A gene nanodrug for enhancing T cell anti-tumor immune effect and a preparation method and application thereof

Abstract

The application discloses a kind of gene nanomedicine for enhancing T cell antitumor immune effect and its preparation method and application.The gene nanomedicine can be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc.The gene nanomedicine can not only be loaded into cationic lipid-assisted polymer nanoparticles by ultrasound double emulsification etc

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a gene nanomedicine for enhancing the anti-tumor immune effect of T cells, its preparation method, and its application. Background Technology

[0002] Cancer is a global public health problem and has become the leading cause of death in my country, seriously threatening the health of the Chinese population. In recent years, tumor immunotherapy has developed rapidly, with T cells playing a crucial role. T cells primarily kill tumors by recognizing tumor antigens presented by antigen-presenting cells, mobilizing and migrating to the tumor site, recognizing tumor cells, activating, and exerting cytotoxic effects. Researchers have developed various strategies to enhance the anti-tumor effects of T cells, such as immune checkpoint antibody blockade and CAR-T cell therapy, but the clinical efficacy of these immunotherapies is not optimistic. This is because most solid tumors are "cold" tumors where T cells have difficulty infiltrating, antibody drugs struggle to efficiently enter tumor tissue, and non-specific drug effects can trigger immune-related adverse reactions, greatly limiting the effectiveness of T cell anti-tumor immune function. Therefore, there is an urgent need to develop new strategies to enhance the anti-tumor immune effects of T cells.

[0003] During tumor killing, T cell infiltration (mainly effector T cells) primarily relies on the chemokine receptor CXCR3 on the cell surface to receive signals from chemokines expressed in tumor tissue, including chemokine ligand 9 (CXCL9), chemokine ligand 10 (CXCL10), and chemokine ligand 11 (CXCL11), thus infiltrating into solid tumors. However, many solid tumors downregulate the expression of these chemokines, inhibiting T cell infiltration into solid tumors. Researchers have already enhanced T cell infiltration into tumors by intratumoral injection of recombinant chemokine proteins or systemic injection of chemokine viral expression vectors. Immune checkpoint antibody drugs can effectively relieve the inhibition of T cell killing function by immune checkpoint signaling pathways, but traditional antibody drug administration is systemic, leading to low utilization in tumor tissues and adverse reactions in non-tumor sites. Researchers have already utilized local or targeted drug delivery systems to enhance the tumor accumulation of antibody drugs. Increasing T cell chemotaxis and activation to enhance the anti-tumor effect of T cells is a key aspect of tumor immunotherapy.

[0004] CRISPR-mediated gene activation tools, such as the CRISPRa expression plasmid, recruit transcription activators to transcription initiation sites by expressing the inactivated endonuclease dCas9 and binding to different guide RNAs (gRNAs), can activate the expression of multiple endogenous genes thousands of times. Currently, numerous studies have used CRISPRa to upregulate the expression of specific genes for the treatment of various diseases. Gene overexpression plasmids can simultaneously increase the levels of one or more functional proteins by overexpressing one or more specific genes. It is a powerful gene therapy tool and has shown some effectiveness in treating malignant tumors, genetic diseases, infectious diseases, cardiovascular diseases, and autoimmune diseases. Upregulating the expression of tumor cell chemokines using CRISPRa or driving tumor cells to efficiently express chemokines and immune checkpoint inhibitors using gene overexpression plasmids holds promise for enhancing the targeted invasion and activation of T cells into solid tumors, thereby improving anti-tumor efficacy. However, many limitations still exist in the practical application of CRISPRa plasmids and gene overexpression plasmids.

[0005] First, there are several commonly used CRISPRa systems. Taking the dCas9-SAM system, which has the strongest activation effect, as an example, the entire system includes a 165kDa dCas9 transcription activation domain fusion protein dCas9-VP64, a 161bp endogenous gene-specific MS2gRNA, and a 46kDa transcription activation accessory protein MS2-p65-HSF1. The total number of nucleotides in the system exceeds 5.8kb, while the packaging capacity of commonly used viral vectors is limited, mostly not exceeding 4.7kb. Viral vector delivery requires disassembling this system and packaging it as a three-plasmid configuration, which significantly reduces gene expression efficiency. Furthermore, viral elements are immunogenic and can induce immune-related responses; viruses pose a risk of random integration into the genome and oncogenicity; and viral design is complex and difficult to scale up. Gene overexpression plasmids also face these problems when expressing multiple genes. Secondly, commonly used plasmid expression backbones all use strong promoters such as CMV, CBh or EF1α promoters to drive the expression of target genes. This method is not cell or tissue specific, and once in vivo, it will also perform non-specific gene manipulation on normal tissues other than tumor tissues, thereby causing adverse reactions.

[0006] In summary, further breakthroughs are needed to achieve efficient, specific, and safe enhancement of the expression of tumor cell chemokines and immune checkpoint inhibitors using gene activation tools such as CRISPRa or gene overexpression plasmids, thereby enhancing T cell chemotaxis and activation and improving T cell-mediated anti-tumor immune effects. Summary of the Invention

[0007] To overcome the aforementioned shortcomings of existing technologies, the purpose of this invention is to develop a gene nanomedicine capable of efficiently delivering the gene activation tool CRISPRa plasmid or gene overexpression plasmid to tumor cells and achieving tumor-specific gene expression, significantly enhancing the targeted infiltration and activation killing effect of T cells into tumors, thereby strengthening the efficacy of T cell-mediated anti-tumor immunotherapy. Furthermore, this method is simple, convenient, efficient, specific, and safe, possessing excellent application potential.

[0008] The specific technical solution is as follows:

[0009] A gene nanomedicine for enhancing the anti-tumor immune effect of T cells includes a plasmid and cationic lipid-assisted nanoparticles, wherein the plasmid is an expression plasmid for genes related to enhancing the immune phase effect of T cells, and the cationic lipid-assisted nanoparticles are cationic lipid-assisted polymer nanoparticles.

[0010] Furthermore, the plasmid is at least one of the gene activation tool CRISPRa plasmid and the gene overexpression plasmid. CRISPRa is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats Activation, which is a gene transcription activation tool for regularly clustered interspaced short palindromic repeats.

[0011] Furthermore, the plasmid promoter is at least one of a strong promoter or a tumor-specific promoter.

[0012] Furthermore, the strong promoter is one of the following: cytomegalovirus (CMV) promoter, chicken β-actin CBh promoter, and elongation factor-1αEF1α promoter; the tumor-specific promoter is at least one of the following: tyrosinase (Tyr) promoter and survivin (Sur) promoter. Gene expression driven by strong promoters is non-selective and has gene expression effects in various cell types; tumor-specific promoters selectively drive gene transcription and expression in tumor cells, while having no effect on other normal cells. Tyrosinase promoters can achieve melanoma-specific gene expression, while survivin promoters can achieve tumor-specific gene expression in various other tumors.

[0013] Furthermore, the gene activation tool CRISPRa plasmid is at least one of the following: a CRISPRa plasmid targeting and activating chemokine ligand 9 (CXCL9), a CRISPRa plasmid targeting and activating chemokine ligand 10 (CXCL10), and a CRISPRa plasmid targeting and activating chemokine ligand 11 (CXCL11); the gene overexpression plasmid is at least one of the following: chemokine ligand 9 (CXCL9) and an anti-programmed cell death-ligand 1 single-stranded variable region fragment (anti-PD L1 scFv) expression plasmid.

[0014] Furthermore, the plasmid contains at least one human or mouse gene.

[0015] Furthermore, the gene activation tool CRISPRa plasmid comprises two plasmids. One plasmid includes a guide RNA (MS2gRNA) with a hairpin structure of phage MS2 coat protein dimer targeting chemokine ligand 9 (CXCL9), chemokine ligand 10 (CXCL10), and chemokine ligand 11 (CXCL11), as well as a fusion expression plasmid of inactivated endonuclease dCas9 and transcription activator VP64 (i.e., plasmid element dCas9-VP64). The other plasmid includes a fusion expression plasmid of transcriptional coactivator protein phage MS2 coat protein, NF-κB transcription factor p65 protein, and heat shock regulatory protein HSF1 protein (i.e., plasmid element MS2-p65-HSF1). The gene overexpression plasmid sequence is a co-expression plasmid formed by linking chemokine ligand 9 (CXCL9) and a single-stranded variable region fragment of anti-programmed cell death-ligand 1 (anti-PD-L1 scFv) through a P2A cleavage peptide. The plasmid element dCas9-VP64 was used to construct the pgCXCL-dCas9 plasmid with MS2gRNA. The plasmid element MS2-p65-HSF1 was used to construct the pMPH plasmid.

