sequence of interest between the 5'LTR and the 3' LTR, and a promoter sequence and a translation initiation sequence operably linked to the gene sequence of interest, the gene sequences of interest being the coding nucleotide sequences of IL-10, IL-13 and IL-4.

In some embodiments, the lentiviral vector comprises the following 3 plasmids: (1) A vector plasmid containing at least 5'LTR and 3' LTR, a ψ sequence and a target gene; (2) Packaging plasmids containing gag and pol necessary for packaging and optionally regulatory genes rev and tat; and (3) envelope plasmids containing env genes.

In some embodiments, the lentiviral vector comprises the following 4 plasmids: (1) A vector plasmid containing at least 5'LTR and 3' LTR, a ψ sequence and a target gene; (B) a packaging plasmid containing gag and pol necessary for packaging; (C) envelope plasmid containing env gene; and (D) rev expression plasmid containing rev.

In some embodiments, the lentiviral vector comprises 3 plasmids: (1) A vector plasmid containing at least 5'LTR and 3' LTR, a ψ sequence and a target gene; (B) a packaging plasmid containing gag and pol necessary for packaging; and (C) a plasmid containing env expression units and rev expression units.

In some embodiments, the 3'ltr and 5' ltr of the vector plasmid comprise one or more modifications.

In some embodiments, U3 of the 5'ltr and 3' ltr may be deleted or mutated.

Preferably, the 3' LTR is a 3' LTR (ΔU3/3' LTR) lacking the U3 region.

In some embodiments, the 5' ltr is a deleted form of the 5' ltr (Δ5' ltr).

In some embodiments, the promoter of the 5' ltr of the vector plasmid is replaced with a heterologous promoter selected from the group consisting of: a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a simian virus 40 (SV 40) promoter.

In some embodiments, the promoter operably linked to the coding nucleotides for IL-10, IL-13 and IL-4 is selected from the group consisting of: short elongation factor 1 alpha (EF 1 alpha) promoters or transcriptionally active fragments thereof, RSV promoters and simian virus 40 (SV 40) promoters.

In some embodiments, the RSV promoter, EF 1. Alpha. Promoter and Kozak translation initiation sequence are operably linked to the coding nucleotides for IL-10, IL-13 and IL-4.

In some embodiments, the vector plasmid further comprises a selectable marker.

In some embodiments, the selectable marker is a luciferase, an enhanced green fluorescent protein, a streptavidin binding peptide, a puromycin resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, and/or a neomycin resistance gene.

In some embodiments, the selectable marker is an Enhanced Green Fluorescent Protein (EGFP)/puromycin resistance gene (Puro) dual reporter marker.

In some embodiments, the vector plasmid further comprises SV40 early pA.

In some embodiments, the post-transcriptional regulatory element of the vector plasmid comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In some embodiments, the vector plasmid comprises a retroviral export element. "retroviral export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm. Preferably, the retroviral export elements include, but are not limited to, the Human Immunodeficiency Virus (HIV) Rev Responsive Element (RRE) and the hepatitis b virus posttranscriptional regulatory element (HPRE).

In some embodiments, the vector plasmid may comprise a central polypurine region (cPPT) or a Central Termination Sequence (CTS) as cis-acting elements, the cPPT/CTS sequence may be the cPPT/CTS of HIV1, capable of increasing vector integration and transduction efficiency.

In some embodiments, the vector plasmid comprises, in order from the 5'ltr region to the 3' ltr region: RSV promoter, 5'LTR, ψ sequence, RRE, cPPT, EF 1. Alpha. Promoter, kozak translation initiation sequence, the coding nucleotides for IL-10, IL-13 and IL-4 shown by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, SV40, kozak translation initiation sequence, EGFR/Puro, WRPE, ΔU3/3' LTR and SV40 early pA.

The sequence of the coding nucleotides for IL-10, IL-13 and IL-4 may be changed as long as the coding nucleotides for IL-10, IL-13 and IL-4 are connected in series.

In some embodiments, the IL-10, IL-13 and IL-4 encoding nucleotides, IL-10, IL-13 and IL-4 are separated from each other by a nucleotide sequence encoding a 2A peptide.

In some embodiments, the 2A peptide is a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide. Preferably, the 2A peptide is a T2A or P2A peptide.

The 2A peptide can avoid producing fusion proteins of IL-10, IL-13 and IL-4.

