Quadruple shRNA combination and application thereof

Abstract

The invention relates to a quadruple shRNA (short hairpin ribonucleic acid) combination and application thereof. The combination comprises a first transcription unit, a second transcription unit, a third transcription unit and a fourth transcription unit which are located on the same expression vector and respectively silence four different target genes in a targeted manner. Each transcription unit comprises an RNA (Ribonucleic Acid) polymerase III promoter, an shRNA (Short Hairpin Ribonucleic Acid) coding sequence and a transcription termination signal; the promoters for driving the four transcription units are different from one another and are all selected from a promoter group consisting of hU6, mU6, hH1, h7SK, minihU6, H1-7SK and hH1-core; and the four transcription units are connected in series in an end-to-end manner. According to the design, by optimizing combination and arrangement of the promoters, expression interference and homologous recombination are effectively avoided, and efficient, stable, balanced and lasting silencing of the four target genes is achieved at the same time.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to quadruple shRNA combinations and their applications, and more specifically to nucleic acid constructs, expression vectors, host cells, pharmaceutical compositions, and methods for enhancing the function of immune cells. Background Technology

[0002] Gene-modified cell immunotherapy, especially based on CAR-T and CAR-NK technologies, has made breakthroughs in cancer treatment. However, the efficacy of this type of therapy in the treatment of solid tumors is still not ideal, mainly due to the complex immunosuppressive properties of the tumor microenvironment (TME) (such as the upregulation of multiple immune checkpoints, inhibitory signal transduction, metabolic stress, and apoptosis induction) and the insufficient persistence of infused cells in vivo.

[0003] To significantly improve therapeutic efficacy, it is necessary to perform multi-target deep modification of immune cells and simultaneously silence multiple key negative regulatory genes, such as immune checkpoint molecules (e.g., TIGIT, TIM3), intracellular inhibitory signaling proteins (e.g., TIPE2, CISH), apoptosis receptors (e.g., FAS), and microenvironmental stress factors (e.g., HIF1A, A2AR). However, achieving efficient, balanced, and durable simultaneous silencing of four genes within the same cell remains a significant challenge for current technology.

[0004] Gene editing technologies such as CRISPR / Cas9 suffer from off-target effects, DNA damage risks, and potential safety issues, limiting their clinical application. RNA interference-based technologies, such as shRNA, hold greater promise due to their high specificity and safety. However, existing shRNA technologies have significant limitations in multi-gene silencing: while the construction of dual-shRNA or triple-shRNA vectors using multiple different promoters has been explored, current technologies struggle to guarantee efficient, stable, and durable silencing of all target genes simultaneously for four or more genes. The genetic stability of the vector becomes even more critical when four genes are involved. Even with combinations of different promoters, inappropriate promoter selection or improper arrangement of transcription units can easily lead to expression interference, uneven efficiency, or increased risk of homologous recombination, failing to meet the high reliability and consistency requirements of clinical applications.

[0005] Therefore, there is an urgent need in this field to develop a new shRNA design strategy that can achieve efficient, stable, balanced and persistent silencing of four target genes on the same vector, and to verify its clear effect on enhancing immune cell function. Summary of the Invention

[0006] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application provides a quadruple shRNA combination and its application.

[0007] This application is based on the following discoveries of the inventors: The inventors discovered that when constructing a quadruple shRNA combination targeting four different genes on the same vector, simply using a two- or three-gene strategy, or arbitrarily combining four promoters, often fails to achieve optimal silencing of all four target genes simultaneously. Some genes are silenced efficiently, while others are silencing inefficiently, and the silencing effect tends to decline or be lost after long-term culture. This problem becomes extremely complex in the silencing of four genes, representing a key bottleneck hindering its clinical application.

[0008] Unexpectedly, through extensive experimental screening and optimization, the inventors discovered that by selecting four different promoters from a specific set of RNA polymerase III promoters (hU6, mU6, hH1, h7SK, minihU6, H1-7SK, hH1-core) and combining them in a specific head-to-tail tandem arrangement, they could synergistically and effectively ensure the stable existence of four shRNA transcription units in the vector, while simultaneously achieving efficient, balanced, and persistent silencing of four target genes. This invention successfully represents a technological leap from three shRNAs to four shRNAs, and this discovery provides a novel, previously undisclosed solution for achieving stable and efficient simultaneous silencing of four genes on a single vector.

[0009] In one aspect of this application, a quadruple shRNA combination is proposed. According to an embodiment of this application, the quadruple shRNA combination comprises a first transcription unit, a second transcription unit, a third transcription unit, and a fourth transcription unit. These four transcription units are disposed on the same expression vector and target and silence four different target genes, respectively. Each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence, and a transcription termination signal. The promoters driving the first, second, third, and fourth transcription units are respectively a first promoter, a second promoter, a third promoter, and a fourth promoter, which are different from each other and selected from any four of hU6, mU6, hH1, h7SK, minihU6, H1-7SK, and hH1-core. The quadruple shRNA combination according to the embodiments of this application, by employing specific combinations and arrangements of different promoters, effectively avoids expression interference and homologous recombination, achieving simultaneous, efficient, stable, and persistent silencing of four target genes.

[0010] In another aspect, this application proposes a nucleic acid construct. According to an embodiment of this application, the nucleic acid construct comprises the aforementioned quadruple shRNA combination. The nucleic acid construct according to the embodiment of this application integrates the quadruple shRNA combination into a single operable genetic unit, facilitating molecular cloning, vector construction, and large-scale production.

[0011] In another aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector comprises the aforementioned nucleic acid construct. The expression vector according to the embodiment of this application can efficiently deliver the quadruple shRNA combination into host cells and achieve its long-term, stable expression, making it a key tool for realizing gene modification of immune cells.

[0012] In another aspect, this application proposes a host cell. According to embodiments of this application, the host cell comprises the aforementioned quadruple shRNA combination, or a nucleic acid construct or expression vector comprising the quadruple shRNA combination. The host cell according to embodiments of this application, by stably expressing the quadruple shRNA combination, can simultaneously downregulate four key target genes, thereby achieving significantly enhanced killing activity, proliferation capacity, resistance to apoptosis, and adaptability to the complex tumor microenvironment.

[0013] In another aspect, this application provides a pharmaceutical composition. According to embodiments of this application, the pharmaceutical composition comprises the aforementioned host cells and a pharmaceutically acceptable carrier. The pharmaceutical composition according to embodiments of this application provides a formulation suitable for clinical administration of the enhanced immune cells, which can be directly used for the treatment of diseases.

[0014] In another aspect, this application proposes the use of the aforementioned host cells or pharmaceutical compositions in the preparation of medicaments for treating tumors, autoimmune diseases, chronic inflammation, or aging-related diseases. The uses described in the embodiments of this application clarify the broad application prospects of the technical solutions of this invention in the treatment of various major diseases.

[0015] In another aspect, this application proposes a method for enhancing the function of immune cells. According to embodiments of this application, the method includes introducing the aforementioned quadruple shRNA combination, nucleic acid construct, or expression vector into the immune cells to simultaneously silence the four different target genes. According to the method of embodiments of this application, through a single gene manipulation, multiple key functions of immune cells can be synergistically enhanced, specifically manifested as: significantly enhanced killing activity against tumor cells, improved cytokine proliferation signal response and long-term amplification capacity, enhanced resistance to apoptosis-inducing factors such as FASL, and enhanced survival rate and functional maintenance capacity under hypoxic or high adenosine stress microenvironments.

[0016] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0017] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This diagram shows the design and screening results of the shRNAs with the best silencing efficiency for the four target genes TIGIT, TIPE2, FAS, and A2AR in Example 1.

[0018] Figure 2 The diagram shows the design of the quadruple shRNA combination strategy in Example 2, illustrating different promoter combinations and connection methods.

[0019] Figure 3 The figure shows the comparison results of the silencing efficiency of four target genes by different combinations of quadruple shRNA designs in Example 3.

[0020] Figure 4 The graph shows the results of detecting the silencing persistence of the four target genes by the quadruple shRNA combination in Example 4.

[0021] Figure 5 The graph shows the detection results of the killing activity of NK cells against K562 cells after quadruple shRNA combination modification in Example 5.

[0022] Figure 6 The graph shows the results of detecting the proliferation capacity of NK cells after quadruple shRNA combination modification in Example 6 under low concentration of cytokines.

[0023] Figure 7 The graph shows the detection results of the ability of NK cells to resist FASL-induced apoptosis after quadruple shRNA combination modification in Example 7.

[0024] Figure 8 The graph shows the detection results of the cytotoxic activity of NK cells after quadruple shRNA combination modification in the adenosine-inhibiting microenvironment in Example 8.

[0025] Figure 9 The graph shows the detection results of the knockdown effect of the quadruple shRNA combination ([407-2]) on different target gene combinations (TIM3, CISH, FAS, HIF1A) in Example 9.

[0026] Figure 10The flow cytometry results show the expression of TIM3 ligands Galectin-9 and CEACAM1 on the surface of various tumor cell lines (HepG2, HCT116, MDA-MB-231) in Example 9.

[0027] Figure 11 The graph shows the detection results of the killing activity of NK cells modified with the quadruple shRNA combination ([407-2]) in Example 9 against different tumor cell lines. Detailed Implementation

[0028] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0033] In this paper, the term "shRNA" refers to a small RNA molecule with a stem-loop hairpin structure that can be processed into small interfering RNA (siRNA) within cells, thereby achieving gene silencing through RNA interference mechanisms that specifically degrade target mRNA or inhibit its translation. The shRNA in this application is transcribed by the RNA polymerase III promoter.

[0034] In this paper, the term "quadruple shRNA combinatorial" refers to a construct containing four shRNA expression units on the same vector, each shRNA targeting a different gene.

[0035] In this document, the term "different RNA polymerase III promoters" refers to promoters with different nucleotide sequences that can all be recognized by RNA polymerase III and initiate transcription. In this application, it specifically refers to any combination of four of the specific promoter groups selected from hU6, mU6, hH1, h7SK, minihU6, H1-7SK, and hH1-core, whose sequence differences are sufficient to avoid homologous recombination.