[0016] Furthermore, the terminator of the plasmid is bGH polyA.

[0017] Furthermore, the lipids in the cationic lipid-assisted nanoparticles are at least one of cationic cholesterol derivatives and alkyl chain cationic lipids.

[0018] Furthermore, the cationic cholesterol derivative is at least one of N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteroloxycarbonylaminoethyl)ammonium bromide (BHEM-Chol), laboratory-synthesized cationic cholesterol derivative CL3, and laboratory-synthesized cationic cholesterol derivative CL7, and the alkyl chain cationic lipid is at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), laboratory-synthesized alkyl chain cationic lipid AL6, and laboratory-synthesized alkyl chain cationic lipid AL7.

[0019] Furthermore, the polymer in the cationic lipid-assisted polymer nanoparticles is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) and poly(glycolic acid-co-lactide) (PLGA).

[0020] Furthermore, the polymers in the cationic lipid-assisted polymer nanoparticles are polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) and poly(glycolic acid-co-lactide) (PLGA), with the polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) accounting for 60%-100% of the total polymer.

[0021] Furthermore, the average particle size of the cationic lipid-assisted nanoparticles is 50 nm-200 nm.

[0022] A method for preparing the gene nanomedicine that enhances the anti-tumor immune effect of T cells includes the following steps:

[0023] Step 1: Mix the plasmid that enhances the anti-tumor immune effect of T cells with an aqueous solution; mix the lipid, polyethylene glycol-modified polyester, and polyester with chloroform organic solvent to obtain the oil phase;

[0024] Step 2: Prepare an oil-in-water emulsion from the oil phase and the aqueous phase;

[0025] Step 3: Concentrate the emulsion to obtain gene nanomedicine.

[0026] Furthermore, the concentration of plasmids enhancing T-cell anti-tumor immune response in the aqueous phase was 1-4 mg / ml; the concentration of polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) or poly(glycolic acid-co-lactide) (PLGA) in the oil phase was 62.5 mg / ml; and the concentration of lipids in the oil phase was 10-30 mg / ml.

[0027] Furthermore, the method for preparing an oil-in-oil-in-water emulsion from the oil phase and the aqueous phase is at least one of ultrasonic emulsification, microfluidics, and microchannel reaction; the method for concentrating the emulsion is at least one of rotary evaporation and freeze drying.

[0028] A gene nanomedicine for enhancing the anti-tumor immune response of T cells improves the efficacy of T cell-mediated anti-tumor immunotherapy by enhancing T cell chemotaxis and activation.

[0029] A gene nanomedicine for enhancing the anti-tumor immune response of T cells is combined with immune checkpoint blockade therapy or CAR-T cell therapy to enhance the efficacy of anti-tumor immune therapy.

[0030] Plasmids can potently enhance the expression levels of specific target genes. Plasmids are driven by strong promoters or tumor-specific promoters. T cells primarily rely on their surface chemokine receptor CXCR3 to sense signals from chemokine ligands 9 (CXCL9), 10 (CXCL10), and 11 (CXCL11) in tumor tissue, and then migrate and infiltrate the tumor tissue along the concentration gradient of chemokines between peripheral and tumor tissues. The gene activation tool CRISPRa can simultaneously activate the expression of chemokine ligands 9 (CXCL9), 10 (CXCL10), and 11 (CXCL11) in tumor cells, forming a significant chemokine concentration gradient, inducing a large number of T cells to infiltrate the tumor and thus kill the tumor. By simultaneously enhancing the expression of tumor cell chemokine ligand 9 (CXCL9) and the single-chain variable region fragment of anti-programmed cell death-ligand 1 (anti-PD-L1 scFv) using gene overexpression plasmids, not only can the targeted infiltration of T cells into tumor sites be enhanced, but the PD-L1 immune checkpoint on the tumor cell surface can also be effectively blocked by anti-PD-L1 scFv, thereby enhancing the activation and tumor-killing function of tumor-infiltrating T cells. In preclinical studies, a murine gene was used, while in actual clinical applications, if for human treatment, a human gene will be used. Compared to the previous lentiviral expression system using the dCas9-SAM three-plasmid, the gene activation tool CRISPRa dual-plasmid expression system offers higher expression efficiency, greater application flexibility, and reduced immunogenicity of the plasmid itself. The gene overexpression plasmid sequence co-expresses chemokine ligand 9 (CXCL9) and the anti-programmed cell death-ligand 1 single-chain variable region fragment (anti-PD-L1 scFv), which can simultaneously enhance T cell chemotaxis and activation, exhibiting a synergistic effect. Furthermore, the sequences of chemokine ligand 9 (CXCL9) and the anti-programmed cell death-ligand 1 single-chain variable region fragment (anti-PD-L1 scFv) are linked by a P2A cleavage peptide, without affecting their respective expression efficiency or protein function. The plasmid terminator is bGH polyA, which can achieve more effective polyadenylation and transcription termination in eukaryotic cells. The cationic lipid is preferably DOTAP, which helps form nanoparticles with higher plasmid encapsulation efficiency, better cellular uptake, and stronger plasmid transfection effect. The polyethylene glycol-modified polyester accounts for 60%-100% of the total polymer. A 100% ratio is preferred, as it has the highest polyethylene glycol (PEG) density, long-lasting circulation time in vivo, and rapid intracellular plasmid release. The nanoparticles have an average particle size of 50 nm to 200 nm. Preferably, the particle size range is 80 nm to 120 nm, allowing the nanoparticles to accumulate more efficiently at the tumor site.

[0031] This invention relates to a gene nanomedicine for enhancing the anti-tumor immune response of T cells. It can increase the expression of chemokines and immune checkpoint inhibitors in tumor cells, thereby recruiting T cells to infiltrate tumor tissues, relieving the inhibition of T cells by immune checkpoints, and enhancing the killing effect of T cells on tumors. This gene nanomedicine is tumor-specific and can enhance the anti-tumor immune response of T cells against specific tumors or various solid tumors.

[0032] The gene nanomedicine of this invention, used to enhance T cell chemotaxis and activation, can also be used in combination with other tumor immunotherapies to achieve synergistic effects. When used in combination with immune checkpoint blockade antibody drugs, it can increase T cell infiltration into tumor sites, thereby enhancing the responsiveness of ICB antibody drugs. Furthermore, when used in combination with CAR-T cell therapy, it can improve the problem of insufficient CAR-T cell infiltration in solid tumors, thereby enhancing the killing effect of CAR-T cells on solid tumors.

[0033] A plasmid system designed for efficient activation of chemokine expression and convenient non-viral vector delivery was developed. This plasmid system eliminates the viral transduction element, replacing it with a short linker peptide, thus optimizing and simplifying a three-plasmid system into a two-plasmid system. Simultaneously, the reporter gene is ligated to the activation element using a T2A-cleaved peptide sequence, achieving co-expression of the activation element and the reporter gene. Furthermore, the reporter gene is ligated after the activation element during the design process, facilitating the characterization of the activation element's expression without affecting its function.

[0034] A plasmid system designed for efficient simultaneous enhancement of the expression of chemokines and immune checkpoint inhibitors, and convenient for delivery via non-viral vectors, was developed. This plasmid utilizes a P2A cleavage peptide to link multiple gene sequences, including chemokine ligand 9 (CXCL9) and a single-stranded variable region fragment of anti-programmed cell death-ligand 1 (anti-PD-L1 scFv), achieving multi-gene expression within a single plasmid.

[0035] By utilizing tumor-specific promoters such as tyrosinase promoters or survivin promoters to drive gene activation, the CRISPRa plasmid or gene overexpression plasmid can be used to specifically express tumor cells, efficiently and specifically enhancing the expression of immune effector molecules such as chemokine ligand 9 (CXCL9), chemokine ligand 10 (CXCL10), chemokine ligand 11 (CXCL11), and the single-chain variable region fragment of anti-programmed cell death-ligand 1 (anti-PD-L1 scFv).

[0036] We used laboratory-developed cationic lipid-assisted nanoparticles (CLAN) to deliver tumor-specific gene activation tools or gene overexpression plasmids in vivo.

[0037] Building upon existing CLAN fabrication processes, a series of CLAN carrier libraries with varying charges and surface polyethylene glycol (PEG) densities were prepared by controlling the types and amounts of cationic lipids and the ratio of polyethylene glycol-modified polyester to polyester. Through high-throughput screening of these material libraries, CLAN nanoparticles containing CRISPRa plasmids (for efficient tumor gene delivery and activation) or gene overexpression plasmids were selected for optimal efficacy.

