Gene modification carrying immune enhancement element and application thereof

Abstract

The invention discloses gene modification carrying an immunological enhancement element and application of the gene modification, and relates to the technical field of biological medicine and gene engineering. B, at least one coding nucleic acid sequence IE of an immunological enhancement element, wherein the immunological enhancement element is used for enhancing antigen presentation, immunological co-stimulation signal transmission or positive regulation of a local immune microenvironment; c, at least one of a signal peptide segment SP and a membrane anchoring localization domain TM; the d expression regulation unit comprises a promoter Pr for driving the expression of Ag and IE and a transcription termination signal pA; the e-site strategy related sequence organization comprises a homologous segment left side HS-L and a homologous segment right side HS-R; wherein the Ag and the IE are assembled in the same expression framework, the IE is located at one of the upstream and the downstream of the Ag, antigen presentation and costimulatory signals are synchronous and stable, the immune response intensity and durability are improved, and inter-individual and inter-batch fluctuation is reduced.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and genetic engineering, specifically to a gene modification carrying immune-enhancing elements and its application. Background Technology

[0002] The core of tumor immunotherapy lies in inducing and maintaining an effective specific immune response. Current technologies, through strategies combining antigen expression constructs with immune adjuvants, can enhance antigen presentation and cytotoxic responses, but there is still room for improvement in balancing safety and response intensity, as well as in the synergistic activation of multiple pathways. Publicly available data on therapeutic gene vaccines show that employing the juxtaposition of tumor-associated antigens and immune co-units, membrane anchoring, and enhanced antigen presentation can yield better immune response phenotypes and safety trends in animal models.

[0003] On the other hand, ideas for traceless genetic modification of the host genome have been published, including using homologous sequences at specific sites in combination with site-specific endonucleases to achieve precise replacement or insertion of target fragments, thereby reducing the risk of interference from residual markers to subsequent multi-site modifications.

[0004] Furthermore, gene editing practices at the immune cell level have shown that the removal of negative regulatory receptors and the rational assembly of functional units can help enhance anti-tumor effects. Published texts emphasize the need for in vitro and in vivo functional evaluation after obtaining specific immune cell populations. Therefore, designing a gene-modified organism carrying immune-enhancing elements and its application is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a gene modification carrying immune-enhancing elements and its application. By configuring one or more types of immune-enhancing elements in the target gene construct and achieving robust integration and expression control through a verifiable site strategy, the invention can improve antigen presentation efficiency and cellular immune response intensity without reducing safety, thereby expanding its application value in anti-tumor and immune regulation and solving the problems mentioned in the background art.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a gene modification carrying an immune-enhancing element, wherein the gene modification comprises... The coding nucleic acid sequence Ag for the a antigen unit; b. A nucleic acid sequence encoding at least one immune-enhancing element IE, wherein the immune-enhancing element is used to enhance antigen presentation, immune co-stimulation signal transmission, or positive regulation of the local immune microenvironment; At least one of the c signal peptide region SP and the membrane anchoring domain TM; The expression regulatory unit includes the promoter Pr, which drives Ag and IE expression, and the transcription termination signal pA; e-site strategy-related sequence organization, including the left HS-L of the homologous segment and the right HS-R of the homologous segment; In this process, Ag and IE are assembled within the same expression frame, with IE located upstream or downstream of Ag. HS-L and HS-R are located on either side of the Ag–IE expression frame for insertion or replacement at predetermined genomic locations, and no coding sequence for permanent selection markers is retained after the modification is completed.