[0036] In this document, the term "transcription unit" refers to a complete DNA functional unit capable of independently performing shRNA transcription. Each transcription unit contains at least one RNA polymerase III promoter, an shRNA coding sequence, and a transcription termination signal arranged in the following order from 5' to 3'. In this application, four of the transcription units are designed to be tandemly linked on the same expression vector using a specific spatial arrangement (e.g., head-to-head, tail-to-tail, or head-to-tail) to form the aforementioned quadruple shRNA combination.

[0037] In this paper, the term "head-to-tail" refers to the arrangement of two adjacent transcription units, that is, a tandem arrangement where the termination signal of the preceding transcription unit and the promoter of the following transcription unit are close to each other, with a 20-50 bp spacer sequence in between as a transition, and restriction enzyme sites can be designed in the spacer sequence. This application demonstrates that this arrangement, in conjunction with specific combinations of different promoters, is key to achieving efficient and persistent silencing of four genes.

[0038] In this paper, the term "head-to-head" refers to two transcription units whose terminators (tails) are close to each other, the transcription directions are opposite, and there is a 20-50 bp spacer sequence in between as a transition, in which restriction enzyme sites are designed.

[0039] In this paper, the term "tail-to-tail" refers to two transcription units whose promoters (heads) are close to each other, the transcription direction is opposite, and there is a 20-50 bp spacer sequence in between as a transition, in which restriction enzyme sites are designed.

[0040] In this paper, the term "silencing efficiency" refers to the degree of reduction in the mRNA expression level of the target gene as detected by qRT-PCR relative to the control group. The preferred shRNA combination in this application achieves a silencing efficiency of over 70% for all four target genes.

[0041] In this article, the term "immune cells" refers to cell types that can be used for immunotherapy, including but not limited to NK cells, T cells, NKT cells, γδT cells, and macrophages.

[0042] In this article, the term "tumor microenvironment" refers to the special local environment in which tumor cells exist, characterized by low oxygen, acidity, nutrient deficiency, high adenosine levels, and containing a variety of immunosuppressive factors.

[0043] In this paper, the term "homological recombination" refers to the phenomenon in which the presence of repetitive sequences during vector construction or cell passage leads to the unexpected exchange and rearrangement of genetic material, resulting in the loss or destruction of shRNA expression cassettes.

[0044] In this document, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0045] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0046] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The chimeric antigen receptor, nucleic acid molecule, expression vector, or transgenic immune cell or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0047] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of drugs or transgenic immune cells to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administration of drugs containing cells with chimeric antigen receptors as described herein to an individual in need.

[0048] In this document, the term "host cell" refers to a cell containing the quadruple shRNA combination, nucleic acid construct, or expression vector described in this application. Specifically, it refers to genetically modified immune cells.

[0049] In this article, the term "pharmaceuticalally acceptable carrier" refers to various excipients, diluents, adjuvants, etc., used in the preparation of pharmaceutical compositions that do not produce significant adverse reactions and are compatible with the active ingredient.

[0050] In this document, the term “linked” refers to the connection of two or more nucleic acid sequences (such as promoters, coding sequences, and terminators) together by covalent bonds (primarily phosphodiester bonds) to form a continuous, single DNA molecular chain. It describes the final physical connection state and result between the elements. For example, “the 3' end of the promoter is linked to the 5' end of the shRNA coding sequence” means that in the final DNA construct, the two elements are consecutive sequences without gaps (except for any possible functional spacer sequences). “Linked” implies not only physical connection but also functional continuity. Linked elements should work together, for example, the promoter should correctly drive the transcription of the coding sequence that follows it. The use of “linked” in this specification is a relatively general and overarching term. It covers all possible ways to achieve this connection state, including but not limited to: (1) direct linking (without additional nucleotide insertion); (2) linking through a fixed, predefined “spacer sequence”; and (3) linking through any conventional molecular cloning method in the art (such as enzyme ligation, Gateway cloning, Gibson assembly, etc.).

[0051] In this paper, the term "ligation" refers to the process or specific structural layout of assembling or arranging two or more originally separate nucleic acid elements together through artificial manipulation (experimental methods) to ultimately form a "ligated" state. It emphasizes the ligation operation, method, or the final specific spatial arrangement (architecture). When appearing in the specific implementation section of the specification, "ligation" refers to an operational step, such as "ligating the first transcription unit to the second transcription unit," which describes an experimental action in constructing a vector. When used to describe the relative arrangement of multiple transcription units, it is synonymous with "ligation method," a specific realization of the "ligated" state. For example, "ligating head-to-tail" explicitly indicates the specific arrangement and orientation of the four transcription units on the vector (promoter A - shRNA A - terminator A - promoter B - shRNA B - terminator B - promoter C - shRNA C - terminator C - promoter D - shRNA D - terminator D). "Ligation" (especially specific ligation methods such as "head-to-tail") refers to one or more specific technical means to achieve the "ligated" state. The scope of protection is further narrowed and clarified by defining the specific “connection method” (i.e. how to “connect”), thus defining the preferred or specific embodiments of the present invention.

[0052] This application proposes a quadruple shRNA combination, a nucleic acid construct, an expression vector, a host cell, a pharmaceutical composition, and related uses, as well as a method for enhancing immune cell function, which will be described in detail below.

[0053] Quad shRNA combination In a first aspect, this application proposes a quadruple shRNA combination. According to an embodiment of this application, the quadruple shRNA combination comprises a first transcription unit, a second transcription unit, a third transcription unit, and a fourth transcription unit. These four transcription units are disposed on the same expression vector and target and silence four different target genes, respectively. Each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence, and a transcription termination signal. The promoters driving the first, second, third, and fourth transcription units are respectively a first promoter, a second promoter, a third promoter, and a fourth promoter. These promoters are selected from the promoter group composed of hU6, mU6, hH1, h7SK, minihU6, H1-7SK, and hH1-core, and are all distinct from each other. The quadruple shRNA combination according to the embodiments of this application, by employing specific combinations and arrangements of different promoters, effectively avoids expression interference and homologous recombination, achieving simultaneous, efficient, stable, and persistent silencing of four target genes.

[0054] According to an embodiment of this application, the first transcription unit contains a first shRNA molecule, the second transcription unit contains a second shRNA molecule, the third transcription unit contains a third shRNA molecule, and the fourth transcription unit contains a fourth shRNA molecule.

[0055] According to embodiments of this application, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, hH1, mU6, and h7SK, respectively; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hH1, and h7SK, respectively; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are all selected from hU6; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hU6, and mU6, respectively; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hH1-core, and H1-7SK, respectively; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, minihU6, hH1-core, and H1-7SK, respectively.

[0056] According to embodiments of this application, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hH1-core, and H1-7SK, respectively; or, the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, minihU6, hH1-core, and H1-7SK, respectively.

[0057] According to embodiments of this application, the first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit are connected in at least one of the following ways: head-to-head, tail-to-tail, and head-to-tail.

[0058] According to an embodiment of this application, the first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit are connected in a head-to-tail manner.

[0059] According to embodiments of this application, the four different target genes are selected from one of the following combinations: (a) TIGIT, TIPE2, FAS and A2AR; (b) TIM3, CISH, FAS and HIF1A.

[0060] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the first transcription unit is connected head-to-head with the second transcription unit, the second transcription unit is connected tail-to-tail with the third transcription unit, and the third transcription unit is connected head-to-head with the fourth transcription unit; or, the first transcription unit, the second transcription unit, and the third transcription unit are connected head-to-tail, and the third transcription unit is connected head-to-head with the fourth transcription unit; or, the first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit are connected head-to-tail.

[0061] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the transcription termination signal of the second transcription unit, and the 5' end of the second promoter is directly or through a spacer sequence connected to the 5' end of the third promoter, and the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the transcription termination signal of the fourth transcription unit; or, the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 5' end of the second promoter, and the transcription termination signal of the second transcription unit is directly or through a spacer sequence connected to the 5' end of the third promoter, and the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the transcription termination signal of the fourth transcription unit; or, the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 5' end of the second promoter, and the transcription termination signal of the second transcription unit is directly or through a spacer sequence connected to the 5' end of the third promoter, and the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 5' end of the fourth promoter.

[0062] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the hH1 promoter, the third transcription unit is driven by the mU6 promoter, and the fourth transcription unit is driven by the h7SK promoter; and the first transcription unit and the second transcription unit are connected head-to-head, the second transcription unit and the third transcription unit are connected tail-to-tail, and the third transcription unit and the fourth transcription unit are connected head-to-head; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, and the third transcription unit is driven by the h7SK promoter. The first transcription unit is driven by the hH1 promoter, the second transcription unit is driven by the h7SK promoter, and the first transcription unit and the second transcription unit are connected head-to-head, the second transcription unit and the third transcription unit are connected tail-to-tail, and the third transcription unit and the fourth transcription unit are connected head-to-head; or, the first, second, third, and fourth transcription units are all driven by the hU6 promoter, and the first transcription unit and the second transcription unit are connected head-to-head, the second transcription unit and the third transcription unit are connected tail-to-tail, and the third transcription unit and the fourth transcription unit are connected head-to-head; or, the first transcription unit is driven by the hU6 promoter. The second transcription unit is driven by the mU6 promoter, the third transcription unit by the hH1 promoter, and the fourth transcription unit by the h7SK promoter. The first, second, and third transcription units are head-to-tail connected, and the third transcription unit is head-to-head connected with the fourth transcription unit. Alternatively, the first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hU6 promoter, and the fourth transcription unit by the mU6 promoter. The first and second transcription units are head-to-head connected, and the second and third transcription units are tail-to-tail connected. The first transcription unit is head-to-head connected to the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the first, second, third, and fourth transcription units are head-to-tail connected; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the minihU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the first, second, third, and fourth transcription units are head-to-tail connected.