[0038] A tumor-specific gene-activating nanomedicine or gene nanomedicine was designed. After systemic administration, this type of drug can specifically enhance the expression of chemokines and immune checkpoint inhibitors in tumor sites in vivo, while having almost no effect in other normal tissues, thereby effectively inducing T cell migration, infiltration, and activation for killing.

[0039] This invention provides a gene nanomedicine for enhancing T cell chemotaxis and activation, significantly improving the efficacy of tumor immunotherapy, immune checkpoint blockade therapy, and CAR-T cell therapy.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] Nanoparticles, non-viral delivery vectors with sizes ranging from 1 to 1000 nm, can help plasmids overcome in vivo delivery barriers, increase the biocompatibility and safety of plasmid drugs, and facilitate transport and distribution within tumor cells to ultimately exert gene expression effects. However, nanoparticles can only relatively enhance the accumulation of plasmid drugs in tumors and can also deliver plasmids to organs and tissues such as the liver, kidneys, and lungs. This non-specific plasmid drug delivery can lead to low drug utilization and trigger immune-related adverse reactions in normal organs.

[0042] Tumor-specific promoters exhibit specific transcriptional activity in tumor cells but lack it in normal cells, making it possible to efficiently and specifically express therapeutic genes in tumor cells. Tyrosinase, the rate-limiting enzyme in melanin synthesis, is specifically expressed in pigment-producing cells; by using tyrosinase promoters, transgenic melanoma-specific expression can be achieved. Survivin plays an important role in the growth and progression of various cancers, and its promoter can achieve gene expression in various tumor cells.

[0043] This invention utilizes cationic lipid-assisted polymer nanoparticles for plasmid delivery. Through hydrophilic-hydrophobic interactions, water-in-oil-in-water dual-emulsion nanoparticles are formed, encapsulating the plasmid within a core. This process exhibits excellent stability. The polyethylene glycol (PEG) shell allows for long-term circulation in vivo. The nanoscale (approximately 100 nm) size enhances particle accumulation in tumors due to the high permeability and retention effect (EPR effect) of solid tumors. The cationic lipids also facilitate cellular uptake of the nanoparticles. Furthermore, by adjusting the type and amount of cationic lipids, as well as the proportion of PEG-modified polyester, a library of nanoparticles with different potentials and PEG densities can be established. This allows for further screening of nanoparticles that efficiently deliver plasmids to tumor tissues.

[0044] The CRISPRa plasmid of this invention is ingeniously designed, optimizing and simplifying a three-plasmid system into a two-plasmid system, which facilitates the use of non-viral vector delivery and improves expression efficiency and the probability of delivering all activation elements to the same cell. After introducing the reporter gene into the activation element, not only is the structure simplified, but the accuracy of reporter gene expression is also improved.

[0045] The gene-activating CRISPRa plasmids or gene overexpression plasmids in this invention are unique, using tumor-specific promoters such as tyrosinase promoters or survivin promoters to drive plasmid expression. These plasmids are encapsulated within CLAN nanoparticles to form gene-activated nanomedicines or gene nanomedicines. After systemic administration in vivo, the nanomedicines accumulate in tumors, but a small portion also enters normal tissues. Due to the selective transcriptional activity of the tumor-specific promoters, the CRISPRa elements or gene overexpression plasmids are expressed only in tumor cells, endowing the nanomedicines with precise tumor-specific properties and enabling the gene drug to exert its specific effect at the tumor site. Furthermore, this drug can load gene-activating plasmids or gene overexpression plasmids with larger capacities.

[0046] This invention specifically enhances the expression of tumor cell chemokines and immune checkpoint inhibitors, among other immune effector molecules. Tumors specifically secrete large amounts of chemokines, creating a significant chemokine concentration gradient between tumor tissue and peripheral tissues, prompting effector T cells to infiltrate the tumor tissue. Tumor-secreted programmed death ligand 1 (PD-L1) inhibitors can efficiently block PD-L1 immune checkpoints on the tumor surface, enhancing the activation of tumor-infiltrating T cells and their tumor-killing effect. Using this method, the chemotaxis and activation of tumor-infiltrating T cells are simultaneously enhanced, improving T cell-mediated anti-tumor immune efficacy and the tumor microenvironment, thus efficiently and specifically enhancing the efficacy of tumor immunotherapy.

[0047] This invention can enhance the efficacy of tumor immunotherapy in a variety of solid tumors regardless of the heterogeneity of tumor patients, and can be used in combination with immune checkpoint blockade therapy and CAR-T cell therapy to enhance efficacy, showing great promise for clinical application.

[0048] The lipid-assisted nanoparticles of this invention are assembled from FDA-approved high-molecular-weight polyesters and lipids, and have excellent biocompatibility. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 Nanoparticle formulations with different polymer and cationic lipid contents;

[0051] Figure 2 Figure 1 shows the characterization results of the potential and particle size of nanoparticles with different polymer and cationic lipid contents.

[0052] Figure 3 Figure 1 shows the results of transfection efficiency determination for nanoparticles with different polymer and cationic lipid contents.

[0053] Figure 4 Structural formulas of different cationic lipids;

[0054] Figure 5 Figure 1 shows the characterization results of the potential and particle size of nanoparticles of different cationic lipid types.

[0055] Figure 6 Figure 1 shows the results of transfection efficiency determination for nanoparticles of different cationic lipid types.

[0056] Figure 7 pCRISPRa map of gene activation plasmid system;

[0057] Figure 8 CLAN (Gene-Activated Nanomedicine) pCRISPRa Figures showing the results of particle size, potential, and morphology characterization;

[0058] Figure 9 CLAN (Gene-Activated Nanomedicine) pCRISPRa Image showing the results of detecting enhanced mRNA expression levels of CXCL9, CXCL10, and CXCL11 in various tumor cells;

[0059] Figure 10 CLAN (Gene-Activated Nanomedicine)pCRISPRa The results of detecting enhanced protein expression levels of CXCL9, CXCL10, and CXCL11 in various tumor cells are shown in the figure.

[0060] Figure 11 CLAN (Gene-Activated Nanomedicine) pCRISPRa Image of in vitro enhanced T cell migration detection results;

[0061] Figure 12 CLAN (Gene-Activated Nanomedicine) pCRISPRa Image showing the results of in vivo intratumoral injection enhancing T-cell infiltration and inhibiting the growth of various tumors;

[0062] Figure 13 CLAN (Gene-Activated Nanomedicine) pCRISPRa Image showing the distribution of tumors and organs in the body after tail vein injection;

[0063] Figure 14 pTyr-CRISPRa map of the tumor-specific gene activation plasmid system;

[0064] Figure 15 CLAN, a tumor-specific gene-activating nanomedicine pTyr-CRISPRa Figure 1: Results of in vivo detection of tumor-specific enhancement of chemokine expression and T cell infiltration;

[0065] Figure 16 CLAN, a tumor-specific gene-activating nanomedicine pTyr-CRISPRa Image showing the results of in vivo detection of melanoma growth inhibition and specific enhancement of T cell infiltration in tumor tissue;

[0066] Figure 17 CLAN, a tumor-specific gene-activating nanomedicine pTyr-CRISPRa Graph showing the results of testing the anti-tumor efficacy of ICB therapy;

[0067] Figure 18 CLAN, a tumor-specific gene-activating nanomedicine pTyr-CRISPRa Image showing the results of efficacy testing for CAR-T cell therapy against solid tumors;

[0068] Figure 19 Maps of tumor-specific gene expression plasmids pTyr-C9AP and pSur-C9AP;

[0069] Figure 20 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP Figures showing the characterization results of particle size, potential, morphology, and stability;

[0070] Figure 21 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP CLAN pSur-C9APImage of results from quantitative PCR detection of tumor-specific gene expression mediated by PCR;

[0071] Figure 22 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP The results of detection that promote the expression of CXCL9 and αPD-L1scFv proteins in tumor cells are shown in the figure.

[0072] Figure 23 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP Figure showing the results of in vitro enhanced T cell infiltration and killing function detection;

[0073] Figure 24 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP Image showing the results of in vivo specific expression detection;

[0074] Figure 25 CLAN, a tumor-specific gene nanomedicine pTyr-C9AP The results of in vivo detection of melanoma growth inhibition, enhanced T cell infiltration and activation are shown in the figure.

[0075] Figure 26 CLAN, a tumor-specific gene nanomedicine pSur-C9AP The results of in vivo detection of various tumor growth inhibition, enhanced T cell infiltration and activation are shown in the figure.