In some embodiments, the EGFR and Puro encoding nucleotides are separated by a nucleotide sequence encoding a 2A peptide between the EGFR and puromycin resistance genes.

The lentiviral vector carrying IL-10, IL-13 and IL-4 coding nucleotides of the invention can simultaneously overexpress three anti-inflammatory factors, IL-10, IL-13 and IL-4, after transfection of host cells.

The slow virus vector carrying IL-10, IL-13 and IL-4 coding nucleotide does not need to prepare three vector plasmids, only one vector plasmid is needed to carry the coding nucleotide for coding IL-10, IL-13 and IL-4 at the same time, the operation cost is simplified, the damage of a plurality of transfected virus vectors to cells is reduced, and the slow virus vector is favorable for the quality control of the cell pharmaceutical preparation.

In some embodiments, the host cell is capable of expressing a nucleotide of interest carried in the lentiviral vector.

In some embodiments, the host cell is a stem cell.

In some embodiments, the stem cells are mesenchymal stem cells. Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 encoding nucleotides may overexpress three anti-inflammatory factors, IL-10, IL-13 and IL-4 simultaneously. The mesenchymal stem cells are energized mesenchymal stem cells with enhanced anti-inflammatory function.

The mesenchymal stem cells can be used as a cell model of a cell pharmaceutical preparation.

The mesenchymal stem cells can be used for treating inflammatory diseases.

The mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 coding nucleotide and over-expressing the three anti-inflammatory factors of IL-10, IL-13 and IL-4 have over-expressed the three anti-inflammatory factors before transplantation in cell therapy, so that immune response after transplantation is not needed, the anti-inflammatory response and effectiveness of the mesenchymal stem cells after transplantation are greatly improved, and the effects of greatly shortening the effective time and increasing the curative effect in the treatment of inflammation are achieved.

The invention has promoter, regulatory sequence, translation initiation sequence and screening marker, and can obtain slow virus vector carrying IL-10, IL-13 and IL-4 coding nucleotide; the simultaneous over-expression of three anti-inflammatory factors of IL-10, IL-13 and IL-4 by using one lentiviral vector is realized; the lentiviral vector constructed by the invention also realizes high-infection-efficiency repeatability and provides a powerful candidate tool for cell treatment of inflammatory diseases. In particular, the combination of the RSV promoter, EF1 alpha promoter sequence and Kozak translation initiation sequence in the vector plasmid before the IL-10, IL-13 and IL-4 coding nucleotides ensures high expression of the vector plasmid in stem cells; the three anti-inflammatory factors are divided by a 2 peptide sequence to prevent the generation of fusion proteins; meanwhile, the vector plasmid carries enhanced GFP and antibiotic Puro sequences, thereby facilitating the observation of transfection efficiency and drug screening when necessary.

The invention is transfected with slow virus vector carrying IL-10, IL-13 and IL-4 coding nucleotide, and the mesenchymal stem cell over expressing three anti-inflammatory factors of IL-10, IL-13 and IL-4 can be used for treating inflammatory diseases. The mesenchymal stem cells over-expressing three anti-inflammatory factors of IL-10, IL-13 and IL-4 have obviously enhanced anti-inflammatory effect compared with common mesenchymal stem cells which are not subjected to gene editing. In particular, it shows an anti-inflammatory effect in an in vivo mouse model, in particular a significant improvement in the anti-inflammatory effect for acute inflammation.

Detailed Description

In some embodiments, the invention constructs a lentiviral vector over-expressing three anti-inflammatory factors IL-10, IL-13 and IL-4, designated pLV [ Exp ] -EF1 alpha > hIL10[ NM-000572.3 ] (ns): T2A: hIL13[ NM-002188.3 ] (ns): P2A: hIL4[ NM-000589.4 ] -SV40> EGFP: T2A: puro, the constructed vector being provided in FIG. 1. The RSVpromoter, EF alpha promoter sequence and the Kozak translation initiation sequence are combined in the vector at the upstream of three anti-inflammatory factor genes, so that the high expression of virus plasmids in stem cells is ensured, and the three anti-inflammatory factors are divided by a 2A peptide sequence, so that fusion proteins are prevented from being generated. Meanwhile, the vector carries enhanced GFP and antibiotic Puro sequences, so that the observation of transfection efficiency and drug screening when necessary are facilitated.