[0063] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the hH1 promoter and contains sh-TIPE2, the third transcription unit is driven by the mU6 promoter and contains sh-FAS, and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR; and the first transcription unit and the second transcription unit are linked head-to-head, the second transcription unit and the third transcription unit are linked tail-to-tail, and the third transcription unit and the fourth transcription unit are linked head-to-head; or The first transcription unit is driven by the hU6 promoter and contains sh-TIGIT; the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2; the third transcription unit is driven by the hH1 promoter and contains sh-FAS; and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR. The first and second transcription units are linked head-to-head, the second and third transcription units are linked tail-to-tail, and the third and fourth transcription units are linked head-to-head. Alternatively, the first, second, third, and fourth transcription units are all driven by the hU6 promoter and are... Each transcription unit contains sh-TIGIT, sh-TIPE2, sh-FAS, and sh-A2AR, and the first transcription unit is head-to-head connected to the second transcription unit, the second transcription unit is tail-to-tail connected to the third transcription unit, and the third transcription unit is head-to-head connected to the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1 promoter and contains sh-FAS, and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR, and the first transcription unit contains sh-TIGIT, sh-TIPE2, sh-FAS, and sh-A2AR, and the first transcription unit contains sh-TIGIT, sh-TIPE2, sh-FAS, and sh-A2AR, and the second transcription unit is head-to-head connected to the second transcription unit, the second transcription unit is tail-to-tail connected to the third transcription unit, and the third transcription unit is head-to-head connected to the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1 promoter and contains sh-FAS, and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR, and the first transcription unit contains sh-TIGIT, sh-TIPE2, sh-FAS, and sh-A2AR, and the second transcription unit is head-to-head connected to the second transcription unit, the second transcription unit is tail-to-tail connected to the third transcription unit, and the third transcription unit is head-to-head connected to the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIPE2, the second transcription unit is driven by the hU6 promoter and contains sh-TIPE2, the third The first transcription unit, the second transcription unit, and the third transcription unit are connected head-to-tail, and the third transcription unit is connected head-to-head with the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hU6 promoter and contains sh-FAS, the fourth transcription unit is driven by the mU6 promoter and contains sh-A2AR, and the first transcription unit is connected head-to-head with the second transcription unit, the second transcription unit is connected tail-to-tail with the third transcription unit, and the third transcription unit is connected head-to-head with the fourth transcription unit.Alternatively, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1-core promoter and contains sh-FAS, and the fourth transcription unit is driven by the H1-7SK promoter and contains sh-A2AR, with the first, second, third, and fourth transcription units connected head-to-tail; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the minihU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1-core promoter and contains sh-FAS, and the fourth transcription unit is driven by the H1-7SK promoter and contains sh-A2AR, with the first, second, third, and fourth transcription units connected head-to-tail.

[0064] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the hH1 promoter, the third transcription unit is driven by the mU6 promoter, and the fourth transcription unit is driven by the h7SK promoter. The 3' end of the transcription termination signal of the first transcription unit is directly or indirectly connected to the 3' end of the transcription termination signal of the second transcription unit via a spacer sequence. The 5' end of the hH1 promoter is directly or indirectly connected to the 5' end of the mU6 promoter via a spacer sequence. The transcription termination signal of the third transcription unit... The 3' end of the transcription termination signal is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1 promoter, and the fourth transcription unit is driven by the h7SK promoter, and the 3' end of the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the second transcription unit, the 5' end of the mU6 promoter is directly or through a spacer sequence connected to the 5' end of the hH1 promoter, and the transcription termination signal of the third transcription unit... The 3' end of the transcription signal of the first transcription unit is directly or indirectly connected to the 3' end of the transcription termination signal of the fourth transcription unit, either through a spacer sequence or directly connected to the 3' end of the transcription termination signal of the second transcription unit. Alternatively, the first, second, third, and fourth transcription units are all driven by the hU6 promoter, and the 3' end of the transcription termination signal of the first transcription unit is directly or indirectly connected to the 3' end of the transcription termination signal of the second transcription unit, either through a spacer sequence or directly connected to the 5' end of the second hU6 promoter, either through a spacer sequence or directly connected to the 5' end of the third hU6 promoter. Furthermore, the 3' end of the transcription termination signal of the third transcription unit is directly or indirectly connected to the 3' end of the transcription termination signal of the fourth transcription unit, either through a spacer sequence or directly connected to the 3' end of the transcription termination signal of the fourth transcription unit. The transcription units are connected in columns; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1 promoter, and the fourth transcription unit is driven by the h7SK promoter, and the 3' end of the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 5' end of the mU6 promoter, the 3' end of the transcription termination signal of the second transcription unit is directly or through a spacer sequence connected to the 5' end of the hH1 promoter, and the 3' end of the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit.Alternatively, the first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hU6 promoter, and the fourth transcription unit by the mU6 promoter, with the 3' end of the transcription termination signal of the first transcription unit directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the second transcription unit, and the 5' end of the second mU6 promoter directly or through a spacer sequence connected to the 5' end of the third hU6 promoter, and the 3' end of the transcription termination signal of the third transcription unit directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hH1-core promoter, and the fourth transcription unit by the H1-7SK promoter, with the 3' end of the transcription termination signal of the first transcription unit directly or through a spacer sequence connected to the 5' end of the mU6 promoter. The transcription termination signal of the second transcription unit is directly or indirectly connected to the 5' end of the hH1-core promoter via a spacer sequence, and the transcription termination signal of the third transcription unit is directly or indirectly connected to the 5' end of the H1-7SK promoter via a spacer sequence; or, the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the minihU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the transcription termination signal of the first transcription unit is directly or indirectly connected to the 5' end of the minihU6 promoter via a spacer sequence, the transcription termination signal of the second transcription unit is directly or indirectly connected to the 5' end of the hH1-core promoter via a spacer sequence, and the transcription termination signal of the third transcription unit is directly or indirectly connected to the 5' end of the H1-7SK promoter via a spacer sequence.

[0065] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the hH1 promoter and contains sh-TIPE2, the third transcription unit is driven by the mU6 promoter and contains sh-FAS, and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR, and the 3' end of the transcription termination signal of the first transcription unit is parallel to the transcription termination signal of the second transcription unit. The 3' ends of the hH1 promoter are directly or indirectly connected to the 5' ends of the mU6 promoter, and the 3' ends of the transcription termination signals of the third transcription unit and the fourth transcription unit are directly or indirectly connected to the 3' ends of the transcription termination signals of the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1 promoter and contains sh-FAS, and the fourth... The transcription units are driven by the h7SK promoter and contain sh-A2AR. The 3' end of the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the second transcription unit. The 5' end of the mU6 promoter is directly or through a spacer sequence connected to the 5' end of the hH1 promoter. The 3' end of the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit. Alternatively, the first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit... Each unit is driven by the hU6 promoter and contains sh-TIGIT, sh-TIPE2, sh-FAS and sh-A2AR respectively. The 3' end of the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the second transcription unit. The 5' end of the second hU6 promoter is directly or through a spacer sequence connected to the 5' end of the third hU6 promoter. The 3' end of the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit.Alternatively, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1 promoter and contains sh-FAS, and the fourth transcription unit is driven by the h7SK promoter and contains sh-A2AR. The 3' end of the transcription termination signal of the first transcription unit is directly or via a spacer sequence connected to the 5' end of the mU6 promoter, and the 3' end of the transcription termination signal of the second transcription unit is directly connected to the 5' end of the hH1 promoter. Alternatively, the transcription units may be linked by a spacer sequence, and the 3' end of the transcription termination signal of the third transcription unit may be directly or indirectly linked to the 3' end of the transcription termination signal of the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hU6 promoter and contains sh-FAS, and the fourth transcription unit is driven by the mU6 promoter and contains sh-A2AR, and the 3' end of the transcription termination signal of the first transcription unit may be linked by a spacer sequence. The 3' end of the transcription termination signal of the second transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the second transcription unit; the 5' end of the second mU6 promoter is directly or through a spacer sequence connected to the 5' end of the third hU6 promoter; and the 3' end of the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 3' end of the transcription termination signal of the fourth transcription unit; or, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the mU6 promoter and contains sh-TIPE2, and the third transcription unit is driven by the hU6 promoter and contains sh-TIPE2. The fourth transcription unit is driven by the hH1-core promoter and contains sh-FAS, and is driven by the H1-7SK promoter and contains sh-A2AR. The 3' end of the transcription termination signal of the first transcription unit is directly or through a spacer sequence connected to the 5' end of the mU6 promoter. The 3' end of the transcription termination signal of the second transcription unit is directly or through a spacer sequence connected to the 5' end of the hH1-core promoter. The 3' end of the transcription termination signal of the third transcription unit is directly or through a spacer sequence connected to the 5' end of the H1-7SK promoter.Alternatively, the first transcription unit is driven by the hU6 promoter and contains sh-TIGIT, the second transcription unit is driven by the minihU6 promoter and contains sh-TIPE2, the third transcription unit is driven by the hH1-core promoter and contains sh-FAS, and the fourth transcription unit is driven by the H1-7SK promoter and contains sh-A2AR. The 3' end of the transcription termination signal of the first transcription unit is directly or indirectly connected to the 5' end of the minihU6 promoter via a spacer sequence; the 3' end of the transcription termination signal of the second transcription unit is directly or indirectly connected to the 5' end of the hH1-core promoter via a spacer sequence; and the 3' end of the transcription termination signal of the third transcription unit is directly or indirectly connected to the 5' end of the H1-7SK promoter via a spacer sequence.

[0066] According to embodiments of this application, when the four different target genes are TIGIT, TIPE2, FAS, and A2AR: the sense strand sequence of the first shRNA molecule targeting TIGIT has a nucleotide sequence as shown in at least one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; the sense strand sequence of the second shRNA molecule targeting TIPE2 has a nucleotide sequence as shown in at least one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; the sense strand sequence of the third shRNA molecule targeting FAS has a nucleotide sequence as shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 22. The nucleotide sequence of the fourth shRNA molecule targeting A2AR has at least one of the nucleotide sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58; the sense strand sequence of the fourth shRNA molecule targeting A2AR has at least one of the nucleotide sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74.

[0067] According to an embodiment of this application, when the four different target genes are TIM3, CISH, FAS and HIF1A: the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the first, second, third and fourth transcription units are connected head to tail.

[0068] According to embodiments of this application, when the four different target genes are TIM3, CISH, FAS, and HIF1A: the first transcription unit is driven by the hU6 promoter and contains sh-TIM3, the second transcription unit is driven by the mU6 promoter and contains sh-CISH, the third transcription unit is driven by the hH1-core promoter and contains sh-FAS, and the fourth transcription unit is driven by the H1-7SK promoter and contains sh-HIF1A, and the first, second, third, and fourth transcription units are connected head-to-tail.