[0007] According to the above technical solution, the IE is selected from at least one of the following categories: a co-stimulatory ligand or its domain; b cytokines or cytokine functional fragments; c. Membrane localization and delivery enhancement unit; d antigen dimerization or oligomerization enhancement unit; e is a functional unit associated with immune metabolism or immune-related transcriptional regulation; The assembly order of Ag and IE is as follows: One of the SP–Ag–linking units Lk–IE–TM, or one of the SP–IE–Lk–Ag–TM, where Lk is an amino acid linking unit; The connecting unit Lk is selected from at least one of flexible linking peptides and peptide chain self-cleaving sequences; The membrane anchor positioning domain TM is selected from at least one of the transmembrane structural domain and the glycosylphosphatidylinositol anchor positioning signal; The immune costimulatory ligand or its domain is selected from at least one of the extracellular domains, intracellular signal transduction domains, and fusion forms of the costimulatory receptor family. The cytokine or its functional fragment is selected from at least one member of the cytokine family that stimulates dendritic cell maturation, promotes T cell proliferation, or promotes NK cell activation. The antigen dimerization or oligomerization enhancing unit is selected from at least one of the oligomerization-promoting multimerization domains and immunoglobulin Fc-associated domains.

[0008] According to the above technical solution, Ag is a nucleic acid sequence encoding a tumor-associated antigen or its immune epitope fragment, or a nucleic acid sequence encoding a pathogen-associated antigen or its immune epitope fragment. The expression framework further includes an internal ribosome entry site (IRES) or a peptide self-cleavage sequence, for expressing Ag and IE respectively on the same transcript.

[0009] According to the above technical solution, the expression regulation unit further includes at least one expression enhancement element, which is selected from at least one of intron enhancers, enhancer elements and splice acceptor or splice donor sites. The HS-L and HS-R in the site strategy related sequence organization are homologous to the upstream and downstream sequences of the predetermined genomic site, and are used to guide the insertion or replacement of the Ag-IE expression framework at the predetermined site.

[0010] According to the above technical solution, the genetically modified vector has eukaryotic expression capability. The vector is selected from at least one of eukaryotic expression plasmids, adeno-associated virus vectors, and lentiviral vectors. It also includes genetically modified cells capable of expressing Ag and IE. The cells are immune cells selected from at least one of T cells, natural killer cells, dendritic cells, and macrophages. The immune cells further include editing of the inhibitory receptor pathway. The editing is selected from at least one of knockout of the inhibitory receptor coding gene and loss-of-function modification of key sites in inhibitory receptor signal transduction.

[0011] An application of genetically modified organisms carrying immune-enhancing elements, including S1. Design the encoding sequences for Ag and IE and determine the assembly order; S2. Based on the site strategy, HS-L and HS-R are determined and an Ag–IE expression framework is formed; S3. Introduce the gene construct consisting of the Ag-IE expression framework and HS-L and HS-R into recipient cells; S4. The insertion or replacement of the gene construct is achieved at a predetermined genomic location through site-specific endonuclease-mediated homology-directed repair. S5. Obtain the modified cells and confirm the expression and localization of Ag and IE; The modified sequence does not contain a permanent selection marker encoding sequence.

[0012] According to the above technical solution, the site-specific endonuclease is selected from at least one of CRISPR-related endonucleases, zinc finger endonucleases, and transcription activator-like effector endonucleases. The predetermined genomic location is a safe site for exogenous expression. The safe site does not overlap with the coding region of the cell's intrinsic key physiological function. The introduction method of S3 is selected from at least one of electroporation, viral vector transduction, and non-viral vector transfection. The confirmation of S5 includes at least one of the confirmation of the existence of Ag membrane localization and the confirmation of the existence of signal activation of the IE action pathway.