[0076] Figure 27 This is a schematic diagram of the design scheme of the gene nanomedicine that enhances the anti-tumor immune effect of T cells according to the present invention. Detailed Implementation

[0077] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0078] Sources of raw materials used in the examples:

[0079] The polyester material was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0080] DOTAP was acquired from Aivito Pharmaceutical Technology Co., Ltd.

[0081] chloroform was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0082] B16-F10, CT26, Panc02, 4T1, C2C12, NIH-3T3, DC2.4, and RAW264.7 cells were purchased from ATCC;

[0083] CD8 sorting magnetic beads were purchased from Miltenyi Biotec.

[0084] BV510-anti-mouse CD45, APC anti-mouse CD3, FITC anti-mouse CD8, BV785 anti-mouse CD8a, and BV605 anti-mouse CD69 flow cytometry antibodies were purchased from Biolegend.

[0085] Anti-His tag antibody was purchased from Abcam.

[0086] Anti-βactin antibody was purchased from Biosharp.

[0087] The mouse CXCL9, CXCL10, and CXCL11 ELISA kits were purchased from Abcam.

[0088] DiI cell membrane dye was purchased from Meilun Biotechnology Co., Ltd.

[0089] Instrument models and manufacturers used in the examples:

[0090] Ultrasonic cell disruptor: Model VCX130, Sonics, USA;

[0091] Benchtop micro-refrigerated centrifuge: Model Microfuge 20R, Beckman Coulter, USA;

[0092] Nano particle size and Zeta potential meter: Model Nano ZSE, Malvern, UK;

[0093] Rotary evaporator: Model RV10 digital V, manufactured by IKA GmbH, Germany;

[0094] Transmission electron microscope: Model Talos L120C, Thermo Fisher Scientific, USA;

[0095] Analytical flow cytometer: Model FACSCelesta, BD Biosciences, USA;

[0096] Laser confocal microscope: Model LSM 880, ZEISS GmbH, Germany;

[0097] Real-time PCR instrument: LightCycler 96, Roche, USA;

[0098] Chemiluminescence imager: Model ChemiDoc MP, Bio-RAD Corporation, USA;

[0099] Microplate reader: Model 800TS, BioTek, USA.

[0100] Example 1, Different PEGs 5k -b-PLGA 11k Preparation and characterization of cationic lipid-assisted nanoparticles with varying mass fractions and cationic lipid contents, and the construction and characterization of nano libraries.

[0101] Formulating polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG) 5k -b-PLGA 11k Solutions of chloroform at concentrations of 25, 22.5, 20, and 15 mg / mL were used to prepare poly(glycolic acid-co-lactide) (PLGA) 11k A chloroform solution helps dissolve the polymer material and achieve more complete emulsification. The concentrations are 0, 2.5, 5, and 10 mg / mL. The two components are mixed to form PEG. 5k -b-PLGA 11k Polymer chloroform solutions with mass fractions of 100%, 90%, 80%, and 60% were prepared. Cationic lipid BHEM-Chol chloroform solutions with concentrations of 1, 1.5, 2, and 3 mg / mL were also prepared. 400 μL of each PEG was used. 5k -b-PLGA 11k A polymeric chloroform solution and 100 μL of different concentrations of BHEM-Chol chloroform solutions were added to a 50 mL centrifuge tube. 100 μg of a plasmid enhancing the anti-tumor immune effect of T cells was dissolved in 25 μL of aqueous solution and added to the centrifuge tube. Emulsification was performed using an ultrasonic cell disruptor; the ultrasonic power was 65 W, the amplitude was 30%, and the sonication time was 5 s with a 2 s pause, for a total sonication time of 1 min. Then, 5 mL of DEPC water was added, and sonication continued at 65 W, the amplitude was 30%, and the sonication time was 10 s with a 2 s pause, for a total sonication time of 2 min. After sonication, the emulsion was transferred to a 50 mL round-bottom flask and removed using a rotary evaporator. The vacuum levels were sequentially increased to 120, 100, 80, 60, and 40 mbar, and maintained for 2 min at each level to remove chloroform. Finally, the vacuum level was reduced to 25 mbar, and the mixture was concentrated to 1-2 mL in a 37°C water bath. The cationic-assisted nanoparticles were then collected for later use. We used this nanoparticle library to... Figure 1 Naming conventions.

[0102] The nanoparticles from Example 1 were used, and their hydration diameter was measured in a sample cell using a nanoparticle size analyzer and a Zeta potential analyzer. The corresponding nanoparticle size and Zeta potential distribution are shown in the figure below. Figure 2 As shown. PEG 5k -b-PLGA11k Changes in the mass fraction of the polymer have no significant effect on the particle size, and the particle potential is directly proportional to the content of cationic lipids.

[0103] B16-F10 melanoma cells were seeded into plates and transfected when the cell density reached approximately 70%-80%. The original culture medium was replaced with basal medium, and nanoparticles with different properties from a nanoparticle library containing 1 μg of plasmid were added. Six hours after transfection, the medium was changed to complete culture medium. Forty-eight hours after transfection, B16-F10 cells were digested and collected for flow cytometry analysis. The results are as follows: Figure 3 As shown, the difference in transfection efficiency mainly comes from the content of cationic lipids.

[0104] Example 2: Preparation and characterization of cationic lipid-assisted nanolibraries of different cationic lipid types

[0105] The above results show that cationic lipids have a significant impact on the cell transfection of nanoparticles. Next, a cationic lipid nanolibrary will be constructed using different cationic lipids. Following the preparation method in Example 1, 400 μL of PEG with a concentration of 62.5 mg / mL was added to a 50 mL centrifuge tube. 5k -b-PLGA 11k A chloroform solution, 100 μL of different cationic lipids with a concentration of 20 mg / mL, such as Figure 4 As shown, a chloroform solution containing BHEM-Chol, laboratory-synthesized cationic cholesterol derivatives CL3 and CL7, alkyl chain cationic lipids DOTAP, and laboratory-synthesized alkyl chain cationic lipids AL6 and AL7 was added, and 25 μL of a plasmid aqueous solution with a concentration of 4 μg / μL was added. A gene nanomedicine library containing different types of cationic lipids was constructed using an ultrasonic double emulsification method.

[0106] The above-mentioned particles were taken, and the hydration diameter of the nanoparticles was measured in the sample cell using a nanoparticle size analyzer and a zeta potential analyzer. The corresponding nanoparticle size and zeta potential distribution are shown in the figure below. Figure 5 As shown.

[0107] B16-F10 cells were seeded into plates and transfected when the cell density reached approximately 70%-80%. The original culture medium was replaced with basal medium, and nanoparticles containing different cationic lipid species from a nanoparticle library (1 μg plasmid) were added. After 6 hours of transfection, the medium was changed to complete medium. Cells were harvested for flow cytometry analysis 48 hours after transfection. Results are as follows: Figure 6 As shown, DOTAP cationic lipid-assisted nanoparticles exhibit the strongest plasmid delivery capability.

[0108] Example 3: Construction of CRISPRa gene activation plasmid for chemokine expression delivered by a non-viral vector

[0109] The inactivated endonuclease and transcription activator dCas9-VP64 element (Addgene, 61422) and the guide RNA MS2gRNA element (Addgene, 61424) containing the hairpin structure of the bacteriophage MS2 coat protein dimer, as described in the literature Konermann S, et al. Nature. 2015 Jan 29; 517(7536):583-8, were constructed into an expression plasmid. The transcriptional auxiliary activator bacteriophage MS2 coat protein, NF-κB transcription factor p65 protein, and heat shock regulator HSF1 element MS2-p65-HSF1 (Addgene, 61426) were constructed into another plasmid with the pX330 plasmid backbone (Addgene, 42230). This resulted in a simplified plasmid structure for gene activation dual-plasmid expression system for polymer vector delivery. The components required for constructing the gene activation plasmid were synthesized by Shanghai Sangon Biotech Co., Ltd. Using AflIII, KpnI, AgeI, EcoRI restriction endonucleases and T4 ligase, the dCas9-VP64 element and the MS2gRNA element were constructed into the same plasmid to obtain the pgCXCL-dCas9 plasmid. Figure 7 A. The main components of this expression plasmid include the Ori replication origin, U6 promoter, MS2gRNA expressed by activating chemokine ligand 9 (CXCL9), chemokine ligand 10 (CXCL10), and chemokine ligand 11 (CXCL11), the CBh promoter, dCas9-VP64, T2A, the reporter gene EGFP, and the ampicillin resistance gene. Using AflIII, KpnI, AgeI, EcoRI restriction endonucleases, and T4 ligase, the linker and MS2-p65-HSF1 used for structural simplification were constructed into another plasmid, resulting in the pMPH plasmid. Figure 7 B. The main components of this expression plasmid include the Ori replication origin, CBh promoter, MS2-p65-HSF1, T2A, the reporter gene tdTomato, and the ampicillin resistance gene. The MS2gRNA that can activate the expression of CXCL9, CXCL10, and CXCL11 was designed using the website https: / / zlab.bio / guide-design-resources. The pgCXCL-dCas9 plasmid and the pMPH plasmid together constitute the pCRISPRa gene activation system plasmid.

