Search

CN-121975802-A - Quadruple shRNA combination and application thereof

CN121975802ACN 121975802 ACN121975802 ACN 121975802ACN-121975802-A

Abstract

The invention relates to a quadruple shRNA combination and application thereof. The combination comprises a first, a second, a third and a fourth transcription unit, which are positioned on the same expression vector and respectively target to silence four different target genes. Each transcription unit comprises an RNA polymerase III promoter, an shRNA coding sequence and a transcription termination signal, the promoters driving the four transcription units are mutually different 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. The design effectively avoids expression interference and homologous recombination by optimizing the combination and arrangement of the promoters, and realizes the simultaneous efficient, stable, balanced and durable silencing of four target genes.

Inventors

  • ZHOU YINGLI
  • WANG TING
  • ZHANG CAI
  • CHEN MINHUA
  • XUE WEI

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 quadruple shRNA combination, which is characterized by comprising a first transcription unit, a second transcription unit, a third transcription unit and a fourth transcription unit, wherein the four transcription units are arranged on the same expression vector and respectively target to silence four different target genes; The transcription units comprise an RNA polymerase III promoter, an shRNA coding sequence and a transcription termination signal, wherein the promoters driving the first transcription unit, the second transcription unit, the third transcription unit and the fourth transcription unit are respectively a first promoter, a second promoter, a third promoter and a fourth promoter, the first promoter, the second promoter, the third promoter and the fourth promoter are selected from a promoter group consisting 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 mutually different.
  2. 2. The quadruple shRNA combination of claim 1, wherein the first transcription unit comprises a first shRNA molecule, the second transcription unit comprises a second shRNA molecule, the third transcription unit comprises a third shRNA molecule, and the fourth transcription unit comprises a fourth shRNA molecule.
  3. 3. The quadruple shRNA combination of claim 1 or 2, wherein the first promoter, the second promoter, the third promoter and the fourth promoter are selected from the group consisting of hU6, hH1, mU6 and h7SK, respectively, The first promoter, the second promoter, the third promoter and the fourth promoter are respectively selected from hU6, mU6, hH1 and h7SK, 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 respectively selected from hU6, mU6, hU6 and mU6, or, The first promoter, the second promoter, the third promoter and the fourth promoter are respectively selected from hU6, mU6, hH1-core and H1-7SK, or, The first promoter, the second promoter, the third promoter and the fourth promoter are respectively selected from hU6, minihU6, hH1-core and H1-7SK.
  4. 4. A quadruple shRNA combination according to any of claims 1-3, wherein the first transcription unit, the second transcription unit, the third transcription unit and the fourth transcription unit are joined in at least one of head-to-head, end-to-end and end-to-end.
  5. 5. The quadruple shRNA combination of claim 4, wherein said four different target genes are selected from one of the following combinations: (a) TIGIT, tige 2, FAS, and A2AR; (b) TIM3, CISH, FAS, and HIF1A.
  6. 6. The quadruple shRNA combination of claim 5, wherein when the four different target genes are TIGIT, TIPE2, FAS, and A2 AR: 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, the third transcription unit is connected with the fourth transcription unit head to head, or, The first transcription unit, the second transcription unit and the third transcription unit are connected in a head-to-tail mode, the third transcription unit is connected with the fourth transcription unit in a head-to-head mode, 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 mode.
  7. 7. The quadruple shRNA combination of claim 6, wherein when the four different target genes are TIGIT, TIPE2, FAS, and A2 AR: 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, the fourth transcription unit is driven by a h7SK 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, the third transcription unit is connected with the fourth transcription unit head to head, 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 fourth transcription unit is driven by a h7SK promoter, 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, 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 all driven by an hU6 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, the third transcription unit is connected with the fourth transcription unit head to head, 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 fourth transcription unit is driven by a h7SK promoter, the first transcription unit, the second transcription unit and the third transcription unit are connected end to end, the third transcription unit is connected end to end with the fourth 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 hU6 promoter, the fourth transcription unit is driven by a mU6 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, the third transcription unit is connected with the fourth transcription unit head to head, 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-core promoter, the fourth transcription unit is driven by a H1-7SK promoter, and the first, second, third and fourth transcription units are connected end to end, or, The first transcription unit is driven by an hU6 promoter, the second transcription unit is driven by a minihU promoter, the third transcription unit is driven by an hH1-core promoter, the fourth transcription unit is driven by an H1-7SK promoter, and the first, second, third and fourth transcription units are connected end to end.
  8. 8. The quadruple shRNA combination of claim 7, wherein when the four different target genes are TIGIT, TIPE2, FAS, and A2 AR: The sense strand sequence of the first shRNA molecule targeted to TIGIT has a nucleotide sequence shown as 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 targeted to TIPE2 has a nucleotide sequence shown as 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 that targets FAS has a nucleotide sequence as set forth 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 as 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. 9. The quadruple shRNA combination of claim 5, wherein when the four different target genes are TIM3, CISH, FAS, and HIF 1A: 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-core promoter, the fourth transcription unit is driven by a H1-7SK promoter, and the first transcription unit, the second transcription unit, the third transcription unit and the fourth transcription unit are connected end to end.
  10. 10. The quadruple shRNA combination of claim 9, wherein when the four different target genes are TIM3, CISH, FAS, and HIF 1A: 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 that targets CISH has a nucleotide sequence as shown in SEQ ID No. 78; the sense strand sequence of the third shRNA molecule that targets FAS has a nucleotide sequence as set forth 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 HIF1A has a nucleotide sequence shown as SEQ ID NO. 80.
  11. 11. A nucleic acid construct comprising the quadruple 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 quadruple 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 four different target genes.