[0013] According to the above technical solution, it also includes a reagent kit, the reagent kit containing... a. A vector carrying the gene construct as described in any one of claims 1 to 12; b. Primer pairs corresponding to HS-L and HS-R; c. Nucleic acid editing components compatible with site-specific endonuclease systems; It is accompanied by written instructions for performing insertion or replacement operations at predetermined genomic locations; The immune-enhancing element (IE) comprises two or more elements, arranged in a parallel or sequential expression manner, wherein the parallel expression is achieved by IRES or peptide self-cleavage sequences respectively, and the sequential expression is achieved by the same open reading frame via a linker unit. The expression frame further includes a signal sequence for subcellular localization regulation, the signal sequence being selected from at least one of secretion signals, endoplasmic reticulum retention signals, and endosome localization signals. Ag contains at least one validated immune epitope fragment and is presented in a multi-epitope splicing manner, the multi-epitopes being separated by linker units Lk. Cells are expanded in vitro to form a cell preparation, the cell preparation being formulated with the pharmaceutical composition of claim 18 for subsequent use, further comprising: S6. Set up the second set of HS-L and HS-R for the second predetermined genomic location, and complete the insertion or replacement of the second Ag–IE expression frame; S7. Without changing the existing Ag-IE expression framework functionality, complete multi-site co-assembly; where, after S6 and S7, each site does not retain the encoding sequence of the permanent selection marker.

[0014] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: Antigen presentation and co-stimulatory signals are synchronized and stable, resulting in enhanced immune response strength and persistence, and reduced inter-individual and batch-to-batch fluctuations. Modular combination enables synergistic enhancement of multiple pathways (presentation, co-stimulation, and microenvironment regulation), allowing rapid adaptation to different indications and population heterogeneity under the same framework, thus improving coverage. Pre-defined site integration leads to long-term, predictable expression, significantly reducing the risks of random integration and insertion, and improving reproducibility and consistency. The entire process is free of permanent selection markers, reducing non-target background and potential immunogenicity, providing a clean background for subsequent multiple rounds of modification and long-term follow-up. Multi-site synergistic assembly does not interfere with each other, enabling superimposed strategies such as "presentation enhancement plus co-stimulation plus microenvironment regulation" to produce additive or synergistic effects. Better functional phenotypes (activation, cytotoxicity tendency, memory tendency) are obtained across multiple immune cell platforms, and the effect can be further enhanced through inhibitory pathway editing. A clear path for confirming presence and localization trends facilitates early identification of structural or localization abnormalities, forming a risk mitigation loop of quality-by-dosage (QbD). Standardized carrier interfaces and cascaded slots reduce the workload of reconstruction, accelerate version iteration and verification, and make it easier to establish unified standards for manufacturing and release. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Overall structure block diagram of the building block; Figure 2 Ag / IE assembly strategies and expression methods are expressed separately and sequentially in diagrams; Figure 3 A diagram showing the relative relationship between HS-L / HS-R and predetermined sites; Figure 4 Logic and shielding zone diagram for selecting safe sites; Figure 5 A hierarchical organization diagram for multi-site collaborative assembly; Figure 6 The vector implementation and modular multiple cloning site layout diagram; Figure 7 This is a flow chart of the receptor cell. Figure 8 This is an indicative readout of the signaling pathway's operational trends and phenotypic dimensions. Detailed Implementation

[0016] 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.