[0110] Example 4: Preparation and Morphology Characterization of Gene-Activated Nanomedicines

[0111] Following the preparation method in Example 1, 400 μl of a solution with a concentration of 62.5 mg / ml was added to a 50 ml centrifuge tube. -1 PEG 5k-b-PLGA 11k A 100 μl solution of chloroform contains 20 mg / mL of the solution. -1 BHEM-Chol cationic lipids were added, and 25 μl of the solution was added to a concentration of 4 μg / μl. -1 Gene-activated nanomedicine CLAN was prepared from pCRISPRa aqueous solution using an ultrasonic double emulsification method. pCRISPRa The prepared gene-activated nanomedicine was concentrated to 0.5 ml using rotary evaporation.

[0112] Take the prepared CLAN pCRISPRa In the sample cell, the nanoparticles were characterized using a nanoparticle size analyzer and a zeta potential analyzer. The hydration diameter of the nanoparticles was measured, and the corresponding nanoparticle size and zeta potential distribution are shown in the figure below. Figure 8 As shown in Figure A, this gene-activating drug is nanoscale in size, with a uniform particle size distribution and positive charge.

[0113] Take the prepared CLAN pCRISPRa The particles were diluted 50 times with ultrapure water and then ultrasonically dispersed. 10 μl of the particle solution was dropped onto a copper grid, left for 1 minute, and then the liquid was blotted out with filter paper. Next, 10 μl of 2% phosphotungstic acid solution was added to the copper grid, stained for 30 seconds, and then the liquid was blotted out. After thorough air drying, cryo-transmission electron microscopy was performed. Figure 8 As shown in B, this gene-activated nanomedicine is a nanoscale spherical structure with a polyethylene glycol (PEG) shell.

[0114] Example 5: In vitro activation of chemokine expression by gene-activated nanomedicines

[0115] In a 24-well plate, inoculate 1×10⁻⁶ cells per well. 5 Transfect the prepared gene-activated nanomedicine CLAN using B16-F10, Panc02, CT26, or 4T1 tumor cells until the cells reach 50% confluence. Discard the old culture medium in the 24-well plate and replace it with antibiotic-free basal medium. Administer the prepared CLAN gene-activated nanomedicine to 10×PBS. pCRISPRa Adjust the solution to isotonic, and add 500 ng of CLAN plasmid to each well. pCRISPRa After gently shaking, incubate in an incubator for 6 hours, then replace with fresh complete culture medium. After 72 hours, collect the culture supernatant and cells for subsequent testing.

[0116] mRNA level characterization: Collected cells were treated with RNAiso plus to extract total RNA. The total RNA was then reverse transcribed into cDNA using the PrimeScript RTreagent Kit with gDNA Eraser. The cDNA was then detected by qRT-PCR using the gene-activated nanomedicine CLAN. pCRISPRaActivate the mRNA expression levels of CXCL9, CXCL10, and CXCL11 in tumor cells. Figure 9 As shown, CLAN pCRISPRa It can efficiently activate the mRNA expression of B16-F10, Panc02, CT26, and 4T1 tumor cell chemokines CXCL9, CXCL10, and CXCL11.

[0117] Protein level characterization: After centrifuging the collected culture supernatant to remove cell debris, the expression levels of CXCL9, CXCL10, and CXCL11 in tumor cells activated by gene-activated nanomedicine were quantitatively detected using a mouse ELISA kit. Results are as follows: Figure 10 As shown, CLAN pCRISPRa It can efficiently activate the protein expression of chemokines in B16-F10, Panc02, CT26, and 4T1 tumor cells. However, the CXCL11 gene in B16-F10 and Panc02 cells derived from C57BL / 6 cells is mutated, therefore its protein level was not further analyzed.

[0118] Example 6: In vitro induction of T cell migration function by gene-activated nanomedicines

[0119] B16-F10, Panc02, CT26, or 4T1 tumor cells were seeded into 24-well plates at a rate of 1 × 10⁶ cells per well. 5 Transfect the cells when the cell confluence reaches 50%. Discard the old culture medium in the 24-well plate and replace it with antibiotic-free basal medium. Transfect the prepared gene-activating nanomedicine CLAN with 10×PBS. pCRISPRa Adjust the solution to isotonic, and add 500 ng of CLAN plasmid to each well. pCRISPRa After gently shaking, incubate in an incubator for 6 hours, then replace with fresh complete culture medium. Collect the culture supernatant after 72 hours for subsequent testing.

[0120] Primary CD3 cells from mice were obtained using magnetic beads. + T cells were then stimulated with αCD3 and αCD28 antibodies to activate CD3. + T cells were collected after 72 hours by centrifugation, yielding activated CD3+. + T cells were labeled with DiI fluorescent dye, and then washed with complete culture medium to remove residual DiI fluorescent dye and adhering debris from the cell surface.

[0121] Add 0.5 ml of transfected tumor culture supernatant to the lower chamber of the Transwell, and add 0.1 ml containing 2 × 10⁻⁶ cells / mL. 6 CD3 labeled with DiI fluorescent dye +T cell culture medium was added to the upper chamber of Transwell, and then CD3 was observed in real time over 3 hours using a high-content imaging system. + T cell migration. Results as follows: Figure 11 As shown, CLAN pCRISPRa Promoting the secretion of chemokines by B16-F10, Panc02, CT26, or 4T1 cells can effectively induce activated T cells to migrate from the upper chamber to the lower chamber of the Transwell.

[0122] Example 7: Gene-activated nanomedicine enhances T-cell infiltration and tumor growth inhibition in solid tumors in vivo.

[0123] To detect CLAN pCRISPRa To investigate the induction of T-cell infiltration and anti-tumor effects in various solid tumors, 10 C57BL / 6 mice implanted with B16-F10 melanoma, 20 C57BL / 6 mice implanted with Panc02 pancreatic cancer, 20 mice implanted with CT26 colorectal cancer, and 20 BALB / c mice implanted with 4T1 orthotopic breast cancer were randomly divided into two groups for each tumor model. Each group received an intratumoral injection of 50 μl of CLAN containing 5 μg of plasmid. pCRISPRa Alternatively, administer an equal volume of CLANNA solution every other day for a total of three doses. During treatment, measure the tumor size daily using calipers. The tumor volume is calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 After treatment, mouse tumor tissue was isolated, and flow cytometry was used to analyze the T cells infiltrating the tumor tissue. Figure 12 In various solid tumor models, CLAN pCRISPRa It significantly increased T cell infiltration and effectively inhibited tumor growth in mouse models.

[0124] Example 8: Design and preparation of tumor-specific gene-activating nanomedicines

[0125] To utilize intravenous administration, a more clinically friendly method of drug delivery than intratumoral injection, for the administration of the designed gene-activated nanomedicine, we labeled the previously prepared gene-activated nanomedicine with the fluorescent dye Rhodamine B. Then, 250 μl of the labeled drug was intravenously injected into a C57BL / 6 mouse model implanted with B16-F10 melanoma to evaluate whether the gene-activated nanomedicine could effectively reach the tumor site and exert its effect after tail vein injection. Figure 13 As shown, due to the high permeability and retention effect (EPR effect) of solid tumors, CLAN pCRISPRaIt can effectively accumulate at tumor sites. However, significant accumulation of the fluorescently labeled gene-activating drug is also observed in other normal organs, such as the liver. Further testing showed that CLAN... pCRISPRa It also activated the expression of chemokines in organs with significant fluorescent enrichment, such as the liver, leading to the accumulation of activated T cells in the liver. Therefore, we envision whether it is possible to develop a tumor-specific gene-activating nanomedicine that can act only at the tumor site.

[0126] Most current tumor-specific nanomedicines are prepared by conjugating ligands that can bind to tumor cell biomarkers to the drug. However, nanomedicines designed using this method may still be taken up by normal tissue cells after intravenous injection and release the drug loaded in the carrier, thus producing off-target effects.