[0069] According to embodiments of this application, when the four different target genes are TIM3, CISH, FAS, and HIF1A: the first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter. The 3' end of the transcription termination signal of the first transcription unit is directly or indirectly connected to the 5' end of the mU6 promoter via a spacer sequence. The 3' end of the transcription termination signal of the second transcription unit is directly or indirectly connected to the 5' end of the hH1-core promoter via a spacer sequence. The 3' end of the transcription termination signal of the third transcription unit is directly or indirectly connected to the 5' end of the H1-7SK promoter via a spacer sequence.

[0070] According to embodiments of this application, when the four different target genes are TIM3, CISH, FAS, and HIF1A: the first transcription unit is driven by the hU6 promoter and contains sh-TIM3, the second transcription unit is driven by the mU6 promoter and contains sh-CISH, the third transcription unit is driven by the hH1-core promoter and contains sh-FAS, and the fourth transcription unit is driven by the H1-7SK promoter and contains sh-HIF1A. Furthermore, the 3' end of the transcription termination signal of the first transcription unit is directly or indirectly connected to the 5' end of the mU6 promoter via a spacer sequence; the 3' end of the transcription termination signal of the second transcription unit is directly or indirectly connected to the 5' end of the hH1-core promoter via a spacer sequence; and the 3' end of the transcription termination signal of the third transcription unit is directly or indirectly connected to the 5' end of the H1-7SK promoter via a spacer sequence.

[0071] According to embodiments of this application, when the four different target genes are TIM3, CISH, FAS, and HIF1A: the 3' end of the hU6 promoter is connected to the 5' end of sh-TIM3, the 3' end of sh-TIM3 is close to the 5' end of the mU6 promoter, with a spacer sequence in between; the 3' end of the mU6 promoter is connected to the 5' end of sh-CISH, the 3' end of sh-CISH is close to the 5' end of the hH1-core promoter, with a spacer sequence in between; the 3' end of the hH1-core promoter is connected to the 5' end of sh-FAS, the 3' end of sh-FAS is close to the 5' end of the H1-7SK promoter, with a spacer sequence in between; and the 3' end of the H1-7SK promoter is connected to the 5' end of sh-HIF1A.

[0072] According to embodiments of this application, when the four different target genes are TIM3, CISH, FAS, and HIF1A: the sense strand of the first shRNA molecule targeting TIM3 has the nucleotide sequence shown in SEQ ID NO: 76; the sense strand of the second shRNA molecule targeting CISH has the nucleotide sequence shown in SEQ ID NO: 78; the sense strand sequence of the third shRNA molecule targeting FAS has the nucleotide sequence shown in at least one of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58; and the sense strand sequence of the fourth shRNA molecule targeting HIF1A has the nucleotide sequence shown in SEQ ID NO: 80.

[0073] According to embodiments of this application, the nucleotide sequence of the spacer sequence is shown in at least one of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90.

[0074] Nucleic acid constructs In a second aspect, this application proposes a nucleic acid construct. According to an embodiment of this application, the nucleic acid construct comprises the aforementioned quadruple shRNA combination. The nucleic acid construct according to the embodiment of this application integrates the quadruple shRNA combination into a single operable genetic unit, facilitating molecular cloning, vector construction, and large-scale production.

[0075] expression carrier In a third aspect, this application proposes an expression vector. According to embodiments of this application, the expression vector comprises the aforementioned nucleic acid construct. The expression vector according to embodiments of this application can efficiently deliver the quadruple shRNA combination into host cells and achieve its long-term, stable expression, making it a key tool for realizing gene modification of immune cells.

[0076] host cells In a fourth aspect, this application provides a host cell. According to embodiments of this application, the host cell comprises a nucleic acid construct or expression vector containing the quadruple shRNA combination. The host cell according to embodiments of this application, by stably expressing the quadruple shRNA combination, can simultaneously downregulate four key target genes, thereby achieving significantly enhanced killing activity, proliferation capacity, resistance to apoptosis, and adaptability to the complex tumor microenvironment.

[0077] According to embodiments of this application, the host cell is selected from at least one of NK cells, T cells, NKT cells, γδT cells, and macrophages.

[0078] Pharmaceutical Composition In a fifth aspect, this application provides a pharmaceutical composition. According to embodiments of this application, the pharmaceutical composition comprises the aforementioned host cells and a pharmaceutically acceptable carrier. The pharmaceutical composition according to embodiments of this application provides a formulation suitable for clinical administration of the enhanced immune cells, which can be directly used for the treatment of diseases.

[0079] use In a sixth aspect, this application proposes the use of the aforementioned host cells or pharmaceutical compositions in the preparation of medicaments for treating tumors, autoimmune diseases, chronic inflammation, or aging-related diseases. The uses described in the embodiments of this application clarify the broad application prospects of the technical solutions of this invention in the treatment of various major diseases.

[0080] According to embodiments of this application, the tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer, melanoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms.

[0081] According to embodiments of this application, the autoimmune disease includes at least one selected from systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0082] According to embodiments of this application, the chronic inflammation or age-related disease includes at least one selected from neurodegenerative diseases, cardiovascular diseases, metabolic diseases, cancer, immune system diseases, and musculoskeletal diseases.

[0083] According to embodiments of this application, the neurodegenerative disease includes at least one of Alzheimer's disease and Parkinson's disease.

[0084] According to embodiments of this application, the cardiovascular disease includes at least one of coronary artery disease, myocardial infarction, and stroke.

[0085] According to embodiments of this application, the metabolic disease includes at least one of hypertension, type 2 diabetes, and hypercholesterolemia.

[0086] Methods to improve immune cell function In a seventh aspect, this application proposes a method for enhancing the function of immune cells. According to embodiments of this application, the method includes introducing the aforementioned quadruple shRNA combination, nucleic acid construct, or expression vector into the immune cells to simultaneously silence the four different target genes. The method according to embodiments of this application, through a single gene manipulation, can synergistically enhance multiple key functions of immune cells, specifically manifested as: significantly enhanced killing activity against tumor cells, improved cytokine proliferation signal response and long-term amplification capacity, enhanced resistance to apoptosis-inducing factors such as FASL, and enhanced survival rate and functional maintenance capacity under hypoxic or high adenosine stress microenvironments.

[0087] The sequence descriptions involved in this invention are detailed in Table 1.

[0088] Table 1: Amino Acid / Nucleotide Sequence Description

[0089] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0090] Example 1: Design and screening of shRNA sequences for silencing TIGIT, TIPE2, FAS, and A2AR genes respectively. This example describes the shRNA design and screening process for four target genes: TIGIT, TIPE2, FAS, and A2AR.

[0091] This embodiment describes the shRNA design and screening process for four target genes: TIGIT (an NK cell inhibitory receptor that inhibits NK cell activation and effector function after binding to its corresponding ligand on the tumor surface, helping tumor cells evade immune surveillance), TIPE2 (an intracellular immune checkpoint molecule that inhibits the anti-tumor immune response and proliferation of immune cells), FAS (a cell death receptor that induces apoptosis after binding to its ligand FASL), and A2AR (an adenosine receptor on the surface of immune cells that inhibits the activity and function of immune cells after binding to its ligand adenosine). By simultaneously silencing these four target genes (TIGIT, TIPE2, FAS, and A2AR), the aim is to enhance the NK cell killing activity, proliferation capacity, and resistance to apoptosis, enabling NK cells to maintain good survival, proliferation, and effector function even in the tumor immunosuppressive microenvironment.

[0092] 1. RNAi target sequence design (1) The gene sequence number of Homo sapiens T cell immunoreceptor with Ig and ITIM domains (TIGIT) was found on the NCBI website as NM_173799.4. The coding sequence (CDS) of TIGIT mRNA is shown in SEQ ID NO: 2. Six RNAi target sequences were designed and screened from the TIGIT gene coding sequence (nucleotide sequence as shown in SEQ ID NO: 2, amino acid sequence as shown in SEQ ID NO: 1).

[0093] Based on the designed RNAi target sequence, shRNA sequences for constructing shRNA lentiviral vectors were designed. The coding sequences of the designed TIGIT shRNA (TIGIT-sh01 to TIGIT-sh06, nucleotide sequences as shown in SEQ ID NO:11~16) and the nonsense control sequence (Non-targeting control, NTC, the DNA coding sequence of which is shown in SEQ ID NO:9, and its transcribed sense strand RNA sequence as shown in SEQ ID NO:10) have the structure of sense strand, loop, antisense strand, and termination sequence.

[0094] (2) The gene sequence number of TNF alpha induced protein 8 like 2 (TNFAIP8L2, TIPE2) was found on the NCBI website as NM_024575.5. The coding sequence (CDS) of TIPE2 mRNA is shown in SEQ ID NO: 4. Seven RNAi target sequences were designed and screened from the TIPE2 gene coding sequence (amino acid sequence as shown in SEQ ID NO: 3, nucleotide sequence as shown in SEQ ID NO: 4).

[0095] Based on the designed RNAi target sequence, an shRNA sequence for constructing the shRNA lentiviral vector was designed. The coding sequence of the designed TIPE2 shRNA (TIPE2-sh01 to TIPE2-sh07, nucleotide sequences as shown in SEQ ID NO:23~29) has a structure of sense strand, loop, antisense strand, and termination sequence.

[0096] (3) The gene sequence number of Homo sapiens Fas cell surface death receptor (FAS) was found on the NCBI website as NM_000043.6, and the coding sequence (CDS) of FAS mRNA is shown in SEQ ID NO: 6. Eleven RNAi target sequences were designed and screened from the FAS gene coding sequence (amino acid sequence as shown in SEQ ID NO: 5, nucleotide sequence as shown in SEQ ID NO: 6).

[0097] Based on the designed RNAi target sequence, an shRNA sequence for constructing the shRNA lentiviral vector was designed. The coding sequence of the designed FASshRNA (FAS-sh01 to FAS-sh11, nucleotide sequences as shown in SEQ ID NO:37~47) has a structure of sense strand, loop, antisense strand, and termination sequence.

[0098] (4) The gene sequence number of Homo sapiens adenosine A2a receptor (ADORA2A, A2AR) was found on the NCBI website as NM_000675.6, and the coding sequence (CDS) of A2AR mRNA is shown in SEQ ID NO: 8. Eight RNAi target sequences were designed and screened from the A2AR gene coding sequence (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 8).