[0017] Please see Figures 1-8 This invention provides a technical solution: a gene modification carrying an immune-enhancing element and its application, wherein the gene modification includes... The coding nucleic acid sequence Ag for the a antigen unit; b. At least one immune-enhancing element encoding a nucleic acid sequence IE, wherein the immune-enhancing element is used to enhance antigen presentation, immune co-stimulation signal transduction, or positive regulation of the local immune microenvironment; At least one of the c signal peptide region SP and the membrane anchoring domain TM; The expression regulatory unit includes the promoter Pr, which drives Ag and IE expression, and the transcription termination signal pA; e-site strategy-related sequence organization, including the left HS-L of the homologous segment and the right HS-R of the homologous segment; In this process, Ag and IE are assembled within the same expression frame, with IE located upstream or downstream of Ag. HS-L and HS-R are located on either side of the Ag–IE expression frame for insertion or replacement at predetermined genomic locations, and no coding sequence for permanent selection markers is retained after the modification is completed. IE is selected from at least one of the following categories: a co-stimulatory ligand or its domain; b cytokines or cytokine functional fragments; c. Membrane localization and delivery enhancement unit; d antigen dimerization or oligomerization enhancement unit; e is a functional unit associated with immune metabolism or immune-related transcriptional regulation; The assembly order of Ag and IE is as follows: One of the SP–Ag–linking units Lk–IE–TM, or one of the SP–IE–Lk–Ag–TM, where Lk is an amino acid linking unit; The linker unit Lk is selected from at least one of flexible linker peptides and peptide chain self-cleaving sequences; The membrane anchoring domain TM is selected from at least one of the transmembrane domain and the glycosylphosphatidylinositol anchoring signal; The immune costimulatory ligand or its domain is selected from at least one of the extracellular domains, intracellular signal transduction domains, and fusion forms of the costimulatory receptor family; Cytokines or functional fragments thereof are selected from at least one member of the cytokine family that stimulates dendritic cell maturation, promotes T cell proliferation, or promotes NK cell activation; The antigen dimerization or oligomerization enhancing unit is selected from at least one of the oligomerization-promoting multimerization domain and the immunoglobulin Fc-associated domain; Ag is a nucleic acid sequence encoding a tumor-associated antigen or an immune epitope fragment thereof, or a nucleic acid sequence encoding a pathogen-associated antigen or an immune epitope fragment thereof. The expression framework further includes an internal ribosome entry site (IRES) or a peptide self-cleavage sequence for expressing Ag and IE separately on the same transcript. The expression regulation unit further includes at least one expression enhancement element, which is selected from at least one of intron enhancers, enhancer elements and splice acceptor or splice donor sites. The HS-L and HS-R in the site strategy related sequence organization are homologous to the upstream and downstream sequences of the predetermined genomic site, respectively, and are used to guide the insertion or substitution of the Ag–IE expression framework at the predetermined site. The genetically modified vector has eukaryotic expression capability. The vector is selected from at least one of eukaryotic expression plasmids, adeno-associated virus vectors, and lentiviral vectors. It also includes genetically modified cells that can express Ag and IE. The cells are immune cells selected from at least one of T cells, natural killer cells, dendritic cells, and macrophages. The immune cells further include editing of the inhibitory receptor pathway. The editing is selected from at least one of knockout of the inhibitory receptor coding gene and loss-of-function modification of key sites of inhibitory receptor signal transduction. An application of genetically modified organisms carrying immune-enhancing elements, including S1. Design the encoding sequences for Ag and IE and determine the assembly order; S2. Based on the site strategy, HS-L and HS-R are determined and an Ag–IE expression framework is formed; S3. Introduce the gene construct consisting of the Ag-IE expression framework and HS-L and HS-R into recipient cells; S4. Homologous targeted repair mediated by site-specific endonucleases enables the insertion or replacement of gene constructs at predetermined genomic locations; S5. Obtain the modified cells and confirm the expression and localization of Ag and IE; The modified sequence does not contain a permanent selection marker. The site-specific endonuclease is selected from at least one of CRISPR-associated endonucleases, zinc finger endonucleases, and transcription activator-like effector endonucleases. The predetermined genomic location is a safe site for exogenous expression. The safe site does not overlap with the coding region of the cell's intrinsic key physiological function. The introduction method of S3 is selected from at least one of electroporation, viral vector transduction, and non-viral vector transfection. The confirmation of S5 includes at least one of the confirmation of the existence of membrane localization of Ag and the confirmation of the existence of signal activation of the IE action pathway. It also includes a kit, which contains a. A vector carrying any one of the gene constructs from rights 1 to 12; b. Primer pairs corresponding to HS-L and HS-R; c. Nucleic acid editing components compatible with site-specific endonuclease systems; It is accompanied by written instructions for performing insertion or replacement operations at predetermined genomic locations; The immune-enhancing element (IE) comprises two or more elements, arranged in a parallel or sequential expression manner. The parallel expression is achieved by IRES or peptide self-cleavage sequences, respectively, while the sequential expression is achieved by the same open reading frame via a linker unit. The expression frame further includes a signal sequence for subcellular localization regulation, the signal sequence being selected from at least one of secretory signals, endoplasmic reticulum retention signals, and endosome localization signals. The Ag element contains at least one validated immune epitope fragment and is presented in a multi-epitope splicing manner, with linker units Lk separating the multi-epitopes. Cells are expanded in vitro to form a cell preparation, which is then formulated with the drug composition of claim 18 for subsequent applications, further comprising: S6. Set up the second set of HS-L and HS-R for the second predetermined genomic location, and complete the insertion or replacement of the second Ag–IE expression frame; S7. Without changing the existing Ag-IE expression framework functionality, complete multi-site co-assembly; where, after S6 and S7, each site does not retain the encoding sequence of the permanent selection marker.