In the preparation of mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 encoding nucleotides according to the present invention, preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the cell seeding density in the culturing step is 0.5X10 ⁵ ～2.0×10 ⁵ Preferably 1.2X10 ⁵ The method comprises the steps of carrying out a first treatment on the surface of the And/or in the transfection step, the viral transfection complex MOI is 10 to 30, preferably 15 to 25, more preferably 17 to 23, e.g. 17, 18, 19, 20, 21, 22 or 23. Although the higher the multiplicity of viral transfection (MOI), the higher the transfection efficiency, the corresponding toxicity increases with increasing MOI, thus balancing the transfection efficiency with toxicity, the present application found that a higher transfection efficiency of greater than 80% can be achieved with MOI selection of greater than 15 when the lentiviral vector of the present invention is used to transfect mesenchymal cells, and a transfection efficiency of 90% can be achieved with MOI of 20.

The protein expression levels of IL-13, IL-10 and IL-4 in the human umbilical cord mesenchymal stem cells obtained by the preparation method of the mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 coding nucleotides according to the present invention can be respectively increased by 100 times, 50000 times and more than 40000 times relative to the baseline level.

The mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 encoding nucleotides according to the invention can be used for the preparation of a cell pharmaceutical composition.

The cell pharmaceutical composition can be used for preventing, treating or alleviating inflammatory diseases.

The cytopharmaceutical compositions are useful for preventing, treating or alleviating diseases associated with IL-10, IL-13 and IL-4.

The lentiviral vector of the invention carries three genes, does not need three vector plasmids, simplifies the operation and the cost, reduces the damage of a plurality of virus transfection to cells, and is beneficial to the quality control of subsequent pharmaceutical preparations.

In addition, the human umbilical cord mesenchymal stem cells are transfected by the lentiviral vector which over-expresses the anti-inflammatory factors, so that the cells over-express the three anti-inflammatory factors IL-10, IL-13 and IL-4, and the cells are expressed in a large amount before the transplantation, so that immune response is not needed after the transplantation, the anti-inflammatory response and effectiveness of the human umbilical cord stem cells after the transplantation are greatly improved, and the method has the characteristics of shortening the effective time and increasing the curative effect on the treatment of inflammation related diseases.

In addition, compared with the BMSCs of the mesenchymal stem cells of which the anti-inflammatory factors TGF beta are over-expressed in the prior art, the invention has the synergistic effect of promoting the transformation of macrophages to an M2 (anti-inflammatory) phenotype, and the polarization effect is more obvious.

The mesenchymal stem cells transfected with the lentiviral vector carrying IL-10, IL-13 and IL-4 coding nucleotides simultaneously and over-expressing three anti-inflammatory factors of IL-10, IL-13 and IL-4 simultaneously can be used for treating inflammatory diseases. The mesenchymal stem cells over-expressing three anti-inflammatory factors of IL-10, IL-13 and IL-4 have obviously enhanced anti-inflammatory effect compared with common mesenchymal stem cells which are not subjected to gene editing. In particular, it shows an anti-inflammatory effect in an in vivo mouse model, in particular a significant improvement in the anti-inflammatory effect for acute inflammation.

Noun interpretation:

lentiviral vector systems include packaging plasmids, envelope plasmids and vector plasmids, and may be two plasmid systems, three plasmid systems or four plasmid systems. The three plasmid system separates the cis-acting sequence structure and the sequence encoding the trans-acting protein required for packaging, reverse transcription and integration in the lentiviral genome, and clones into three independent plasmids, with all helper sequences removed. The four plasmid system was modified on the basis of a three plasmid system, the first variation being the placement of the rev gene on a separate expression plasmid, the addition of a plasmid further increasing the safety of the system, the second variation being the removal of the tat gene and the addition of chimeric 5' LTR fused to a heterologous promoter on the vector plasmid to initiate expression of the vector plasmid. In addition, the three-plasmid system and the four-plasmid system have a vector in which a desired gene sequence can be placed, which is called a vector plasmid.

IL-4 is produced mainly by activated T cells, and can inhibit synthesis and secretion of inflammatory cytokines such as IL-1, IL-6, TNF-alpha and the like by endothelial cells and monocytes, thereby inhibiting synthesis of proinflammatory cytokines and inhibiting inflammatory response. IL-10 is secreted by Th2 cells, activated monocytes and epithelial cells, and has well-defined immunosuppressive activity. IL-13 is a novel cytokine discovered in 1993 to be produced by activated T cells, which has homology with the receptor alpha chain of IL-4, and also has very similar signal transduction pathways and biological activities, and has the function of inhibiting inflammatory responses.