[0099] Based on the designed RNAi target sequence, an shRNA sequence for constructing the shRNA lentiviral vector was designed. The coding sequence of the designed A2AR shRNA (A2AR-sh01 to A2AR-sh08, nucleotide sequences as shown in SEQ ID NO:59~66) has a structure of sense strand, loop, antisense strand, and termination sequence.

[0100] 2. Lentiviral packaging and viral fluid concentration Take 5 × 10⁵ 293T cells in the logarithmic growth phase 6Cells were seeded in 10 cm cell culture dishes, and 10 mL of DMEM complete medium was added. The dishes were incubated overnight at 37°C with saturated humidity and 5% CO2. When the cell density reached 80%–90%, 10 mL of fresh DMEM complete medium was added, and the dishes were kept in the incubator for later use. Lentiviral packaging systems were prepared by adding 6 μg of PSPAX2 and 3 μg of PMD2.G lentiviral packaging helper plasmids, and 6 μg of lentiviral vector plasmids, to 250 μL of OPTI-MEM serum-free medium to prepare plasmid mixtures. 15 μL of PEI MAX (Polysciences) transfection reagent was added to 235 μL of OPTI-MEM serum-free medium and mixed thoroughly. The PEI MAX (Polysciences) mixture was then added to the plasmid mixtures, mixed, and incubated at room temperature for 20 min. Finally, the mixtures were added to 293T cell culture medium. 24 h after transfection, the medium was changed, the old medium was aspirated, and 10 mL of fresh DMEM complete medium was replaced. The culture dishes were returned to an incubator at 37℃, saturated humidity, and 5% CO2. After 48 h, the cell supernatant was collected, centrifuged at 400×g for 5 min to remove cell debris, and the supernatant was filtered through a 0.45 μm needle filter into 50 mL centrifuge tubes to obtain viral solutions of different TIGIT shRNA (TIGIT-sh01~sh06), TIPE2 shRNA (TIPE2-sh01~sh07), FAS-shRNA (FAS-sh01~sh11), A2AR shRNA (A2AR-sh01~sh08), and nonsense control (NTC) lentiviruses. The viral solutions were concentrated using a lentivirus concentration kit. The concentrated virus was used to infect cells or stored at -80℃.

[0101] 3. Detection of silencing efficiency of shRNA for different target genes Viral infection: NKG cells (NK cell line, from the University of Science and Technology of China) were infected with concentrated lentiviruses containing different TIGIT shRNAs (TIGIT-sh01~sh06), TIPE2 shRNAs (TIPE2-sh01~sh07), FAS shRNAs (FAS-sh01~sh11), A2AR shRNAs (A2AR-sh01~sh08), and nonsense control sequences (NTC). The expanded NKG cells were harvested by centrifugation at 300×g for 5 min, resuspended in an appropriate amount of NKG cell culture medium, and the cell density was adjusted to 5×10⁶ cells / year. 5 Cells / mL. 5 × 10⁵ cells / mL were added to each well of a 24-well plate. 5One NKG cell, 0.2 mL of virus concentrate prepared in Example 1, "2. Packaging and Concentration of Lentiviral Fluid", 0.8 mL of NKG cell culture medium and infection-promoting reagent were mixed thoroughly. The mixed 24-well plate was placed in an incubator at 37°C, saturated humidity, and 5% CO2. After 24 hours of viral infection of NKG cells, the NKG cell status was observed, and an equal volume of fresh NKG cell culture medium was added. The NKG cells were then transferred to cell culture flasks for further culture. Fresh NKG cell culture medium was added every 2 days, along with NKG cell growth-promoting factors such as IL2 (final concentration 200 IU / mL).

[0102] NKG cells were infected with different lentiviruses containing TIGIT shRNA (TIGIT-sh01~sh06), TIPE2 shRNA (TIPE2-sh01~sh07), FAS shRNA (FAS-sh01~sh11), A2AR shRNA (A2AR-sh01~sh08), and nonsense control (NTC) sequences. Stable cell lines were established after Puromycin screening treatment. On day 7 post-infection, the expression of different target genes in the infected NKG cells was detected by quantitative real-time PCR (qPCR).

[0103] qRT-PCR: 2 × 10⁻⁶ stable NKG cells established after Puromycin screening were used. 6 The cell pellet was transferred to a 15 mL centrifuge tube, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in PBS and washed. The pellet was then centrifuged again at 300×g for 5 min, the supernatant was discarded, and 1 mL of Trizol reagent was added to lyse the cell pellet. The lysate was then transferred to a 1.5 mL RNase-free EP tube. Total RNA was extracted from the cells and reverse transcribed into cDNA. Real-time quantitative PCR was then performed using target gene-specific primers to detect the expression level of the transcribed mRNA.

[0104] The results are as follows Figure 1As shown in Figure A, compared with the nonsense control group (NTC), the expression levels of TIGIT mRNA in cells infected with different TIGIT shRNAs (TIGIT-sh01~TIGIT-sh06) varied. TIGIT-sh01, TIGIT-sh02, TIGIT-sh03, and TIGIT-sh04 showed significant silencing effects, while TIGIT-sh05 and TIGIT-sh06 did not show significant silencing effects. Among them, TIGIT-sh01 and TIGIT-sh04 sequences showed the most significant silencing effects, with a silencing efficiency of about 80%, and were able to maintain a stable silencing effect.

[0105] The results are as follows Figure 1 As shown in Figure B, compared with the nonsense control (NTC) sequence group, the expression levels of TIPE2 mRNA in cells infected by different TIPE2 shRNAs (TIPE2-sh01~TIPE2-sh07) varied. Some shRNAs showed significant silencing effects, while others had insignificant or no silencing effects. Among them, the TIPE2-sh01, sh02, sh03, and sh06 sequences had high silencing efficiencies, reaching approximately 80%, and maintained stable silencing effects.

[0106] The results are as follows Figure 1 As shown in Figure C, compared with the nonsense control group (NTC), the expression levels of FAS mRNA in cells infected by different FAS shRNAs (FAS-sh01~FAS-sh11) varied. Some shRNAs showed significant silencing effects, while others had insignificant or no silencing effects. Among them, the FAS-sh01, sh02, sh06, sh07, sh08, and sh10 sequences had high silencing efficiencies, reaching approximately 80%, and maintained stable silencing effects.

[0107] The results are as follows Figure 1 As shown in Figure D, compared with the nonsense control (NTC) sequence group, the expression levels of A2AR mRNA in cells infected by different A2AR shRNAs (A2AR-sh01~A2AR-sh08) varied. Some shRNAs showed significant silencing effects, while others had insignificant or no silencing effects. Among them, the A2AR-sh01, sh02, sh05, and sh06 sequences had high silencing efficiencies, reaching approximately 80%, and maintained stable silencing effects.

[0108] Example 2: Design of a quadruple shRNA combinatorial strategy This embodiment describes a strategy for designing quadruple shRNA combinations. To address the limitations of existing technologies that cannot simultaneously silence four target genes in the same cell or that using four identical promoters can easily lead to homologous recombination events and reduced silencing efficiency, we selected different promoters (hU6, mU6, hH1, h7SK, minihU6, H1-7SK, hH1-core) that drive high transcriptional efficiency of shRNAs and combined them to optimize the promoter combinations that can guarantee a durable and effective simultaneous silencing of four target genes. The use of identical promoter combinations with four hU6 promoters was compared as a proportion of existing technologies.

[0109] Four shRNA combinations were designed within the same vector, considering the use of different RNA polymerase III promoters (hU6, mU6, hH1, h7SK, minihU6, H1-7SK, hH1-core) and different shRNA transcription unit ligation methods ("head-to-head", "tail-to-tail", "head-to-tail"). Combination information is as follows: Figure 2 As shown.

[0110] Keeping the target gene shRNA sequence unchanged, #1 includes the shRNA coding sequence targeting TIGIT and its matching transcription termination signal (TIGIT-shRNA, using the TIGIT-sh04 sequence), #2 includes the shRNA coding sequence targeting TIPE2 and its matching transcription termination signal (TIPE2-shRNA, using the TIPE2-sh01 sequence), #3 includes the shRNA coding sequence targeting FAS and its matching transcription termination signal (FAS-shRNA, using the FAS-sh01 sequence), and #4 includes the shRNA coding sequence targeting A2AR and its matching transcription termination signal (A2AR-shRNA, using the A2AR-sh02 sequence). Different RNA polymerase III promoters and their linkage methods are compared to screen for the optimal combination strategy of the four shRNAs.

[0111] In combination

[401] , different RNA polymerase III promoters (hU6, hH1, mU6, h7SK) were used, wherein the first hU6-shRNA transcription unit was connected to the second hH1-shRNA transcription unit in a head-to-head manner, the second hH1-shRNA transcription unit was connected to the third mU6-shRNA transcription unit in a tail-to-tail manner, the third mU6-shRNA transcription unit was connected to the fourth h7SK-shRNA transcription unit in a head-to-head manner, and the transcription units were connected by spacer sequences. In combination

[402] , different RNA polymerase III promoters (hU6, mU6, hH1, h7SK) were used, wherein the first hU6-shRNA transcription unit was connected to the mU6-shRNA transcription unit in a head-to-head manner, the mU6-shRNA transcription unit was connected to the hH1-shRNA transcription unit in a tail-to-tail manner, the hH1-shRNA transcription unit was connected to the h7SK-shRNA transcription unit in a head-to-head manner, and the transcription units were connected by spacer sequences. In combination

[403] , four identical RNA polymerase III promoters (hU6) were used, wherein the first hU6-shRNA transcription unit was connected to the second hU6-shRNA transcription unit in a head-to-head manner, the second hU6-shRNA transcription unit was connected to the third hU6-shRNA transcription unit in a tail-to-tail manner, the third hU6-shRNA transcription unit was connected to the fourth hU6-shRNA transcription unit in a head-to-head manner, and the transcription units were connected by spacer sequences. In the comparative example

[404] combination, four identical RNA polymerase III promoters (hU6) were used, and the four hU6-shRNA transcription units were connected in a “head-to-tail” manner, with the transcription units connected by spacer sequences. In combination