[0018] Example 1: Constructing the overall architecture and module mapping, aiming to build an engineered combination of Ag and IE within the same expression framework, and to provide homologous organization for site-level insertion / replacement. The overall structure is as follows: Figure 1 The expression framework (EF) consists of a promoter, open reading frame (ORF), linker units, optional localization / anchoring signals, and transcription termination signals in sequence. Ag and IE are organized within the EF using either of the following strategies: separate expression: two translation units are formed on the same transcript using IRES or self-cleaving peptide sequences; sequential expression: linked by a linker peptide Lk within a single open reading frame. Localization and presentation: SPs can be placed within the EF to support secretion trends, or TMs can be placed to support membrane presentation trends; combinations can also be used to accommodate different scenarios. Site strategy interfaces: HS-L and HS-R are placed on both sides of the EF for pairing with upstream and downstream sequences at predetermined sites to form a structural docking interface. Figure 3 The beneficial effect is that the structural coupling of antigen presentation and immune synergy is achieved in a single EF, leaving room for subsequent site-level stable expression and multiple modifications.

[0019] This paper discusses the selection and assembly of immune-enhancing elements (IEs), aiming to provide optional IE types and Ag assembly strategies to ensure that the types listed in the claims have a practical implementation path. The IE type library allows for the selection or arbitrary combination of the following types: co-stimulatory ligand-related domains can be extracellular, intracellular, or fused; cytokines or functional fragments that tend to promote DC maturation, T cell expansion, or NK activation; membrane localization and presentation enhancement units that complement the configuration of TMs; dimerization / oligomerization-promoting domains, including domains with a tendency to polymerize; and immune metabolism / transcriptional regulation-related functional units. Assembly methods include the relative position of Ag and IE: IEs can be located upstream or downstream of Ag, or tandemly linked with Ag via Lk in a single open reading frame; expression methods: expression of IRES / self-cleaving peptides or sequential expression of a single ORF; subcellular localization: endoplasmic reticulum retention or endosome localization signals can be added to guide the subcellular distribution trend of Ag or IE. Figure 2 The beneficial effects achieve a structured combination of presentation intensity, signal link, and microenvironment regulation, taking into account both scalability and customizability.

[0020] Site Strategy and Safe Site Selection: The aim is to provide predetermined sites and homologous tissues for stable exogenous expression of EF without altering key cellular functions. Key Points: HS-L / HS-R sequences must be homologous to the upstream and downstream sequences of the predetermined site; the sequences must be compatible with site-specific endonuclease target recognition regions to facilitate insertion or replacement via homology guidance. Figure 3 The selection of safe sites follows the principles of "exogenous expression friendly, far from key coding regions, and reusable". Figure 4 Label-free expression avoids permanent selection marker residues through process and structural organization, retaining only the regulatory elements necessary for expression. Beneficial effects include facilitating subsequent multi-site stacking and long-term stable expression, reducing the risk of perturbation to core host physiological processes.