The ψ sequence is the minimum packaging signal required for encapsidation of the lentiviral genome.

The 2A peptide is a short peptide of viral origin, typically 18 to 25 amino acids long, commonly referred to as a self-cleaving peptide, that enables one transcript to produce multiple proteins.

"Stem cells" refers to undifferentiated cells that are capable of self-renewal over a long period of time, or are capable of producing at least one identical copy of the original cells; differentiation into multiple, and in some cases, only one particular cell type at the single cell level; achieving in vivo functional regeneration of tissue. Stem cells are subdivided into totipotent stem cells, sub-totipotent stem cells, pluripotent stem cells and oligopotent stem cells according to their fine developmental potential.

Mesenchymal Stem Cells (MSCs) have been widely used in clinical researchers for regenerative medicine, autoimmune diseases, because of their multipotent differentiation and immunoregulatory function. Unlike other stem cells, such as hematopoietic stem cells, MSCs are a class of stem cells that can be expanded in vitro. In the present application hUC-MSCs are mesenchymal stem cells obtained from the umbilical cord, in particular human umbilical cord mesenchymal stem cells. The human umbilical cord mesenchymal stem cells are derived from neonatal umbilical cord mesenchymal stem cells, and have strong proliferation and multidirectional differentiation capacity. However, those skilled in the art will appreciate that the source of mesenchymal stem cells is not limited to human umbilical cord mesenchymal stem cells, and that other sources of mesenchymal stem cells may be used to practice the present invention.

Macrophages are phenotypically heterogeneous immune cells that play an important role during inflammation (initiation and regression). Macrophages can be polarized to two phenotypes upon stimulation: (1) One is the classical activation (inflammatory) phenotype M1, which can be induced by Lipopolysaccharide (LPS) or interferon gamma (IFN-gamma) etc., to produce pro-inflammatory cytokines such as TNF alpha, IL-1 beta etc.; (2) Another alternative is the alternate activation (wound healing) phenotype M2, which can be induced by IL-4, IL-13, etc., to produce anti-inflammatory cytokines such as IL-10, IL-13, arg1, etc. The balance of M1/M2 macrophage polarization determines the fate of an organ in inflammation or injury. M1 exerts a pro-inflammatory effect against stimulation in the early stages of inflammation, but persists to cause tissue damage; m2 exerts an anti-inflammatory effect, promoting tissue repair and revascularization.

Drawings

FIG. 1 shows a lentiviral overexpression vector profile.

FIG. 2 shows the effect of lentiviral transfection on hUC-MSCs confocal microscopy.

FIG. 3 shows immunofluorescence and flow detection of eGFP expression.

The upper graph in FIG. 4 shows the effect of different cell seeding densities on cell fusion, and the lower graph shows the effect of different Polybrene working concentrations on growing cells.

Figure 5 shows that qPCR detects a significant increase in over-expressed factor secretion 72h after the hic-MSCs transfection of virus.

FIG. 6 shows that ELISA detects a significant increase in the secretion of overexpressed factors after 72h/96h transfection of hUC-MSCs into the virus.

FIG. 7 shows the reproducibility of transfection efficiency.

FIG. 8 shows that hUC-MSCs overexpressing IL-10, IL-13, and IL-4 are capable of inhibiting the M1 phenotype of macrophages and promoting the M2 phenotype of macrophages.

FIG. 9 shows that hUC-MSCs that overexpress IL-10, IL-13, and IL-4 simultaneously act synergistically to promote macrophage transformation to M2 as compared to IL-4 alone.

FIG. 10 shows the effect of bone marrow mesenchymal stem cell BMSCs over-expressing anti-inflammatory factor TGF beta on macrophage polarization to M2 phenotype in the prior art.

Fig. 11 shows lung HE staining patterns (4×) for each group of mice.

Fig. 12 shows an enlarged view of the lung HE staining (second behavior 10×) and labeling analysis of representative mice for each group.

Fig. 13 shows HE staining analysis of the mouse spleen (first row 4×, second row partial enlargement fig. 10×).

The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that modifications and improvements made on the basis of the inventive concept will be within the scope of the present invention for those skilled in the art. The reagents used were conventional products commercially available without the manufacturer's knowledge.