[0127] To enable the designed gene-activated nanomedicines to achieve tumor-specific chemokine activation and expression, we digested the pgCXCL-dCas9 and pMPH plasmids with KpnI, AgeI restriction endonucleases, and T4 ligase in the constructed CRISPRa system expression plasmids. Then, we used T4 ligase to ligate the tumor-specific tyrosinase promoter into the digested plasmids, thus constructing the pgCXCL-Tyr-dCas9 and pTyr-MPH plasmids, respectively. Figure 14 As shown in A and 14B, these two plasmids together form the melanoma-specific pTyr-CRISPRa system.

[0128] Following the preparation method in Example 1, 400 μl of a solution with a concentration of 62.5 mg / ml was added to a 50 ml centrifuge tube. -1 PEG 5k -b-PLGA 11k A 100 μl solution of chloroform contains 20 mg / mL of the solution. -1 BHEM-Chol cationic lipids were added, and 25 μl of the solution was added to a concentration of 4 μg / μl. -1 CLAN, a melanoma-specific gene-activating nanomedicine, was prepared from an enzyme-free aqueous solution of pTyr-CRISPRa using an ultrasonic double emulsification method. pTyr-CRISPRa .

[0129] Example 9: In vivo tumor-specific detection of tumor-specific gene-activated nanomedicines

[0130] To test the in vivo specificity of the designed tumor-specific gene-activated nanomedicine, 250 μl of CLAN containing 25 μg of plasmid was used. pTyr-CRISPRaAlternatively, an equal volume of CLAN was intravenously injected into a C57BL / 6 mouse model implanted with B16-F10 melanoma. After 72 hours, tumor tissue, liver, lungs, spleen, kidneys, heart, skin, and eyes were isolated from the mice for subsequent testing. Figure 15 As shown, you can see CLAN pTyr-CRISPRa This approach specifically activates the expression of chemokines at the tumor site with minimal impact on other normal organs and tissues, and also significantly increases the enrichment and infiltration of T cells at the tumor site. This demonstrates that even when using the more clinically friendly intravenous administration method instead of intratumoral injection, this strategy can achieve precise tumor specificity, improve the safety of tumor immunotherapy, and shows promising clinical application prospects.

[0131] To verify CLAN pTyr-CRISPRa To investigate the antitumor effect, we used 20 C57BL / 6 mice implanted with B16-F10 melanoma and randomly divided them into two groups: CLAN... pTyr-CRISPRa Eleven mice in the control group and nine mice in the CLAN group were injected intravenously with 250 μl of CLAN containing 25 μg of plasmid. pTyr-CRISPRa Alternatively, CLAN can be administered every other day for a total of three doses. Throughout the treatment, tumor size is measured daily using calipers. After treatment, mouse tumor tissue is isolated, and flow cytometry is used to analyze the infiltrating T cells within the tumor tissue. Figure 16 As shown, CLAN pTyr-CRISPRa It significantly inhibited the growth of melanoma in mice and significantly increased T cell infiltration in mouse tumors after administration.

[0132] Example 10: Tumor-specific gene-activated nanomedicines enhance ICB and CAR-T cell therapy

[0133] To validate the tumor-specific gene-activating nanomedicine CLAN pTyr-CRISPRa To enhance the anti-tumor effect of immune checkpoint blockade antibodies, we administered 250 μl of CLAN antibody containing 25 μg of plasmid via tail vein injection. pTyr-CRISPRa 100 μg of αPD-L1 antibody or 50 μg of αCTLA-4 antibody was injected intraperitoneally into C57BL / 6 mouse models implanted with B16-F10 melanoma. The administration was repeated every other day for a total of three doses. Throughout the treatment, the tumor size was measured daily using calipers. Figure 17 As shown, CLAN pTyr-CRISPRa It significantly enhanced the antitumor efficacy of αPD-L1 antibody and αCTLA-4 antibody.

[0134] To validate the gene-activated nanomedicine CLAN pTyr-CRISPRaTo enhance the inhibitory effect of CAR-T cell therapy on solid tumors, we constructed a B16-F10-FAP subcutaneous melanoma mouse model. First, 1×10⁻⁶ cells were infused via the tail vein. 7 10 FAP5CAR-T cells were administered via intraperitoneal injection, along with 2×10 5 Units IL-2 and intravenously injected 250 μl of CLAN containing 25 μg plasmid. pTyr-CRISPRa The medication was administered every other day, for a total of three doses. Throughout the treatment, the tumor size was measured daily using calipers. The tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 .like Figure 18 A, CLAN pTyr-CRISPRa This significantly enhanced the anti-tumor efficacy of CAR-T cells. After treatment, mouse tumor tissue was isolated and fixed, paraffin sections were prepared, and immunofluorescence staining was performed. Confocal microscopy was used for imaging, such as... Figure 18 B, CLAN pTyr-CRISPRa It significantly enhanced the infiltration of CAR-T cells into solid tumors.

[0135] Example 11: Constructing a gene overexpression plasmid that drives the expression of chemokine 9 or anti-PD-L1 scFv using a tumor-specific promoter.

[0136] The αPD-L1 scFv sequence was obtained from patent US20100169296A1, the chemokine ligand 9 (CXCL9) cDNA sequence was obtained from the CCDS database (CCDS39152.1), and the tyrosinase promoter and surpromoter sequences were obtained from the literature Klüppel M, et al. Proc Natl Acad Sci US A. 1991 May 1; 88(9):3777-81 and Zhu ZB, et al. Cancer Gene Ther. 2004 Apr; 11(4):256-62, respectively. All of the above sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and a P2A cleavage peptide sequence was introduced between the αPD-L1 scFv sequence and the CXCL9 cDNA sequence, so that the αPD-L1 scFv protein and the CXCL9 protein can be expressed separately in cells.

[0137] Using molecular cloning technology, restriction endonucleases were used to digest the αPD-L1 scFv-P2A-CXCL9 sequence, the Tyr promoter sequence, the Sur promoter sequence, and the DNA expression vector pU57 plasmid backbone. First, the digested αPD-L1 scFv-P2A-CXCL9 fragment was inserted into the pU57 plasmid backbone. Then, the digested Tyr promoter fragment and Sur promoter fragment were inserted upstream of the αPD-L1 scFv-P2A-CXCL9 fragment, respectively, forming the pTyr-C9AP and pSur-C9AP plasmids. Figure 19 A,B, The main components of this expression plasmid include the Ori replication initiation site, the tyrosinase promoter Tyr or the survivin promoter Sur, the anti-PD-L1 antibody single-chain variable fragment (αPD-L1), the cleavage peptide P2A, the chemokine ligand 9 (CXCL9), and the ampicillin resistance gene Amp.

[0138] Example 12: Preparation, morphology, and stability characterization of tumor-specific gene nanomedicines

[0139] Following the preparation method in Example 1 and the screening results in Examples 1 and 2, 400 μL of PEG with a concentration of 62.5 mg / mL was added to a 50 mL centrifuge tube. 5k -b-PLGA 11k The tumor-specific gene nanomedicine CLAN was prepared by ultrasonic double emulsification using a chloroform solution, 100 μL of DOTAP cationic lipid at a concentration of 20 mg / mL, and 25 μL of pTyr-C9AP or pSur-C9AP aqueous solution at a concentration of 4 μg / μL. pTyr-C9AP or CLAN pSur-C9AP The prepared gene-activated nanomedicine was concentrated to 1 mL using rotary evaporation.

[0140] Take CLAN pTyr-C9AP As representative particles, the hydration diameter of the nanoparticles was measured in the sample cell using a nanoparticle size analyzer and a Zeta potential analyzer. The corresponding nanoparticle size and Zeta potential distribution are shown in the figure below. Figure 20 As shown in Figure A, this gene nanomedicine is nanoscale in size, with a uniform particle size distribution and positive charge.

[0141] Take CLAN pTyr-C9AP The particles were diluted 50 times with ultrapure water and then ultrasonically dispersed. 10 μL of the particle solution was added dropwise onto a copper grid, left for 1 minute, and then the liquid was aspirated. Next, 10 μL of 2% phosphotungstic acid solution was added to the copper grid, stained for 30 seconds, and then the liquid was aspirated. After thorough drying, cryo-transmission electron microscopy was performed. Figure 20 As shown in B, this gene nanomedicine is a nanoscale spherical structure with a PEG shell.