[0021] Vector Implementation and Modular Organization: The aim is to provide a vector implementation path for carrying expression vectors (EF) and to reserve structural interfaces for multi-site extensions. Vector types can include eukaryotic expression plasmids, adeno-associated virus vectors, or lentiviral vectors. Figure 6 The startup subregion, MCS, and control region are presented in a modular format. The modularly organized MCS reserves multiple pairs of restrictive interfaces or synonymous recombination interfaces for easy replacement of Ag, IE, or Lk. "Multi-site cascade slots" are set at the boundary between the vector skeleton V and EF for subsequent access to a second expression framework. Figure 5 Echoing. Beneficial effects include enabling component-level reuse and rapid refactoring through standardized interfaces.

[0022] Recipient cell acquisition and editing scenario: This section aims to illustrate the type of carrier cell, confirm its presence and trend localization after introduction, and demonstrate optional editing of inhibitory receptor pathways. Cell type: Recipient cell C can be T cells, natural killer cells, dendritic cells, or macrophages. Editing scenario: After EF introduction, optional gene-level editing of inhibitory receptor pathways can be performed, including knockout of inhibitory receptor coding genes or loss-of-function modification of key signal transduction sites. Presence / localization confirmation: This aims to confirm the membrane localization trend of Ag and the activation trend of the IE pathway, focusing on "structural in situ and trend localization." Figure 7 , Figure 8 This process does not involve any operational conditions or readout thresholds. The trend towards formulation: Cells can be expanded in vitro into cell preparations for subsequent research and evaluation.

[0023] Example 2: Gene Modification Method Flowchart: Objective To provide a closed-loop flowchart (S1–S5) of claim 6, strengthening the structural / flowchart consistency with claims 1–5. Flowchart S1: Design the coding sequences of Ag and IE and determine their assembly relationship within the EF for separate or sequential expression, and whether they contain SP / TM / localization signals; S2: Determine the homology relationship between HS-L and HS-R and predetermined sites, and assemble them with the EF to form a construct; S3: Introduce the construct into recipient cells using at least one of electroporation, viral vector transduction, or non-viral vector transfection; S4: Under the mediation of site-specific endonucleases, achieve insertion or replacement at predetermined sites through homology-directed repair; S5: Obtain modified cells and confirm the "existence and localization trend"; Boundary conditions: The predetermined site is a safe site; No permanent selection marker coding sequence is retained after the process. Beneficial effects: Forming a closed loop from "design—introduction—in situ—trend confirmation" supports subsequent expansion.

[0024] Multi-site expansion and co-assembly: The goal is to achieve marker-free superposition of a second expression framework without degradation of the existing expression frame. The expansion process S6 sets up a second set of HS-L / HS-R for the second predetermined site and completes the insertion or replacement of the second Ag-IE expression framework; S7 evaluates the hierarchical and trend levels of the expression independence relationships among multiple sites and confirms that no permanent selection marker coding sequences remain at either site. Figure 5 Organizational relationships: Two or more immune enhancers (EFs) can be arranged in parallel through cascaded slots in the vector backbone or different "safe sites" in the genome. Beneficial effects support the synergistic deployment of multiple immune enhancement strategies within the same receptor system.

[0025] The kit and accompanying information elements aim to modularize the key components and information elements of execution rights 6 / 7 for standardized delivery. The interface information between the vector backbone comprising any construct in rights 1–3 and the EF is explicitly stated; primer pairs matching HS-L / HS-R are used for structural-level existence verification; nucleic acid editing components compatible with the selected site-specific endonuclease system are only labeled for system compatibility, not for ratios; written descriptions include: the correspondence between the predetermined site and HS-L / HS-R, the assembly options of EF (sequential / sequential expression), the selection of SP / TM / location signals, the structural boundary description for multi-site co-assembly, and the principle of not retaining permanent selection markers after modification. The beneficial effect is that information is solidified in a "structure-process-verification point" manner, shortening the structure confirmation path.