EXAMPLE 1 construction of lentiviral vectors

Firstly, a lentiviral vector which overexpresses three anti-inflammatory factors IL-10, IL-13 and IL-4 is designed and synthesized, the vector is named pLV [ Exp ] -EF1 alpha ] hIL10[ NM_000572.3] (ns): T2A: hIL13[ NM_002188.3] (ns): P2A: hIL4[ NM_000589.4] -SV40> EGFP: T2A: puro, and the vector design diagram is shown in figure 1. The vector uses RSVpromoter, EF alpha promoter sequence and Kozak translation initiation sequence on the upstream of the inserted anti-inflammatory factor gene, ensures the high expression of virus plasmid in stem cells, and the three anti-inflammatory factors are divided by 2A peptide sequence to prevent fusion protein. The constructed lentiviral vector carries enhanced GFP and antibiotic Puro sequences at the same time, thereby facilitating the observation of transfection efficiency and drug screening when necessary.

Analyzing the insert sequence and restriction enzyme sites on the vector skeleton, selecting enzyme digestion plasmid DNA capable of cutting out proper bands, running agarose gel electrophoresis on the digested DNA, dying with EtBr, judging the size of the DNA fragment, and designing primers for targeting the vector skeleton and/or the insert sequence. The nucleic acid sequences were identified by Sanger sequencing and aligned exactly.

EXAMPLE 2 packaging and transfection of lentiviral vectors

After successful construction of the viral vector plasmid, viral packaging is performed and hUC-MSCs are transfected. Transduction for D0 days, selection of hUC-MSCs of P4-P8 generation, and transformation of cells into 1X 10 ⁵ -2×10 ⁵ Density of wells inoculated into 6 well plates with DMEM/F12+10% FBS at 37deg.C with 5% CO ₂ After 18-20h of culture in an incubator, transfection is performed when the cell density is fused to 30% -50%. On the day of transduction (D1), the virus solution was thawed on ice, gently mixed, the number of viruses was aspirated according to MOI (MOI range 10-30), and gently mixed in culture medium. The amount of the culture medium is preferably 100. Mu.L/mL, and the amount of the culture medium in the 6-well plate is 1 mL/well. The original medium was aspirated and the virus-supplemented medium was added to a 6-well plate in which hUC-MSCs were cultured. Simultaneously adding 5-8 μg/mL Polybrene as auxiliary transfection reagent into each well, mixing to make virus cover each cell, and adding 5% CO at 37deg.C ₂ Is cultured in an incubator for 6-8 hours. Excessive exposure to Polybrene can cause cell poisoning, and therefore transduction times are not likely to be excessive, which may otherwise affect cell status. Viral fluid with empty Vector was also transduced in the same manner as a blank.

The next day, the virus-containing medium was aspirated, fresh DMEM/F12+10% FBS medium was added, and 5% CO at 37 ℃ ₂ Is cultured overnight in an incubator. Typically lentiviral-carried genes begin to express only on day 2 of transduction, and green fluorescence is observed 48-72 hours after transfection, designated 3IL-MSCs. Fluorescent expression was observed daily, and after 72h, confocal microscopy observed strong expression of green fluorescent protein of GFP (see fig. 2), the percentage of green fluorescence was counted, while flow analysis confirmed the infection efficiency (see fig. 3), and the result showed that the effective infection rate exceeded 90% at an MOI of 20.

Experiments were performed at different cell seeding densities and different MOI values, and as a result, it was found that the cell seeding density was 1.2X10 ⁵ Well (see FIG. 4), the transfection complex MOI was 20 and the transfection efficiency was best (see FIG. 3), polybrene was suitably used at a working concentration of 6. Mu.g/mL (see FIG. 4). High density cells and high concentration helper transferStaining agents are detrimental to cell survival and the effect of overexpression is reduced (see figure 4). Specifically, 2×10 ⁵ Is too dense after 72h of transfection at a density of 1.2X10 s, and causes contact inhibition of growth ⁵ The fusion degree on the day of the inoculation transfection is about 30%, the fusion degree of cells after 72 hours of transfection is about 90%, and the growth state is good. Polybrene working concentrations tested 6 μg/mL and 8 μg/mL, fluorescence was not significantly different under the microscope, and excessive concentrations were likely to cause cell poisoning, thus 6 μg/mL was chosen.