[405] , different RNA polymerase III promoters (hU6, mU6, hH1, h7SK) were used, wherein the hU6-shRNA transcription unit was connected to the mU6-shRNA transcription unit in a "head-to-tail" manner, the mU6-shRNA transcription unit was connected to the hH1-shRNA transcription unit in a "head-to-tail" manner, the hH1-shRNA transcription unit was connected to the h7SK-shRNA transcription unit in a "head-to-head" manner, and the transcription units were connected by spacer sequences. In combination

[406] , two identical RNA polymerase III promoters (hU6, mU6) were used, wherein the first hU6-shRNA transcription unit was connected to the second mU6-shRNA transcription unit in a head-to-head manner, the second mU6-shRNA transcription unit was connected to the third hU6-shRNA transcription unit in a tail-to-tail manner, the third hU6-shRNA transcription unit was connected to the fourth mU6-shRNA transcription unit in a head-to-head manner, and the transcription units were connected by spacer sequences. In combination

[407] , different RNA polymerase III promoters (hU6, mU6, hH1-core, H1-7SK) were used, wherein the hU6-shRNA transcription unit was connected to the mU6-shRNA transcription unit in a head-to-tail manner, the mU6-shRNA transcription unit was connected to the hH1-core-shRNA transcription unit in a head-to-tail manner, the hH1-core-shRNA transcription unit was connected to the H1-7SK-shRNA transcription unit in a head-to-tail manner, and the transcription units were connected by spacer sequences. In the

[408] combination, different RNA polymerase III promoters (hU6, minihU6, hH1-core, H1-7SK) were used, wherein the hU6-shRNA transcription unit was connected to the minihU6-shRNA transcription unit in a head-to-tail manner, the minihU6-shRNA transcription unit was connected to the hH1-core-shRNA transcription unit in a head-to-tail manner, the hH1-core-shRNA transcription unit was connected to the H1-7SK-shRNA transcription unit in a head-to-tail manner, and the transcription units were connected by spacer sequences.

[0112] Example 3: Comparison of four shRNA combination strategies This example describes a comparison of the target gene silencing efficiency of different combinations of quadruple shRNA designs.

[0113] Plasmid synthesis: The base sequences of different designed quadruple shRNAs were synthesized through whole-genome synthesis. Different restriction enzyme sites were inserted between different shRNA transcription units to facilitate molecular cloning sequence replacement. The sequences were cloned into lentiviral vectors through the restriction enzyme sites. After sequencing verification, the following plasmids were obtained: sh-

[401] , sh-

[402] , sh-

[403] , sh-

[404] , sh-

[405] , sh-

[406] , sh-

[407] and sh-

[408] , which are the quadruple shRNA combination vector plasmids involved in this invention.

[0114] Packaging and concentration of lentiviruses: 6 μg of lentivirus packaging helper plasmid PSPAX2 and 3 μg of pMD2.G, and 6 μg of quadruple shRNA combination vector plasmid were added to 250 μL of OPTI-MEM serum-free medium to prepare plasmid mixtures and mixed evenly. Lentiviral viruses were packaged and concentrated using the method described in 1.2 of Example 1 to obtain lentiviruses packaged with different quadruple shRNA combination vectors.

[0115] Comparison of target gene silencing efficiency of different quadruple shRNA combinations: NKG cells were infected with lentiviruses containing different packaged quadruple shRNA combinations. Stable NKG cell lines were established by Puromycin screening. The silencing efficiency of target genes (TIGIT, TIPE2, FAS, and A2AR) in NKG cell lines with different quadruple shRNA combinations was detected by qRT-PCR using the method described in 1.3 of Example 1.

[0116] The results are as follows Figure 3 As shown, for the first target gene TIGIT, the expression levels of TIGIT mRNA in cells infected with different quadruple shRNA combinations varied compared to the nonsense control group (NTC). Some quadruple shRNA combinations showed significant silencing effects, while others did not. The sh-

[401] , sh-

[402] , sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] sequences all showed silencing efficiency for TIGIT, with sh-

[407] and sh-

[408] showing higher silencing efficiency, reaching approximately 70%.

[0117] For the second target gene TIPE2, the expression levels of TIPE2 mRNA in cells infected with different quadruple shRNA combinations varied compared to the nonsense control (NTC) sequence group. Some quadruple shRNA combinations showed significant silencing effects, while others did not. The sh-

[401] , sh-

[402] , sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] sequences all showed silencing efficiency for TIPE2, with the sh-

[407] and sh-

[408] sequences exhibiting higher silencing efficiency, reaching approximately 70%.

[0118] For the third target gene FAS, the expression levels of FAS mRNA in cells infected with different quadruple shRNA combinations varied compared to the nonsense control group (NTC). Some quadruple shRNA combinations showed significant silencing effects, while others did not. The sh-

[401] , sh-

[402] , sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] sequences all showed silencing efficiency for FAS, with the sh-

[407] and sh-

[408] sequences showing higher silencing efficiency, reaching approximately 80%.

[0119] For the fourth target gene, A2AR, the expression levels of A2AR mRNA in lentivirally infected cells varied with different quadruple shRNA combinations compared to the nonsense control (NTC) sequence group. Some quadruple shRNA combinations showed significant silencing effects, while others did not. The sh-

[401] , sh-

[402] , sh-

[405] , sh-

[407] , and sh-

[408] sequences all showed silencing efficiency for A2AR, with the sh-

[407] and sh-

[408] sequences exhibiting higher silencing efficiency, reaching approximately 70%.

[0120] Based on the silencing efficiency of different quadruple shRNA combinations on four target genes (TIGIT, TIPE2, FAS, A2AR), the sh-

[407] and sh-

[408] combinations showed high silencing effects on all four target genes, indicating that the promoter combination of sh-

[407] and sh-

[408] has the advantage of effectively silencing four target genes simultaneously.

[0121] Further analysis: (1) When shRNA transcription units are combined in a “head-to-tail” manner (sh-

[404] , sh-

[407] , sh-

[408] ), the silencing effect of the four target genes in the comparative sh-

[404] combination using the four identical promoters hU6 is lower than that of other combinations, while the silencing effect of the four target genes in the sh-

[407] and sh-

[408] combinations using different promoters is higher, indicating that using different promoters is more effective for simultaneously targeting and silencing four different target genes.

[0122] (2) By comparing sh-

[401] , sh-

[402] , sh-

[403] , sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] , it is shown that the combination of the four promoter transcription units connected in a "head-to-tail" manner (sh-

[407] and sh-

[408] ) is more effective.

[0123] Example 4: Determination of the persistence of silencing effect of quadruple shRNA combination modification This example describes the testing of the persistence of silencing effects on four target genes using a quadruple shRNA combination design.

[0124] The inventors tested the persistence of silencing four target genes in cells constructed using a quadruple shRNA combination design. NKG cell lines stably transfected with the sh-

[401] , sh-

[402] , sh-

[404] , sh-

[405] , sh-

[407] , and sh-

[408] combinations established in Example 3 were used. The silencing efficiency of target genes (TIGIT, TIPE2, FAS, A2AR) in these NKG cell lines was detected using qRT-PCR, following the method described in 1.3 of Example 1. Testing began on day 6 post-infection and was repeated every 3-4 days until day 24 post-infection.

[0125] The results are as follows Figure 4 As shown, within the continuous detection time range, the comparative design of sh-

[404] using four identical hU6 promoters showed a silencing efficiency of only 20%~30% for the four target genes TIGIT, TIPE2, FAS, and A2AR, indicating that the silencing effect of sh-

[404] on the four target genes was low and could not achieve the expected high silencing effect. The silencing efficiency of the first target gene TIGIT in the sh-

[401] , sh-

[402] , and sh-

[405] combinations was 40%~60%, the silencing efficiency of the second target gene TIPE2 was 50%~70%, the silencing efficiency of the third target gene FAS was 50%~70%, and the silencing efficiency of the fourth target gene A2AR was 40%~60%. The silencing effect and silencing persistence were significantly improved compared with the comparative design sh-

[404] . The sh-

[407] and sh-

[408] combinations exhibited high and stable silencing effects. Specifically, the silencing efficiency of the first target gene TIGIT remained stable at 60%–90%, the second target gene TIPE2 at 60%–80%, the third target gene FAS at 60%–80%, and the fourth target gene A2AR at 50%–70%. Both the silencing effect and persistence were significantly improved compared to the control group sh-

[404] , with the largest increase observed in sh-

[407] , and the improvement was even more significant. These results indicate that the sh-

[407] and sh-

[408] combinations have a greater advantage in simultaneously silencing four target genes.

[0126] Example 5: Enhancement of immune cell killing activity by quadruple shRNA combination modification To verify the effect of quadruple shRNA combination modification on the effector function of immune cells, this example describes the results of the detection of NKG cell killing activity of quadruple shRNA combination modification.

[0127] The inventors tested the cytotoxic activity of cells constructed using a quadruple shRNA combination design. NKG cell lines stably transfected with NTC, the comparative sh-

[404] , sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] combinations established in Example 3, and the control NKG cell line with TIGIT silenced alone were used to test the cytotoxicity of target cells K562.

[0128] The results are as follows Figure 5 As shown, compared with the nonsense control (NTC) group, the sh-TIGIT control group and the combinations of sh-

[405] , sh-

[406] , sh-

[407] , and sh-

[408] all exhibited significantly enhanced NKG cell killing activity. This indicates that the modification of NKG cells by the quadruple shRNA combination significantly improved the NKG cell killing efficiency. Among them, the sh-

[407] combination had the highest killing efficiency and was also significantly improved compared with the control group that silenced TIGIT alone. This suggests that this combination simultaneously silences four genes, especially knocking down TIGIT expression, which can reverse TIGIT-mediated inhibitory signals and enhance the killing function of NK cells. Other quadruple shRNA combinations with high silencing efficiency also showed the same enhanced efficacy.