[0142] Take CLAN pTyr-C9AP The particles were added to a culture medium containing 10% fetal bovine serum (FBS) and incubated at 37°C. Using a nanoparticle size analyzer and a Zeta potential analyzer, samples were taken at 3, 12, 24, 48, 72, 96, and 120 h and added to a sample cell to determine the hydration diameter of the nanoparticles. The corresponding changes in nanoparticle size and PDI stability are shown in the figure. Figure 20 As shown in Figure C. The results show that this gene nanomedicine remains stable in serum-containing culture medium.

[0143] Example 13: In vitro specific expression of tumor-specific gene nanomedicines

[0144] To verify CLAN pTyr-C9AP To express tumor-specific genes in vitro, we seeded B16-F10, CT26, Panc02, 4T1, C2C12, NIH-3T3, DC2.4, RAW264.7 cells, or SkMC, T cells, BMDC, and BMDM cells into six-well plates. When the cell density reached approximately 50%-70%, we prepared for transfection. We adjusted the particles to isotonicity with 10×PBS, removed the original culture medium from the six-well plates, and replaced it with a medium containing CLANNAD. pTyr-C9AP or CLAN pSur-C9AP The basal medium was prepared with plasmid at a concentration of 2 μg / well. After 6 hours of incubation, the medium was replaced with Opti-MEM. After 72 hours, the supernatant and cells were collected for subsequent assays.

[0145] mRNA level characterization: Cells transfected with the particles were removed, the original culture medium was discarded, and residual culture medium was washed away with PBS. Total RNA was extracted using RNAiso plus, and the total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser. CLANNA was detected by qRT-PCR. pTyr-C9AP or CLAN pSur-C9AP The mRNA expression levels of αPD-L1scFv and CXCL9 in various cells after treatment. Figure 21 As shown in A, CLAN pTyr-C9AP It can specifically enhance the expression of αPD-L1 scFv and CXCL9 mRNA in B16-F10 cells, without affecting other tumor cells or other cell types, making it a melanoma-specific gene nanomedicine; Figure 21 As shown in B, CLAN pSur-C9AP It can significantly enhance the expression of αPD-L1scFv and CXCL9 mRNA in various tumor cells, while having almost no effect on other cell types, making it a universal tumor-specific gene nanomedicine.

[0146] Protein level characterization: (I) Detection of αPD-L1 scFv-6×His protein: Cell supernatant was collected after transfection, and purified after removing cell debris. Nickel beads were rinsed with pre-chilled 1×PBS, and the cell supernatant was added to the nickel beads. The mixture was incubated at 4℃ for 1 hour by rotational adsorption. After centrifugation to remove impurity proteins, the nickel beads were resuspended in 1×PBS and washed by centrifugation. 500 mM (pH 7.0) imidazole solution was added to fully elute the His-tagged protein from the nickel column, and the filtrate was collected by centrifugation. The filtrate was concentrated by ultrafiltration in an ultrafiltration tube with a molecular weight cutoff of 10 kDa to remove small-molecule imidazole from the solution, with a final concentration volume of 100 μL. The protein concentration was determined using the BCA protein quantification method, and protein denaturation was performed after adjusting the loading volume. The treated protein samples were electrophoresed using SDS-PAGE gels, and the expression of αPD-L1 scFv-6×His protein was detected by Western blot. (II) CXCL9 Detection: After collecting the supernatant from transfected cells and removing cell debris, the cells were centrifuged at 500g for 5 minutes at 4℃. The supernatant was then collected and stored on ice. The expression level of CXCL9 was quantified using the Abcam mouse CXCL9 ELISA kit. Results are as follows: Figure 22 As shown in A and B, CLAN pTyr-C9AP It can effectively enhance the protein expression levels of αPD-L1 scFv and CXCL9 in B16-F10 cells, thereby improving T cell infiltration and activation.

[0147] Example 14: Tumor-specific gene nanomedicines promote T cell migration and activation in vitro.

[0148] As described in Example 13, B16-F10 cells were seeded in six-well plates and transfected when the cell density reached approximately 50%-70%. The particles were adjusted to an isotonic solution with 10×PBS, and the original culture medium in the six-well plates was removed and replaced with a medium containing CLAN. pTyr-C9AP The basal medium was used, with plasmid dosage at 2 μg / well. After 6 hours of incubation, the medium was replaced with Opti-MEM, and the supernatant was collected after 72 hours for subsequent detection. Additionally, primary mouse CD8 cells were obtained using magnetic bead sorting. + T cells were then stimulated with αCD3 and αCD28 antibodies to activate CD8 cells. + T cells will be ready for use after 72 hours.

[0149] like Figure 23 A. B16-F10-OVA-EGFP cells were seeded in the lower chamber of a Transwell chamber and cultured overnight. The original culture medium was then replaced with 300 μL of transfected tumor cell culture supernatant. Activated CD8+ cells were then introduced... +Hoechst dye was added to T cells, and the cells were incubated at 37°C for 15 minutes. The cells were then washed three times with 1×PBS. One million dye-labeled CD8⁸ cells were collected. + T cells were added to the upper chamber of the Transwell for co-incubation. Confocal observation was performed after 18 hours of incubation. Figure 23 B, CLAN pTyr-C9AP It can increase T cell infiltration and promote the killing effect of T cells on tumor cells. Collect the supernatant from the lower chamber and centrifuge to collect CD8. + T cells were fluorescently labeled with FITC anti-mouse CD8α flow cytometry antibody. After labeling, the cells were washed twice with 1×PBS, and finally resuspended in 300 μL of pre-chilled 1×PBS. 10 μL of Precision Count Beads were added to the cell suspension, and after mixing, the cells were analyzed and counted by flow cytometry to determine the number of CD8α cells migrating to the lower chamber. + The number of T cells was determined. Tumor cells in the lower chamber were collected after re-digestion, labeled with PI, and analyzed by flow cytometry. Results are as follows: Figure 23 As shown in C and D, CLAN pTyr-C9AP It can simultaneously enhance the infiltration and activation of T cells.

[0150] Example 15: In vivo specific expression of tumor-specific gene nanomedicines

[0151] To verify CLAN pTyr-C9AP To investigate the expression of tumor-specific genes in vivo, we randomly divided 12 C57BL / 6 mice implanted with B16-F10 subcutaneous melanoma into two groups of 6 mice each. Each group received a tail vein injection of 400 μL of CLANNAD. Control or CLAN pTyr-C9AP The dosage was 1 mg plasmid / kg body weight, administered every two days for a total of three doses. After administration, the mice were sacrificed, and tumor tissue, as well as heart, liver, spleen, lung, and kidney tissues, were isolated and obtained for subsequent testing.

[0152] Tumors and normal organs from a subset of mice were isolated and used for flow cytometry analysis: 1 mg / mL collagenase IV, 100 μg / mL nuclease, and 100 μg / mL hyaluronidase were added to each tissue, and the mixture was incubated at 37°C with shaking for 20 min for digestion. After grinding, erythrocyte lysis, and filtration through a 200-mesh nylon mesh, the cells were dispersed into single cells and finally analyzed using an analytical flow cytometer. Results are as follows: Figure 24 As shown in A, CLAN pTyr-C9AP It specifically induces the expression of the pTyr-C9AP plasmid in tumor tissues, while it is almost not expressed in other normal tissues.

[0153] Another group of mouse tumors and normal organs were used for qRT-PCR detection: Each tissue was rapidly frozen in liquid nitrogen and then quickly ground. RNAiso was added to extract total RNA, which was then reverse transcribed into cDNA. The expression levels of αPD-L1scFv and CXCL9 mRNA in each tissue and organ were then detected by qRT-PCR. For example... Figure 24 B, CLAN pTyr-C9AP In the treated group of mice, there was a significant increase in the gene expression of αPD-L1 scFv and CXCL9 in tumors, which were almost undetectable in normal tissues. In summary, CLAN... pTyr-C9AP This is a tumor-specific gene nanomedicine that can specifically enhance the expression levels of αPD-L1 scFv and CXCL9 in vivo. This strategy can achieve precise tumor specificity, improve the safety of tumor immunotherapy, and has promising clinical application prospects.

[0154] Example 16: Tumor-specific gene nanomedicines enhance T cell infiltration and activation at the animal level, exerting anti-tumor effects.

[0155] To verify CLAN pTyr-C9AP To investigate the antitumor effect, we randomly divided 20 C57BL / 6 mice implanted with B16-F10 subcutaneous melanoma into two groups of 10 mice each. Each group received a tail vein injection of 400 μL of CLANNAD. Control or CLAN pTyr-C9AP The dosage was 1 mg plasmid / kg body weight, administered every two days for a total of 5 doses. Throughout the treatment, the tumor size was measured every two days using calipers. The tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 After treatment, the mouse tumor tissue was isolated and weighed, such as... Figure 25 A,CLAN pTyr-C9AP It significantly inhibited the growth of tumors in mice.