To further confirm the increased secretion of the over-expressed factors, baseline levels of anti-inflammatory factor were not even detectable, but post-over-expressed cellular gene transcript levels (FIG. 5) and protein secretion levels in the supernatant (FIG. 6) were significantly increased, as analyzed by real-time fluorescent quantitative PCR (FIG. 5) and ELISA (FIG. 6), where IL-13 protein secretion was increased by more than 100-fold from undetectable (less than 10 pg/mL) or empty vector control (about 20 pg/mL) to more than 2713 pg/mL; anti-inflammatory factors IL-10 and IL-4 increased 50000 and 40000 fold, respectively (see FIG. 6).

FIG. 5 shows that qPCR detected a significant increase in secretion of the overexpressed cytokines IL-4, IL-10 and IL-13 72h after transfection of hUC-MSCs, wherein UP8 VM10 represents the expression result of human umbilical cord mesenchymal stem cells MOI 10 at passage 8; UP4 LM15 represents the expression result of human umbilical cord mesenchymal stem cells MOI of 15 after 4 passages; UP 8L M shows the expression result of 20 MOI of human umbilical cord mesenchymal stem cells passaged 8 times.

FIG. 6 also shows that ELISA detected a significant increase in the secretion of the overexpressed factor after 72h/96h viral transfection of hUC-MSCs with different MOI, and that the overexpression effect of IL-13, IL-10 and IL-4 was significant at a multiplicity of transfection MOI of 20.

FIG. 7 shows reproducibility of infection efficiency, wherein UP 8V M10 represents the expression result of human umbilical cord mesenchymal stem cells MOI of 10 at passage 8 times; UP4 LM15 represents the expression result of human umbilical cord mesenchymal stem cells MOI of 15 after 4 passages; UP8 LM20 represents the expression result of human umbilical cord mesenchymal stem cells MOI of 20 passaged 8 times.

It can be seen that by the optimal combination of vector design and the above transfection steps, high efficiency of infection and success of overexpression are ensured.

EXAMPLE 3 anti-inflammatory Effect of supernatants of hUC-MSCs overexpressing IL-13, IL-10 and IL-4

The lentiviral vectors constructed and packaged in examples 1 and 2 were transfected with hUC-MSCs at MOI of 20, and after 72h of transfection, the culture medium was collected and the supernatant hUC-MSCs-CM was collected by centrifugation at 1000g for 10 min.

Mouse macrophage cell line RAW264.7 was used at 3X 10 ⁵ Is inoculated into 6-well plates, and the culture medium is DMED/F12 (HG) +10% FBS complete culture medium. To induce M1 polarization, lipopolysaccharide (LPS) was added to complete medium at a concentration of 100ng/mL the next day, and induced for 24h. The negative control group (CON) was cultured with complete medium (DMED/F12 (HG) +10% FBS) for 4d; LPS groups were treated with 100ng/mL LPS for 24 and then replaced with complete medium (DMED/F12 (HG) +10% FBS) for further 48h; to understand the effect of hUC-MSCs-CM on RAW264.7 in inflammatory environment, the experimental group was incubated for 48h after 24h of 100ng/mL LPS treatment with various concentrations of hUC-MSCs-CM (1%, 5%, 10%) in the new complete medium (DMED/F12 (HG) +10% FBS). Macrophages were collected for qPCR analysis.

FIG. 8 shows that after polarization of RAW264.7+100ng/mL LPS in macrophages, the macrophages were co-cultured with supernatants over-expressing hUC-MSCs and tested for anti-inflammatory effects, and based on qPCR results, the expression of pro-inflammatory cytokines (TNF-. Alpha.) and anti-inflammatory cytokines (IL-10, IL-13 and Arg 1) was altered in macrophages under different conditions. After LPS stimulation, the mRNA levels of the pro-inflammatory cytokines were elevated compared to the control group, while the mRNA levels of the anti-inflammatory cytokines were not significantly altered. The addition of hUC-MSCs-CM down regulates the level of the proinflammatory cytokines and enhances the expression of the anti-inflammatory cytokines, wherein the effect of adding 5% of hUC-MSCs-CM is most obvious. Experiments prove that hUC-MSCs-CM over-expressing three anti-inflammatory factors of IL-10, IL-13 and IL-4 can inhibit an M1 phenotype and promote an M2 phenotype, and further influence the development of inflammation by influencing macrophage polarization.