[0129] Specific steps of cell killing experiment: Target cell plating: Transfer K562 target cells to a 50 mL centrifuge tube, centrifuge at 300×g for 5 min, discard the supernatant, resuspend the cells in an appropriate volume of 1640 complete culture medium, gently mix with a pipette, count the cells, and adjust the cell concentration to 1×10⁻⁶. 6 / mL. Mix the prepared cells and CFSE solution at a volume ratio of 1:1000 and incubate at 37°C for 15 min. After incubation, add 5 volumes of PBS, centrifuge at 200 g for 5 min, and discard the supernatant. Resuspend the cell pellet in 1640 medium, count the cells, and adjust the cell concentration to 1×10⁹ / mL. 6 / mL. Prepare a cell suspension at a concentration of 100 μL / well and seed it into a 96-well plate with round bottoms.

[0130] Effector cell plating: Transfer the required effector cells to 50 mL centrifuge tubes, centrifuge at 300×g for 5 min, discard the supernatant, resuspend in an appropriate volume of 1640 complete culture medium, gently mix with a pipette, count the cells, and adjust the cell concentration to 1×10⁻⁶. 6 / mL. Perform cell counting and adjust the cell concentration to 1×10⁹ / mL. 6 / mL. Remove the 96-well plate inoculated with target cells from the incubator. At an effector-to-target ratio of 1:1, inoculate 100 μL / well of effector cells, adjusted to the desired cell concentration, into the 96-well plate, mix well by pipetting, and return the cells to the incubator for further culture.

[0131] Flow cytometry analysis: 4 h after cell death, completely aspirate the original culture medium and cells from the wells and add them to 2.0 ml EP tubes. 5–10 min before flow cytometry analysis, add 5 μL of PI solution to each group, mix well, and then analyze.

[0132] Formula for calculating lethality: Kill efficiency = Kill experimental group CFSE + PI + % - Natural death group CFSE + PI + %.

[0133] Example 6: Detection of the Proliferative Capacity of Immune Cells Modified by Quadruple shRNA Combinations This embodiment describes the detection of the proliferation capacity of NKG cells modified by a quadruple shRNA combination.

[0134] The inventors tested the proliferation capacity of cells constructed using a quadruple shRNA combination design. The proliferation capacity of NKG cell lines stably transfected with the NTC, sh-

[407] , and sh-

[408] combination established in Example 3, and the control NKG cell line with TIPE2 silenced alone, was tested under low concentrations of IL2 (10 UI / mL) and IL15 (10 ng / mL).

[0135] Cell proliferation experiment: NTC, sh-TIPE2, sh-

[407] , and sh-

[408] cells in good growth condition were used to stably transform NKG cell lines. These were first cultured in serum-free NKG cell culture medium for 24 h, and then the same number (20 × 10⁶ cells) were separately cultured. 4 NKG cells (number per mL) were resuspended in NKG cell culture medium containing low concentrations of IL2 (10 UI / mL) and IL15 (10 ng / mL) and seeded in 12-well plates. Cells were counted every 48 hours and NKG cell culture medium containing low concentrations of IL2 (10 UI / mL) and IL15 (10 ng / mL) was added. Cell proliferation curves for each group were plotted.

[0136] The results are as follows Figure 6As shown, compared with the nonsense control (NTC) group, the proliferation capacity of the sh-TIPE2 control group, the sh-

[407] and sh-

[408] combinations was significantly improved. This indicates that the modification of NKG cells by the quadruple shRNA combination significantly improved the proliferation capacity of NKG cells, especially by knocking down TIPE2 expression, which blocked the TIPE2-mediated inhibition of cell proliferation signaling and effectively improved the proliferation capacity of NK cells. Other quadruple shRNA combinations with high silencing efficiency also showed the same efficacy improvement.

[0137] Example 7: Detection of the ability of immune cells modified with quadruple shRNA to resist apoptosis This embodiment describes the detection of the apoptosis resistance of NKG cells modified with a quadruple shRNA combination.

[0138] The FASL / FAS pathway is a highly effective NK cell-mediated cell-killing mechanism. NK cell-expressed FASL binds to target cell FAS, transmitting a "death signal" and inducing rapid apoptosis in the target cells. FAS acts as a cell death receptor; the ligand FASL expressed by immune cells interacts with FAS expressed by tumor cells, inducing tumor cell apoptosis. However, during cancer cell progression, FAS expression is downregulated and FASL expression increases, triggering FASL / FAS apoptosis signaling and inducing immune cell apoptosis, allowing tumor cells to evade immune surveillance. Furthermore, immune cells highly express the cell death receptor FAS during culture and expansion, leading to reverse killing by tumor cells or induced apoptosis in the tumor microenvironment. Mutual killing or suicide mediated by FASL / FAS apoptosis signaling may also occur between immune cells. In clinical immunotherapy, donor-derived therapeutic immune cells with high FAS expression may be rejected by the patient's immune system, weakening the killing efficacy and reducing the durability of the treatment. Therefore, it is crucial to design and develop novel immunotherapy strategies targeting FAS to enhance the survival and persistence of immune cells in the complex tumor microenvironment and to stimulate the potential of immune cells in treating tumors.

[0139] The inventors further verified that quadruple shRNA combination modification silencing FAS can resist FASL-mediated apoptosis. The specific method is as follows: Take the same amount (1×10⁻⁶) of each... 5NTC cells (number / mL), a control group with FAS silenced alone, and sh-

[407] cells containing a quadruple shRNA combination containing the FAS target gene were added to 12-well plates, and 100 ng / mL FASL protein (AdipoGen Life Sciences) was added to induce apoptosis. Apoptosis was monitored using an apoptosis detection kit (Annexin V-APC / 7-AAD Apoptosis Kit). Staining was performed according to the kit instructions, and then apoptosis was detected by flow cytometry.

[0140] This embodiment exemplifies how quadruple shRNA combination modification silencing FAS in immune cells can resist FASL-induced apoptosis. Results are as follows... Figure 7 As shown, compared with the nonsense control group (NTC), the apoptosis rate of NKG cells modified with the sh-FAS control group and the quadruple shRNA combination containing the FAS target gene silencing significantly decreased. This indicates that the modification of NKG cells by the quadruple shRNA combination significantly enhances the resistance of NKG cells to apoptotic pressure, especially by knocking down FAS expression, which can block FASL binding-induced apoptosis and effectively improve NK cell survival. Other quadruple shRNA combinations with high silencing efficiency also showed the same efficacy enhancement.

[0141] Example 8: Detection of the ability of four-fold shRNA combination-modified immune cells to adapt to the tumor immunosuppressive microenvironment This embodiment describes the detection of the ability of quadruple shRNA combination modification to adapt to the tumor immunosuppressive microenvironment.

[0142] Adenosine (ADO) is an important signaling molecule that regulates the body's immune response and maintains homeostasis. Under normal body conditions, extracellular adenosine is maintained at a low concentration level (below 1 μM). When the body's tissues are damaged, such as by inflammation, ischemia, or hypoxia, the concentration of extracellular adenosine rises sharply (up to 100 μM), inhibiting the activity and effector function of immune cells, thereby preventing excessive damage to body tissues. However, in the tumor microenvironment of solid tumors, when extracellular adenosine remains at a high concentration level (around 100 μM), adenosine inhibits the function of various immune cells by binding to the adenosine receptor A2AR on the surface of immune cells, leading to a downregulation of the anti-tumor immune response. This is an important mechanism of tumor immune escape and one of the important reasons why immunotherapy is not effective against solid tumors.

[0143] The inventors further verified that the quadruple shRNA combination modification silencing A2AR can resist the inhibition of immune cell function by the tumor microenvironment containing high concentrations of adenosine. The specific method is as follows: 100 μM adenosine was added to the NK cell culture medium to simulate the tumor suppressive microenvironment containing high concentrations of adenosine. NTC cells, the control group that silenced A2AR alone, and sh-

[407] cells containing the quadruple shRNA combination containing the A2AR target gene were cultured in the culture medium containing 100 μM adenosine for 24 h. Then, the same number (2 × 10⁻⁶) of cells were collected. 5 NTC cells (1 × 10⁻⁶ cells / mL) and sh-[40⁷] cells were added to a 96-well round-bottom plate, and then mixed with the same number of NTC cells (1 × 10⁻⁶ cells / mL). 5 K562 cells (number / mL) were co-incubated for 4 hours, and the expression and killing activity of the cytotoxic factor IFN-γ were detected by flow cytometry.

[0144] This embodiment exemplifies how immune cells modified with a quadruple shRNA combination to silence A2AR can adapt to a tumor-suppressive microenvironment containing high concentrations of adenosine. Results are as follows... Figure 8 As shown, the cytotoxic activity and expression of the cytotoxic factor IFN-γ in NKG cells modified with a quadruple shRNA combination containing the A2AR target gene silencing were significantly higher than those in the NTC group. This indicates that the modification of NKG cells by the quadruple shRNA combination significantly enhances the cytotoxic activity of NKG cells in an inhibitory environment. In particular, knocking down A2AR expression allows NKG cells to maintain cytotoxic activity in a high-concentration adenosine-inhibitory microenvironment, thus adapting to the tumor immunosuppressive microenvironment. Other quadruple shRNA combinations with high silencing efficiency also showed the same efficacy enhancement.

[0145] Example 9: Detection of the knockdown effect of a quadruple shRNA combination strategy on different target genes This embodiment explores whether the quadruple shRNA combination strategy is applicable to the knockdown of multiple different target genes, and tests the knockdown effect on other target genes besides TIGIT, TIPE2, FAS, and A2AR.

[0146] In the above-mentioned

[407] promoter combinations with good knockdown effects, the shRNA sequences of target genes at each position were changed to test the universality of the

[407] promoter combinations for knockdown effects on other different target genes. The inventors replaced the target genes TIGIT, TIPE2, FAS, and A2AR shRNAs of the

[407] combination with shRNA sequences targeting the target genes TIM3 (an NK cell inhibitory receptor that inhibits NK cell activation and effector function after binding to its corresponding ligand on the surface of tumor cells, promoting immune escape of tumor cells), CISH (an intracellular negative regulator of IL-15 signaling, which inhibits the signaling of cytokines such as IL15, thereby inhibiting NK cell proliferation and anti-tumor activity), FAS (a cell death receptor that causes apoptosis after binding to its ligand FASL), and HIF1A (hypoxia-inducible factor that inhibits NK cell anti-tumor activity under hypoxic conditions), defining this as the [407-2] combination. This combination aims to improve NK cell killing activity, reverse the immunosuppressive microenvironment, improve in vivo survival and proliferation, resist apoptosis, and enhance survival ability and effector function under hypoxic conditions in vivo by simultaneously silencing four target genes: TIM3, CISH, FAS, and HIF1A.