[0156] 1 mg / mL collagenase IV, 100 μg / mL nuclease, and 100 μg / mL hyaluronidase were added to each tissue, and the mixture was incubated at 37°C with shaking for 20 min for digestion. After grinding, erythrocyte lysis, and filtration through a 200-mesh nylon mesh, the cells were dispersed into single cells. αCD16 / 32 antibody was added to the tissue single cells, and the mixture was incubated at 4°C for 15 min for blocking. A mixture of flow cytometry antibodies (BV510 anti-mouse CD45, APC anti-mouse CD3, BV785 anti-mouse CD8, and BV605 anti-mouse CD69) was then added, and the mixture was incubated at 4°C for 1 h for labeling. After labeling, unlabeled antibodies were washed away with PBS. Finally, immunoassay was performed on the tumor tissue using analytical flow cytometry. Results are as follows: Figure 25 As shown in B, CLAN pTyr-C9AP It significantly enhanced the infiltration of T cells in tumors and increased the number of activated cytotoxic T cells.

[0157] To verify CLAN pSur-C9AP To assess its versatility in treating various solid tumors, we used 12 C57BL / 6 mice implanted with B16-F10 subcutaneous melanoma, 12 BALB / c mice with CT26 colorectal cancer, or 4T1 orthotopic breast cancer. Each tumor model was randomly divided into two groups of six mice each, and each group received a tail vein injection of 400 μL of CLANNAT. Control or CLAN pSur-C9AP The dosage was 1 mg plasmid / kg body weight, administered every two days for a total of 5 doses. Additionally, 12 C57BL / 6 mice implanted with Panc02 subcutaneous pancreatic cancer were used, grouped and given the same dosage, administered every four days for a total of 5 doses. Throughout the treatment, tumor size was measured every two days using calipers. Figure 26 A,CLAN pSur-C9AP It significantly inhibited the growth of various types of tumors.

[0158] After treatment, mouse tumor tissue was isolated and the infiltrating T cells in the tumor tissue were analyzed by flow cytometry. The results are as follows: Figure 26 As shown in B, CLAN pSur-C9AP It significantly enhanced the infiltration and activation of T cells in various tumors.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gene nanomedicine for enhancing the anti-tumor immune response of T cells, characterized in that: The invention includes the gene activation tool CRISPRa plasmid and cationic lipid-assisted polymer nanoparticles; wherein the promoter of the gene activation tool CRISPRa plasmid is a tumor-specific promoter, and the terminator of the gene activation tool CRISPRa plasmid is bGH polyA. The gene activation tool CRISPRa plasmid comprises two plasmids. One plasmid includes a guide RNA with a hairpin structure of phage MS2 coat protein dimer that activates chemokine ligands 9, 10, and 11, as well as a fusion expression plasmid of inactivated endonuclease dCas9 and transcription activator VP64. The other plasmid includes a fusion expression plasmid of transcriptional coactivator protein phage MS2 coat protein, NF-κB transcription factor p65 protein, and heat shock regulatory protein HSF1 protein. The cationic lipid in the cationic lipid-assisted polymer nanoparticles is at least one of N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteroloxycarbonylaminoethyl)ammonium bromide, laboratory-synthesized cationic cholesterol derivative CL3, laboratory-synthesized cationic cholesterol derivative CL7, trimethyl-2,3-dioleoyloxypropylammonium bromide, and laboratory-synthesized alkyl chain cationic lipid AL7. The structural formula of the laboratory-synthesized cationic cholesterol derivative CL3 is as follows: ； The structural formula of the laboratory-synthesized cationic cholesterol derivative CL7 is as follows: ； The structural formula of the laboratory-synthesized alkyl chain cationic lipid AL7 is as follows: ； The polymers in the cationic lipid-assisted polymer nanoparticles are polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide), with the polyethylene glycol-modified poly(glycolic acid-co-lactide) accounting for 60%-100% of the total polymer. The average particle size of the cationic lipid-assisted polymer nanoparticles is 50 nm-200 nm.

2. The gene nanomedicine for enhancing the anti-tumor immune effect of T cells according to claim 1, characterized in that: The tumor-specific promoter is at least one of tyrosinase promoter and survivin promoter.

3. A method for preparing a gene nanomedicine for enhancing the anti-tumor immune effect of T cells as described in claim 1 or 2, characterized in that: Includes the following steps: Step 1: Mix the gene activation tool CRISPRa plasmid with an aqueous solution to obtain an aqueous phase; wherein the concentration of the gene activation tool CRISPRa plasmid in the aqueous phase is 1-4 mg / ml; Lipids, polyethylene glycol-modified poly(glycolic acid-co-lactide), and poly(glycolic acid-co-lactide) are mixed with chloroform organic solvent to obtain an oil phase; wherein the concentration of the polyethylene glycol-modified poly(glycolic acid-co-lactide) or the poly(glycolic acid-co-lactide) in the oil phase is 62.5 mg / ml, and the concentration of the lipids in the oil phase is 10-30 mg / ml; Step 2: Prepare an oil-in-water emulsion from the oil phase and the aqueous phase; Step 3: Concentrate the emulsion to obtain gene nanomedicine.

4. The method for preparing gene nanomedicine for enhancing the anti-tumor immune effect of T cells according to claim 3, characterized in that: The method for preparing an oil-in-water emulsion from an oil phase and an aqueous phase is at least one of ultrasonic emulsification, microfluidics, or microchannel reaction; the method for concentrating the emulsion is at least one of rotary evaporation or freeze drying.

5. A gene nanomedicine for enhancing the anti-tumor immune response of T cells, characterized in that: The invention comprises a gene overexpression plasmid and cationic lipid-assisted polymer nanoparticles; wherein the promoter of the gene overexpression plasmid is a tumor-specific promoter, and the terminator of the gene overexpression plasmid is bGH polyA. The gene overexpression plasmid sequence is a co-expression plasmid formed by linking a single-chain variable region fragment of chemokine ligand 9 and anti-programmed cell death-ligand 1 via a P2A cleavage peptide. The cationic lipid in the cationic lipid-assisted polymer nanoparticles is at least one of N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteroloxycarbonylaminoethyl)ammonium bromide, laboratory-synthesized cationic cholesterol derivative CL3, laboratory-synthesized cationic cholesterol derivative CL7, trimethyl-2,3-dioleoyloxypropylammonium bromide, and laboratory-synthesized alkyl chain cationic lipid AL7. The structural formula of the laboratory-synthesized cationic cholesterol derivative CL3 is as follows: ； The structural formula of the laboratory-synthesized cationic cholesterol derivative CL7 is as follows: ； The structural formula of the laboratory-synthesized alkyl chain cationic lipid AL7 is as follows: ； The polymers in the cationic lipid-assisted polymer nanoparticles are polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide), with the polyethylene glycol-modified poly(glycolic acid-co-lactide) accounting for 60%-100% of the total polymer. The average particle size of the cationic lipid-assisted polymer nanoparticles is 50 nm-200 nm.

6. The gene nanomedicine for enhancing the anti-tumor immune effect of T cells according to claim 5, characterized in that: The tumor-specific promoter is at least one of tyrosinase promoter and survivin promoter.

7. A method for preparing a gene nanomedicine for enhancing the anti-tumor immune effect of T cells as described in claim 5 or 6, characterized in that: Includes the following steps: Step 1: Mix the gene overexpression plasmid with an aqueous solution to obtain an aqueous phase; wherein the concentration of the gene overexpression plasmid in the aqueous phase is 1-4 mg / ml; Lipids, polyethylene glycol-modified poly(glycolic acid-co-lactide), and poly(glycolic acid-co-lactide) are mixed with chloroform organic solvent to obtain an oil phase; wherein the concentration of the polyethylene glycol-modified poly(glycolic acid-co-lactide) or the poly(glycolic acid-co-lactide) in the oil phase is 62.5 mg / ml, and the concentration of the lipids in the oil phase is 10-30 mg / ml; Step 2: Prepare an oil-in-water emulsion from the oil phase and the aqueous phase; Step 3: Concentrate the emulsion to obtain gene nanomedicine.

8. The method for preparing gene nanomedicine for enhancing the anti-tumor immune effect of T cells according to claim 7, characterized in that: The method for preparing an oil-in-water emulsion from an oil phase and an aqueous phase is at least one of ultrasonic emulsification, microfluidics, or microchannel reaction; the method for concentrating the emulsion is at least one of rotary evaporation or freeze drying.

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