Macrophages are readily activated by polarization of LPS towards M1, whereas IL4 or IL13 alone hardly converts cells towards M2. Mouse macrophage cell line RAW264.7 was used at 3X 10 ⁵ Density joint of (2)Seed to 6-well plate, medium was DMED/F12 (HG) +10% FBS. To induce M2 polarization, the next day IL-4 recombinant cytokines were added to complete medium at 10, 20, 50ng/mL and induced for 48h. The negative control group (CON) was cultured with complete medium (DMED/F12 (HG) +10% FBS) for 3d. Macrophages were collected for qPCR analysis.

According to qPCR results (FIG. 9), anti-inflammatory factors were up-regulated in addition to TGF-beta, IL-10, IL-13, arg1 were not significantly altered, and pro-inflammatory factors IL-1 beta were not significantly down-regulated, after addition of IL-4 alone, as compared to the control group. The above results demonstrate that IL-4 alone hardly polarizes macrophages towards M2, while we overexpress three anti-inflammatory factors IL-10, IL-13 and IL-4 simultaneously, which act synergistically and promote macrophage transformation into M2 (anti-inflammatory).

In contrast, the BMSCs in the prior art overexpress the anti-inflammatory factor TGF beta, which can promote macrophage polarization to M2 phenotype (see FIG. 10), but the polarization effect is not obvious because we over-express three anti-inflammatory factors IL-10, IL-13 and IL-4 at the same time.

EXAMPLE 4 construction of acute inflammation mouse model

When DMED/F12+10% FBS complete medium is subcultured to reach the required cells, 3IL-MSCs are collected and diluted to 2.5X10 with physiological saline compared with non-genetically edited MSCs ⁶ The concentration of the solution was kept at a value of/mL.

The mice are randomly divided into four groups of placebo group, model group, control group and treatment group according to random number, which are 3, 5, 4 and 5 respectively; a mouse model of acute inflammation was induced by injecting 100. Mu.L of LPS solution into each group except the placebo group, which had an LPS concentration of 6 mg/mL.

Example 5 stem cell transplantation therapy and histopathological observations

2 hours after molding, the treatment group was intraperitoneally injected with 3IL-MSCs cells 1X 10 ⁶ 200 μl/mouse, control group was injected with unedited MSCs, and the other two groups were injected with equal amounts of physiological saline.

After 6 hours of molding, all mice were sacrificed by cervical removal and the distal ends of the right lung 1 leaf and spleen were harvested and fixed. Tissue was fixed in 40g/L paraformaldehyde solution, paraffin embedded, and 4 μm thick sections were stained with hematoxylin-eosin (HE) (see FIGS. 11-13). Fig. 11-13 show that the MSCs subjected to gene editing prepared by the scheme has better anti-inflammatory treatment effect on acute inflammation of mice induced by LPS by comparing the effects of MSCs before and after gene editing on lungs and spleens of the mice.

As shown in fig. 12, HE staining showed clear lung tissue structure, uniform alveolar distribution, intact alveolar septum, bronchial wall, uniform pulmonary interstitial distribution, and rare inflammatory cell infiltration in placebo group mice. The model group mice have disordered lung tissue structure, partial alveolar atrophy and collapse (shown by arrows), large lung bubbles, bronchi wall thickening (shown by triangles), increased lung interstitial and perivascular extracellular matrix, fibroblast increase and massive inflammatory cell infiltration (shown by boxes); the control group has half restoration of alveolar structure, uneven alveolar distribution, thick alveolar interval, bronchus wall thickness and inflammatory cell infiltration; the recovery of alveolar structure in the treated group was significantly better than that in the control group, with reduced inflammatory cell infiltration of bronchi and alveolar spaces, generally approximating normal lung tissue in the placebo group.

As shown in fig. 13, normal spleen HE staining has normal capsule, spleen trabecula, red pulp and white pulp structure. Inflammatory, highly atrophic white marrow (splenomegaly and periarterial lymph sheath), reduced volume, disintegrated and diffuse (shown by triangles), massive hemorrhage of red marrow, and indistinct cell line; in the control group, the white pulp atrophy and red pulp bleeding are relieved to a certain extent; the treatment group is more full of white marrow, larger in volume, higher in proportion in the whole spleen and the whole body tends to be normal compared with the model group and the control group.