[0147] The virus was synthesized and packaged in its entirety, and used to infect and construct stably knocked-down NKG cell lines through Puromycin screening. Individual NKG cell lines with individual gene knockdowns (sh-TIM3, sh-CISH, sh-FAS, sh-HIF1A) were then revived and cultured. Using the method described in Example 1.3, qRT-PCR was employed to detect the silencing efficiency of target genes (TIM3, CISH, FAS, HIF1A) in NKG cell lines with different quadruple shRNA combinations, and the knockdown efficiency of the corresponding genes in NKG cell lines with individual gene knockdowns. Results are as follows: Figure 9 As shown, compared with the nonsense control sequence group (NTC), the sh-[407-2] combination exhibits higher silencing efficiencies for the target genes TIM3, CISH, FAS, and HIF1A, with silencing efficiencies reaching 60%–80%. Furthermore, the silencing efficiency for each target gene is comparable to the knockdown efficiency of individual target gene knockdown cells. This indicates that the

[407] promoter arrangement can achieve good simultaneous silencing effects for different genes, and this quadruple shRNA promoter combination design is suitable for simultaneously silencing any four different target genes, demonstrating universality in its silencing effect.

[0148] The inventors further tested the cytotoxic activity of NKG cells constructed using the sh-[407-2] combination design. Tumor cells typically express ligands of inhibitory receptors such as TIM-3 (e.g., Galectin-9, CEACAM1), which inhibit the activation and effector function of immune cells such as NK cells by binding to inhibitory receptors like TIM-3 on the surface of immune cells, even leading to immune cell exhaustion. The inventors selected tumor cells known to express the important TIM3 ligands Galectin-9 and CEACAM1 (hepatocellular carcinoma cells (HepG2), colon cancer cells (HCT116), and breast cancer cells (MDA-MB-231)) as target cells for cytotoxicity testing. The flow cytometry results of the expression of the important TIM3 ligands Galectin-9 and CEACAM1 in tumor cells are shown below. Figure 10 As shown, HepG2 liver cancer cells, HCT116 colon cancer cells, and MDA-MB-231 breast cancer cells all highly express the important TIM3 ligands Galectin-9 and CEACAM1. The killing efficiency results are as follows... Figure 11 As shown, compared with the nonsense control sequence group (NTC), the sh-[407-2] combination showed significantly enhanced killing activity, and the killing activity of the sh-[407-2] combination was higher than that of knocking down TIM3 alone. This indicates that the

[407] promoter arrangement significantly improved the killing efficiency of NKG cells, and this quadruple shRNA promoter combination design can achieve the functional effect of corresponding gene silencing, demonstrating universality in terms of function.

[0149] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0150] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A quadruple shRNA combination, characterized in that, The quadruple shRNA combination comprises a first transcription unit, a second transcription unit, a third transcription unit, and a fourth transcription unit. The four transcription units are located on the same expression vector and target and silence four different target genes, respectively. Each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence, and a transcription termination signal; the promoters driving the first, second, third, and fourth transcription units are respectively the first promoter, the second promoter, the third promoter, and the fourth promoter, which are selected from the promoter group composed of hU6, mU6, hH1, h7SK, minihU6, H1-7SK, and hH1-core, and the first promoter, the second promoter, the third promoter, and the fourth promoter are all different from each other.

2. The quadruple shRNA combination according to claim 1, characterized in that, The first transcription unit contains a first shRNA molecule, the second transcription unit contains a second shRNA molecule, the third transcription unit contains a third shRNA molecule, and the fourth transcription unit contains a fourth shRNA molecule.

3. The quadruple shRNA combination according to claim 1 or 2, characterized in that, The first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, hH1, mU6, and h7SK, respectively; or, The first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hH1, and h7SK, respectively; or, The first promoter, the second promoter, the third promoter, and the fourth promoter are all selected from hU6; or, The first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, mU6, hU6, and mU6, respectively; or, The first, second, third, and fourth promoters are selected from hU6, mU6, hH1-core, and H1-7SK, respectively; or, The first promoter, the second promoter, the third promoter, and the fourth promoter are selected from hU6, minihU6, hH1-core, and H1-7SK, respectively.

4. The quadruple shRNA combination according to any one of claims 1-3, characterized in that, The first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit are connected in at least one of the following ways: head-to-head, tail-to-tail, and head-to-tail.

5. The quadruple shRNA combination according to claim 4, characterized in that, The four different target genes are selected from one of the following combinations: (a) TIGIT, TIPE2, FAS and A2AR; (b) TIM3, CISH, FAS and HIF1A.

6. The quadruple shRNA combination according to claim 5, characterized in that, When the four different target genes are TIGIT, TIPE2, FAS, and A2AR: The first transcription unit is connected head-to-head to the second transcription unit, the second transcription unit is connected tail-to-tail to the third transcription unit, and the third transcription unit is connected head-to-head to the fourth transcription unit; or, The first, second, and third transcription units are connected head-to-tail, with the third transcription unit and the fourth transcription unit connected head-to-head; or, The first transcription unit, the second transcription unit, the third transcription unit, and the fourth transcription unit are connected in a head-to-tail manner.

7. The quadruple shRNA combination according to claim 6, characterized in that, When the four different target genes are TIGIT, TIPE2, FAS, and A2AR: The first transcription unit is driven by the hU6 promoter, the second transcription unit by the hH1 promoter, the third transcription unit by the mU6 promoter, and the fourth transcription unit by the h7SK promoter. The first and second transcription units are connected head-to-head, the second and third transcription units are connected tail-to-tail, and the third and fourth transcription units are connected head-to-head; or... The first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hH1 promoter, and the fourth transcription unit by the h7SK promoter. The first and second transcription units are connected head-to-head, the second and third transcription units are connected tail-to-tail, and the third and fourth transcription units are connected head-to-head; or... The first, second, third, and fourth transcription units are all driven by the hU6 promoter, and the first and second transcription units are connected head-to-head, the second and third transcription units are connected tail-to-tail, and the third and fourth transcription units are connected head-to-head; or, The first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hH1 promoter, and the fourth transcription unit by the h7SK promoter. The first, second, and third transcription units are connected head-to-tail, and the third and fourth transcription units are connected head-to-head; or... The first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hU6 promoter, and the fourth transcription unit by the mU6 promoter. Furthermore, the first and second transcription units are connected head-to-head, the second and third transcription units are connected tail-to-tail, and the third and fourth transcription units are connected head-to-head; or... The first transcription unit is driven by the hU6 promoter, the second transcription unit by the mU6 promoter, the third transcription unit by the hH1-core promoter, and the fourth transcription unit by the H1-7SK promoter, and the first, second, third, and fourth transcription units are connected head-to-tail; or, The first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the minihU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the first, second, third, and fourth transcription units are connected head-to-tail.

8. The quadruple shRNA combination according to claim 7, characterized in that, When the four different target genes are TIGIT, TIPE2, FAS, and A2AR: The sense strand sequence of the first shRNA molecule targeting TIGIT has a nucleotide sequence shown in at least one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22; The sense strand sequence of the second shRNA molecule targeting TIPE2 has a nucleotide sequence shown in at least one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; The sense strand sequence of the third shRNA molecule targeting FAS has a nucleotide sequence shown in at least one of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58; The sense strand sequence of the fourth shRNA molecule targeting A2AR has a nucleotide sequence shown in at least one of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO:

74.

9. The quadruple shRNA combination according to claim 5, characterized in that, When the four different target genes are TIM3, CISH, FAS, and HIF1A: The first transcription unit is driven by the hU6 promoter, the second transcription unit is driven by the mU6 promoter, the third transcription unit is driven by the hH1-core promoter, and the fourth transcription unit is driven by the H1-7SK promoter, and the first, second, third, and fourth transcription units are connected head-to-tail.

10. The quadruple shRNA combination according to claim 9, characterized in that, When the four different target genes are TIM3, CISH, FAS, and HIF1A: The sense strand of the first shRNA molecule targeting TIM3 has the nucleotide sequence shown in SEQ ID NO: 76; The sense strand of the second shRNA molecule targeting CISH has the nucleotide sequence shown in SEQ ID NO: 78; The sense strand sequence of the third shRNA molecule targeting FAS has a nucleotide sequence shown in at least one of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58; The positive strand sequence of the fourth shRNA molecule targeting HIF1A has the nucleotide sequence shown in SEQ ID NO:

80.

11. A nucleic acid construct, characterized in that, The nucleic acid construct comprises a quadruple shRNA combination as described in any one of claims 1-10.

12. An expression carrier, characterized in that, The expression vector comprises the nucleic acid construct of claim 11.

13. A host cell, characterized in that, The host cell comprises the nucleic acid construct of claim 11 or the expression vector of claim 12.

14. The host cell according to claim 13, characterized in that, The host cell is selected from at least one of NK cells, T cells, NKT cells, γδT cells, and macrophages.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the host cell as described in claim 13 or 14, and a pharmaceutically acceptable carrier.

16. Use of the host cell of claim 13 or 14, or the pharmaceutical composition of claim 15, in the preparation of a medicament for treating tumors, autoimmune diseases, chronic inflammation, or age-related diseases.

17. The use according to claim 16, characterized in that, The tumors include at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer, melanoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms. And / or, the autoimmune disease includes at least one selected from systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma; And / or, the chronic inflammation or age-related disease includes at least one selected from neurodegenerative diseases, cardiovascular diseases, metabolic diseases, cancer, immune system diseases, and musculoskeletal diseases.

18. The use according to claim 17, characterized in that, The neurodegenerative diseases include at least one of Alzheimer's disease and Parkinson's disease; And / or, the cardiovascular disease includes at least one of coronary artery disease, myocardial infarction, and stroke; And / or, the metabolic disease includes at least one of hypertension, type 2 diabetes, and hypercholesterolemia.

19. A method for enhancing the function of immune cells, characterized in that, The method includes introducing the quadruple shRNA combination of any one of claims 1-10, the nucleic acid construct of claim 11, or the expression vector of claim 12 into the immune cells to simultaneously silence the four different target genes.

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