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CN-121975801-A - Multiplex shRNA combinations and uses thereof

CN121975801ACN 121975801 ACN121975801 ACN 121975801ACN-121975801-A

Abstract

The invention relates to a multiple shRNA combination and application thereof, and belongs to the technical field of biology. The combination comprises a first transcription unit, a second transcription unit and a third transcription unit which are positioned on the same expression vector and respectively target and silence three different target genes. Each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence and a transcription termination signal, and the promoters driving the three transcription units are mutually different and are selected from any three of hU6, mU6, hH1, H7SK, mini-hU6, H1-7SK and hH 1-core. The technology realizes the simultaneous high-efficiency, stable and lasting silencing of three target genes by avoiding homologous recombination, can be constructed into a nucleic acid construct and an expression vector and introduced into immune cells, effectively enhances the function of the immune cells, and has application prospects in the fields of treating tumors, autoimmune diseases, aging-related diseases and the like.

Inventors

  • ZHOU YINGLI
  • WANG TING
  • ZHANG CAI
  • CHEN MINHUA
  • ZHU XIAOPING

Assignees

  • 上海恩凯细胞技术有限公司

Dates

Publication Date
20260505
Application Date
20251211

Claims (19)

  1. 1. A multiple shRNA combination, comprising a first transcription unit, a second transcription unit, and a third transcription unit, wherein the first transcription unit, the second transcription unit, and the third transcription unit are disposed on the same expression vector and are targeted to silence three different target genes, respectively; wherein each of said transcriptional units comprises an RNA polymerase III promoter, an shRNA coding sequence, and a transcription termination signal; And, the promoters driving the first transcription unit, the second transcription unit, and the third transcription unit are a first promoter, a second promoter, and a third promoter, respectively, which are different from each other and are selected from any three of hU6, mU6, hH1, H7SK, mini-hU6, H1-7SK, and hH 1-core.
  2. 2. The multiplex shRNA combination of claim 1, wherein the first transcription unit comprises a first shRNA molecule, the second transcription unit comprises a second shRNA molecule, and the third transcription unit comprises a third shRNA molecule.
  3. 3. The multiplex shRNA combination of claim 1, wherein the first promoter, the second promoter, and the third promoter are selected from the group consisting of hU6, hH1, and mU6, respectively, or, The first promoter, the second promoter and the third promoter are respectively selected from hU6, mU6 and hH1, or, The first promoter, the second promoter and the third promoter are respectively selected from hU6, mini-hU6 and H1-7SK, or, The first promoter, the second promoter and the third promoter are respectively selected from hU6, H7SK and H1-7SK.
  4. 4. A multiplex shRNA combination according to any of claims 1-3, wherein the first transcription unit, the second transcription unit and the third transcription unit are linked in at least one of head-to-head, end-to-end and end-to-end.
  5. 5. The multiplex shRNA combination according to any one of claims 1-4, wherein the three different target genes are selected from one of (a) TIGIT, CISH and HIF1A and (b) TIM3, TIPE2 and FAS.
  6. 6. The multiplex shRNA combination of claim 5, wherein when the three different target genes are TIGIT, CISH, and HIF1A, the first transcription unit is head-to-head linked to the second transcription unit and the second transcription unit is tail-to-tail linked to the third transcription unit, or the first transcription unit, the second transcription unit, and the third transcription unit are head-to-tail linked.
  7. 7. The multiplex shRNA combination of claim 6, wherein when the three different target genes are TIGIT, CISH, and HIF1A, the first transcription unit is driven by a hU6 promoter, the second transcription unit is driven by a hH1 promoter, the third transcription unit is driven by a mU6 promoter, and the first transcription unit is head-to-head linked to the second transcription unit, and the second transcription unit is tail-to-tail linked to the third transcription unit, or, The first transcription unit is driven by a hU6 promoter, the second transcription unit is driven by a mU6 promoter, the third transcription unit is driven by a hH1 promoter, the first transcription unit is connected with the second transcription unit head to head, the second transcription unit is connected with the third transcription unit tail to tail, or, The first transcription unit is driven by a hU6 promoter, the second transcription unit is driven by