By comparing the effects of MSCs before and after gene editing on the lung and spleen of an inflammatory mouse, the MSCs prepared by the scheme have better anti-inflammatory treatment effect on acute inflammation of the mouse induced by LPS.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

SEQUENCE LISTING

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<120> mesenchymal stem cells and anti-inflammatory applications thereof

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<170> PatentIn version 3.5

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cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180

ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240

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Claims

1. Use of a host cell comprising an expression vector carrying coding sequences for the expression of IL-10, IL-13 and IL-4 for the preparation of a medicament for the treatment of an inflammatory disease, which is an acute inflammatory disease, said host cell being a mesenchymal stem cell.

2. The use of claim 1, wherein the expression vector is a lentiviral vector comprising a vector plasmid comprising a 5'ltr, a 3' ltr comprising a ψ sequence, a gene sequence of interest between the 5'ltr and the 3' ltr, and a promoter sequence and a translation initiation sequence operably linked to the gene sequence of interest, the gene sequence of interest being the coding nucleotide sequences of IL-10, IL-13 and IL-4.

3. The use according to claim 2, wherein the 3'ltr and/or 5' ltr comprises one or more modifications.

4. The use according to claim 3, wherein U3 of the 5'ltr and 3' ltr is deleted or mutated.

5. The use according to claim 3, wherein the 3'ltr is a Δu3/3' ltr lacking the U3 region.

6. The use according to claim 3, wherein the 5'ltr is a deletion form of Δ5' ltr.

7. The use according to claim 2, wherein the promoter of the 5' ltr of the vector plasmid is selected from the group consisting of: cytomegalovirus CMV promoter, rous sarcoma virus RSV promoter, and simian virus SV40 promoter;

the promoter operably linked to the coding nucleotides of IL-10, IL-13 and IL-4 is selected from the group consisting of short elongation factor 1A (EF 1. Alpha.) promoters or transcriptionally active fragments thereof, RSV promoters and simian virus SV40 promoters.

8. The use of claim 7, wherein the coding nucleotides for IL-10, IL-13 and IL-4 are operably linked to an EF 1a promoter and a Kozak translation initiation sequence.

9. The use according to claim 2, wherein the vector plasmid further comprises a nucleotide encoding a selection marker selected from one or more of a luciferase, an enhanced green fluorescent protein, a streptavidin binding peptide, a puromycin resistance gene, an ampicillin resistance gene, a kanamycin resistance gene and a neomycin resistance gene.

10. The use according to claim 9, wherein the screening marker is an enhanced green fluorescent protein EGFP/puromycin resistance gene Puro dual reporter marker, the EGFR and puromycin resistance genes being spaced apart by a nucleotide sequence encoding a 2A peptide.

11. The use of claim 10, wherein the coding nucleotide of the EGFP/Puro dual reporter marker is operably linked to an SV40 promoter and a Kozak coding sequence.

12. The use according to claim 2, wherein the vector plasmid further comprises woodchuck hepatitis virus posttranscriptional regulatory element WPRE, retroviral export element, central polypurine region cPPT or central termination sequence CTS.

13. Use according to claim 12, characterized in that said retroviral export element is selected from the group consisting of the human immunodeficiency virus rev responsive element RRE and the hepatitis b virus posttranscriptional regulatory element HPRE.

14. The use according to claim 12, wherein the cPPT or CTS sequence is cPPT or CTS of HIV 1.

15. The use according to claim 2, wherein the vector plasmid comprises, in order from the 5'ltr region to the 3' ltr region: RSV promoter, 5'LTR, ψ sequence, RRE, cPPT, EF 1. Alpha. Promoter, kozak translation initiation sequence, the coding nucleotides for IL-10, IL-13 and IL-4 shown by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, SV40, kozak translation initiation sequence, EGFR/Puro, WRPE, ΔU3/3' LTR and SV40 early pA.

16. The use according to claim 2, wherein the IL-10, IL-13 and IL-4 are separated from each other by a nucleotide sequence encoding a 2A peptide, from the encoding nucleotides of IL-10, IL-13 and IL-4.

17. The use of claim 16, wherein the 2A peptide is a T2A peptide, a P2A peptide, an E2A peptide or an F2A peptide.

18. The use according to claim 16, wherein the 2A peptide is a T2A or P2A peptide.

19. The use according to claim 1, wherein the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.