[0026] This invention achieves synchronous and stable antigen presentation and co-stimulatory signals, enhancing the strength and persistence of the immune response while reducing inter-individual and batch-to-batch variability. Modular combination enables synergistic enhancement of multiple pathways (presentation, co-stimulation, and microenvironment regulation), allowing for rapid adaptation to different indications and population heterogeneity within the same framework, thus improving coverage. Pre-defined site integration leads to long-term, predictable expression, significantly reducing the risks of random integration and insertion, and improving reproducibility and consistency. The entire process is marker-free, reducing non-target background and potential immunogenicity, providing a clean background for subsequent multiple rounds of modification and long-term follow-up. Multi-site synergistic assembly operates without interference, enabling synergistic strategies such as "presentation enhancement plus co-stimulation plus microenvironment regulation," producing additive or synergistic effects. Better functional phenotypes (activation, cytotoxicity tendency, memory tendency) are obtained across multiple immune cell platforms, and the effect can be further enhanced through inhibitory pathway editing. A clearly defined path for confirming presence and localization trends facilitates early identification of structural or localization anomalies, forming a quality-by-dosage (QbD) risk mitigation loop. Standardized carrier interfaces and cascaded slots reduce the workload of reconstruction, accelerate version iteration and verification, and make it easier to establish unified standards for manufacturing and release.

[0027] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0028] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 genetically modified organism carrying an immune-enhancing element, characterized in that: The genetic modification includes The coding nucleic acid sequence Ag for the a antigen unit; b. A nucleic acid sequence encoding at least one immune-enhancing element IE, wherein the immune-enhancing element is used to enhance antigen presentation, immune co-stimulation signal transmission, or positive regulation of the local immune microenvironment; At least one of the c signal peptide region SP and the membrane anchoring domain TM; The expression regulatory unit includes the promoter Pr, which drives Ag and IE expression, and the transcription termination signal pA; e-site strategy-related sequence organization, including the left HS-L of the homologous segment and the right HS-R of the homologous segment; In this process, Ag and IE are assembled within the same expression frame, with IE located upstream or downstream of Ag. HS-L and HS-R are located on either side of the Ag–IE expression frame for insertion or replacement at predetermined genomic locations, and no coding sequence for permanent selection markers is retained after the modification is completed.

2. The genetically modified organism carrying an immune-enhancing element according to claim 1, characterized in that: The IE is selected from at least one of the following categories: a co-stimulatory ligand or its domain; b cytokines or cytokine functional fragments; c. Membrane localization and delivery enhancement unit; d antigen dimerization or oligomerization enhancement unit; e is a functional unit associated with immune metabolism or immune-related transcriptional regulation; The assembly order of Ag and IE is as follows: One of the SP–Ag–linking units Lk–IE–TM, or one of the SP–IE–Lk–Ag–TM, where Lk is an amino acid linking unit; The connecting unit Lk is selected from at least one of flexible linking peptides and peptide chain self-cleaving sequences; The membrane anchor positioning domain TM is selected from at least one of the transmembrane structural domain and the glycosylphosphatidylinositol anchor positioning signal; The immune costimulatory ligand or its domain is selected from at least one of the extracellular domains, intracellular signal transduction domains, and fusion forms of the costimulatory receptor family. The cytokine or its functional fragment is selected from at least one member of the cytokine family that stimulates dendritic cell maturation, promotes T cell proliferation, or promotes NK cell activation. The antigen dimerization or oligomerization enhancing unit is selected from at least one of the oligomerization-promoting multimerization domains and immunoglobulin Fc-associated domains.

3. The genetically modified organism carrying an immune-enhancing element according to claim 2, characterized in that: The Ag is a nucleic acid sequence encoding a tumor-associated antigen or an immunoepidemitope fragment thereof, or a nucleic acid sequence encoding a pathogen-associated antigen or an immunoepidemitope fragment thereof. The expression framework further includes an internal ribosome entry site (IRES) or a peptide self-cleavage sequence for expressing Ag and IE separately on the same transcript.