Description

Quadruple shRNA combination and application thereof Technical Field The invention relates to the field of biotechnology, in particular to a quadruple 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 complex immunosuppressive properties of the Tumor Microenvironment (TME), such as up-regulation of various immune checkpoints, inhibitory signaling, metabolic stress and induction of apoptosis, and insufficient persistence of infused cells in vivo. In order to significantly improve therapeutic effects, multiple target point deep modification of immune cells is required, and simultaneously, multiple key negative regulatory genes are silenced, such as immune checkpoint molecules (e.g., TIGIT, TIM 3), intracellular inhibitory signaling proteins (e.g., TIPE2, CISH), apoptotic receptors (e.g., FAS), and microenvironment stress factors (e.g., HIF1A, A AR). However, achieving efficient, balanced and durable simultaneous silencing of four genes within the same cell is a serious challenge faced by current technology. 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 obvious limitation in the aspect of multi-gene silencing, and although a plurality of different promoters are utilized to construct a double shRNA or triple shRNA vector, synchronous silencing of four or more genes is difficult to ensure that all target genes can obtain efficient, stable and lasting silencing effects in the prior art. The problem of genetic stability of the vector is even more pronounced when four genes are involved. Even if different promoters are used in combination, if the promoters are improperly selected or the arrangement mode of transcription units is not reasonable, the expression interference, the uneven efficiency or the increased risk of homologous recombination still easily occur, and the high requirements of clinical application on reliability and consistency cannot be met. Therefore, there is an urgent need in the art to develop a new strategy for designing shRNA that can achieve efficient, stable, balanced and durable silencing of four target genes on the same vector, and to verify its clear effect in enhancing immune cell function. 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. For this reason, the application provides a quadruple shRNA combination and application thereof. The present application has been completed based on the following findings by the inventors: The inventor finds that when four shRNA combinations targeting four different genes are constructed on the same vector, if a double-gene or three-gene strategy is simply adopted or four promoters are randomly combined, the optimal silencing effect of the four target genes cannot be achieved at the same time. Part of the genes have high silencing efficiency, and part of the genes have low silencing efficiency, and silencing effect is easy to decline or lose after long-term culture. This problem becomes extremely complex in the silencing of four genes, a key bottleneck that prevents them from going to clinical applications. Unexpectedly, the inventors found through extensive experimental screening and optimization that by selecting four different promoters from a group of specific RNA polymerase III promoters (hU 6, mU6, hH1, H7SK, minihU6, H1-7SK, hH 1-core) to combine and adopting a specific tandem arrangement of head-to-tail connection, the stable existence of four shRNA transcription units in a vector can be synergistically and effectively ensured, and meanwhile, the efficient, balanced and durable silencing of four target genes can be realized. The invention successfully realizes the technical crossing from three shRNAs to four shRNAs, and the discovery provides a brand new solution which is not disclosed before for realizing stable and efficient four-gene synchronous silencing on a single vector. In one aspect of the application, the application provides a quadruple shRNA combination. According to the embodiment of the application, the quadruple shRNA combination comprises a first transcription unit, a second transcription unit, a third transcription unit and a fourth transcription unit, wherein the four transcription units are arranged on the same expression vector and respectively target and silence