a mini-hU6 promoter, the third transcription unit is driven by an H1-7SK promoter, and the first transcription unit, the second transcription unit and the third transcription unit are connected in an end-to-end manner, or, The first transcription unit is driven by a hU6 promoter, the second transcription unit is driven by a mU6 promoter, the third transcription unit is driven by a hH1 promoter, and the first transcription unit, the second transcription unit and the third transcription unit are connected in an end-to-end manner, or, The first transcription unit is driven by an hU6 promoter, the second transcription unit is driven by an H7SK promoter, the third transcription unit is driven by an H1-7SK promoter, and the first transcription unit, the second transcription unit and the third transcription unit are connected in a head-to-tail mode.
  8. 8. The multiplex shRNA combination of claim 7, wherein when the three different target genes are TIGIT, CISH, and HIF 1A: The sense strand sequence of the first shRNA molecule targeting TIGIT has a nucleotide sequence shown as at least one of SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16; The sense strand sequence of the second shRNA molecule targeting CISH has a nucleotide sequence shown as at least one of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 and SEQ ID NO. 32; The sense strand sequence of the third shRNA molecule that targets HIF1A has a nucleotide sequence as set forth in at least one of SEQ ID NO: 44、SEQ ID NO: 45、SEQ ID NO: 46、SEQ ID NO: 47、SEQ ID NO: 48、SEQ ID NO: 49、SEQ ID NO: 50、SEQ ID NO: 51、SEQ ID NO: 52、SEQ ID NO: 53 and SEQ ID No. 54.
  9. 9. The multiplex shRNA combination of claim 5, wherein when the three different target genes are TIM3, TIPE2 and FAS, the first transcription unit is driven by a hU6 promoter, the second transcription unit is driven by a mU6 promoter, the third transcription unit is driven by a hH1 promoter, and the first transcription unit, the second transcription unit and the third transcription unit are joined end-to-end.
  10. 10. The multiplex shRNA combination of claim 9, wherein when the three different target genes are TIM3, TIPE2 and FAS: The sense strand of the first shRNA molecule targeting TIM3 has the nucleotide sequence shown as SEQ ID No. 56; the sense strand of the second shRNA molecule that targets TIPE2 has the nucleotide sequence shown as SEQ ID No. 58; The sense strand sequence of the third shRNA molecule that targets FAS has a nucleotide sequence shown as SEQ ID NO. 60.
  11. 11. A nucleic acid construct comprising the multiplex shRNA combination of any one of claims 1-10.
  12. 12. An expression vector comprising the nucleic acid construct of claim 11.
  13. 13. A host cell comprising the nucleic acid construct of claim 11 or the expression vector of claim 12.
  14. 14. The host cell of claim 13, wherein the host cell is selected from at least one of NK cells, T cells, NKT cells, γδ T cells, and macrophages.
  15. 15. A pharmaceutical composition comprising the host cell of claim 13 or 14 and a pharmaceutically acceptable carrier.
  16. 16. Use of a host cell according to claim 13 or 14, or a pharmaceutical composition according to claim 15, for the manufacture of a medicament for the treatment of a tumor, an autoimmune disease, chronic inflammation or a disease associated with aging.
  17. 17. The use according to claim 16, wherein the tumour comprises at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, stomach cancer, oesophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, renal cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer, melanoma, acute myeloid leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia, chronic lymphocytic leukaemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative tumour; and/or the autoimmune disease comprises at least one selected from systemic lupus erythematosus, rheumatoid arthritis, sjogren'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 aging-related disorder includes at least one selected from the group consisting of neurodegenerative disorders, cardiovascular disorders, metabolic disorders, cancer, immune system disorders, and musculoskeletal disorders.
  18. 18. The use according to claim 17, wherein the neurodegenerative disease comprises at least one of alzheimer's disease and parkinson's disease; and/or, the cardiovascular disease comprises at least one of coronary artery disease, myocardial infarction, and stroke; And/or the metabolic disease comprises at least one of hypertension, type 2 diabetes, and hypercholesterolemia.
  19. 19. A method of enhancing immune cell function, comprising introducing into the immune cell the multiplex shRNA combination of any one of claims 1-10, the nucleic acid construct of claim 11, or the expression vector of claim 12 to simultaneously silence the three different target genes.