4. The genetically modified organism carrying an immune-enhancing element according to claim 3, characterized in that: The expression regulation unit further includes at least one expression enhancement element selected from at least one of intron enhancers, enhancer elements, and splice acceptor or splice donor sites. The HS-L and HS-R in the site strategy-related sequence organization are homologous to the upstream and downstream sequences of the predetermined genomic site, respectively, and are used to guide the insertion or replacement of the Ag–IE expression framework at the predetermined site.

5. The genetically modified organism carrying an immune-enhancing element according to claim 4, characterized in that: The genetically modified vector has eukaryotic expression capability. The vector is selected from at least one of eukaryotic expression plasmids, adeno-associated virus vectors, and lentiviral vectors. It also includes genetically modified cells capable of expressing Ag and IE. The cells are immune cells selected from at least one of T cells, natural killer cells, dendritic cells, and macrophages. The immune cells further include editing of the inhibitory receptor pathway. The editing is selected from at least one of knockout of the inhibitory receptor coding gene and loss-of-function modification of key sites in inhibitory receptor signal transduction.

6. An application of a genetically modified organism carrying an immune-enhancing element, characterized in that: include S1. Design the encoding sequences for Ag and IE and determine the assembly order; S2. Based on the site strategy, HS-L and HS-R are determined and an Ag–IE expression framework is formed; S3. Introduce the gene construct consisting of the Ag-IE expression framework and HS-L and HS-R into recipient cells; S4. The insertion or replacement of the gene construct is achieved at a predetermined genomic location through site-specific endonuclease-mediated homology-directed repair. S5. Obtain the modified cells and confirm the expression and localization of Ag and IE; The modified sequence does not contain a permanent selection marker encoding sequence.

7. The application of a genetically modified organism carrying an immune-enhancing element according to claim 6, characterized in that: The site-specific endonuclease is selected from at least one of CRISPR-associated endonucleases, zinc finger endonucleases, and transcription activator-like effector endonucleases. The predetermined genomic location is a safe site for exogenous expression. The safe site does not overlap with the coding region of the cell's intrinsic key physiological function. The introduction method of S3 is selected from at least one of electroporation, viral vector transduction, and non-viral vector transfection. The confirmation of S5 includes at least one of the confirmation of the existence of Ag membrane localization and the confirmation of the existence of signal activation of the IE action pathway.

8. The application of a genetically modified organism carrying an immune-enhancing element according to claim 7, characterized in that: It also includes a kit, which contains a. A vector carrying the gene construct as described in any one of claims 1 to 12; b. Primer pairs corresponding to HS-L and HS-R; c. Nucleic acid editing components compatible with site-specific endonuclease systems; It is accompanied by written instructions for performing insertion or replacement operations at predetermined genomic locations; The immune-enhancing element (IE) comprises two or more elements, arranged in a parallel or sequential expression manner, wherein the parallel expression is achieved by IRES or peptide self-cleavage sequences respectively, and the sequential expression is achieved by the same open reading frame via a linker unit. The expression frame further includes a signal sequence for subcellular localization regulation, the signal sequence being selected from at least one of secretion signals, endoplasmic reticulum retention signals, and endosome localization signals. Ag contains at least one validated immune epitope fragment and is presented in a multi-epitope splicing manner, the multi-epitopes being separated by linker units Lk. Cells are expanded in vitro to form a cell preparation, the cell preparation being formulated with the pharmaceutical composition of claim 18 for subsequent use, further comprising: S6. Set up the second set of HS-L and HS-R for the second predetermined genomic location, and complete the insertion or replacement of the second Ag–IE expression frame; S7. Without changing the existing Ag-IE expression framework functionality, complete multi-site co-assembly; where, after S6 and S7, each site does not retain the encoding sequence of the permanent selection marker.

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