Description

Multiplex shRNA combinations and uses thereof Technical Field The invention relates to the field of biotechnology, in particular to a multiplex shRNA combination and application thereof, and more particularly relates to a nucleic acid construct, an expression vector, a host cell, a pharmaceutical composition and a method for improving immune cell functions. Background Genetically modified cell immunotherapy, in particular based on CAR-T, CAR-NK and the like, has made a breakthrough in cancer treatment. However, this type of therapy is still not ideal in the treatment of solid tumors, mainly due to the immunosuppressive properties of the Tumor Microenvironment (TME), such as hypoxia, immune checkpoint upregulation, inhibitory signaling, and insufficient persistence of the infused cells in the body. To enhance therapeutic effects, it is desirable to modify immune cells at multiple targets while silencing multiple negative regulatory genes, such as immune checkpoint molecules (e.g., TIGIT, TIM 3), inhibitory signaling proteins (e.g., CISH, TIPE 2), and environmental stress factors (e.g., HIF 1A). However, achieving efficient and durable silencing of multiple genes within the same cell remains a technical challenge. The CRISPR/Cas9 isogenic editing technology has the problems of off-target effect, DNA damage risk and potential safety, and has limited clinical application. RNA interference-based techniques, such as shRNA, have the potential for their high specificity and safety. However, the existing shRNA technology has the limitation in the aspect of multi-gene silencing that when a plurality of identical promoters (such as hU 6) are used for constructing a multi-shRNA vector in series, homologous recombination is easily initiated by repeated sequences, so that the gene silencing efficiency is reduced or lost. Although double shRNA vectors have been reported, no reliable solution capable of simultaneously guaranteeing high efficiency, stability and durability is available at present for synchronous silencing of three or more genes. In particular, when three genes are involved, the problem of homologous recombination caused by the use of the same promoter is greatly amplified, and simply combining different promoters but misarranging does not ensure that all three target genes are silenced uniformly and efficiently. Therefore, there is an urgent need in the art to develop a new strategy for shRNA design that can achieve efficient, stable and durable silencing of three or more target genes on the same vector. Disclosure of Invention The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the application provides a multiplex shRNA combination. The present application has been completed based on the following findings by the inventors: The inventors found that when three triple shRNA combinations targeting three different genes are constructed on the same vector, if three identical RNA polymerase III promoters (e.g., hU 6) are used, homologous recombination is rapidly induced due to the high repetition of the promoter sequences, resulting in loss of one or more shRNA expression cassettes, resulting in reduced gene silencing efficiency, unstable effect and inability to persist. This problem becomes particularly pronounced in the silencing of three genes, a key bottleneck that hampers their adoption to clinical applications. Unexpectedly, the inventors found through a large number of experiments that the occurrence of homologous recombination can be synergistically and effectively avoided by using three different sequences of RNA polymerase III promoters (e.g., hU6, mU6 and hH 1) in combination and adopting a specific arrangement. The specific design can ensure that three shRNA transcription units exist stably in a vector, and simultaneously realize efficient, balanced and durable silencing of three target genes. The invention successfully realizes the technical crossing from double shRNA to triple shRNA, and the discovery provides a brand new solution which is not disclosed before for realizing stable and efficient three-gene synchronous silencing on a single carrier. In one aspect of the application, the application provides a multiplex shRNA combination. According to an embodiment of the application, the multiplex shRNA combination comprises a first transcription unit, a second transcription unit and a third transcription unit which are arranged on the same expression vector and respectively target to silence three different target genes, wherein each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence and a transcription termination signal, and the promoters driving the first transcription unit, the second transcription unit and the third transcription unit are respectively a first promoter, a second promoter and a third promoter, and the first promoter, the second promoter and the third promote