Method for promoting expression of stem cell-like memory T cells and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for promoting expression of stem cell-like memory T cells and application of the method. According to the application, a non-phase separation transcription factor is prepared by adopting a non-phase separation engineering reprogramming factor mode, then the non-phase separation transcription factor is introduced into a target cell, and the target cell is subjected to starting stage reprogramming. According to the present invention, through the specific design, the amino acid modification is performed on the specific site so as to weaken the phase separation ability, and then the corresponding codon optimization is performed to prepare the circular RNA with the purity gt; according to the present invention, the three target proteins can be expressed after the cells are transfected, and the proportion of Tscm in the T cell population can be effectively improved after the human primary T cell is transfected;

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to a method for promoting the expression of stem cell-like memory T cells and its application. Background Art

[0002] After being activated by antigens, T cells will produce effector T cells and memory T cells. The former has the ability to kill target cells but has a short survival period, while the latter has a long survival period and can maintain immunity for a long time. Specifically, memory T cells can be divided into Tscm, Tcm and Tem, among which Tscm has a strong ability to self-proliferate and differentiate into other types of memory T cells; while the ability of Tcm and Tem to transform into Tscm is very weak, so Tscm is considered to be the "stem cell" in T cells. According to current studies, after T cell products with a high proportion of memory T cells (such as Tscm) are transfused into individuals, exogenous T cells will remain longer and have better efficacy; however, in the production process of T cell products, due to individual differences in patients, the proportion of memory T cells in the final product is difficult to predict. At present, traditional technologies can usually increase the proportion of memory T cells through IL-7, IL-15 or metabolic reprogramming, and this method has been widely used in the in vitro production of T cells.

[0003] In addition to the above methods, epigenetic changes are important biological changes in the process of T cell activation. Epigenetic changes are indeed important biological changes in the process of T cell activation. These changes involve DNA methylation, histone modification, etc. They play a key role in the development, differentiation, activation and immune memory formation of T cells. After T cells are activated, they undergo transcriptional regulation and epigenetic changes and then enter the differentiation stage. Because they involve long-term changes in chromatin structure and stabilization of gene expression patterns, these changes are generally considered to be difficult to reverse and cannot be applied to T cell-related products at present.

[0004] There are currently no reports on the use of epigenetic reprogramming-based methods for T cell therapy products. Summary of the invention

[0005] Based on this, one embodiment of the present application provides a method for promoting the expression of stem cell-like memory T cells and its application.

[0006] On the one hand, the present application provides a method for promoting the expression of stem cell-like memory T cells, comprising:

[0007] A phase-separation-free engineered reprogramming factor is used to prepare a phase-separation-free transcription factor, which is introduced into a target cell to perform an initial phase reprogramming on the target cell, wherein the target cell includes a T cell.

[0008] The method of engineering reprogramming factors without phase separation includes mutating the regions related to the phase separation function of the transcription factors so that the transcription factors lack the function of inducing phase separation.

[0009] In some embodiments, the phase-separation-free transcription factor is introduced into the target cell to make CCR7 + CD45RA + The proportion of T cell population increased.

[0010] In some of these embodiments, the region associated with the phase separation function of the transcription factor includes the IDR region.

[0011] In some embodiments, the mutation treatment includes mutating the polar charged amino acids or acidic amino acids in the IDR region.

[0012] In some of these embodiments, the polar charged amino acid or the acidic amino acid is replaced with a neutral amino acid.

[0013] In some of these embodiments, the acidic amino acids are mutated to neutral amino acids.

[0014] In some of the embodiments, the amino acid sequence shown in SEQ ID NO.1 is mutated at positions 1 to 140 and / or positions 288 to 360.

[0015] In some of the embodiments, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

[0016] In some embodiments, the invention comprises: mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.1.

[0017] In some embodiments, the amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0018] In some embodiments, before the phase-separation-free transcription factor is introduced into cells, the step of preparing the phase-separation-free transcription factor into an element comprising the phase-separation-free transcription factor is further included.

[0019] In some embodiments, the elements include: one or more of: DNA, RNA, recombinant vector, recombinant protein and recombinant cell.

[0020] In some embodiments, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector, and a lentivirus vector.

[0021] In some embodiments, the RNA comprises one or more of linear mRNA, siRNA, circular RNA, circular mRNA and tRNA.

[0022] In one embodiment, the circular RNA comprises multiple transcription factors connected in series.

[0023] In some embodiments, the transcription factors are linked via a linker peptide.

[0024] Optionally, the nucleotide sequence of the connecting peptide is shown as SEQ ID NO.3 to SEQ ID NO.4.

[0025] In some embodiments, the element comprises one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

[0026] In some embodiments, the nucleotide sequence of the element is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0027] In some embodiments, introducing the phase separation-free transcription factor into the target cell comprises using a delivery system;

[0028] Optionally, the delivery system comprises one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

[0029] On the other hand, the present application provides the use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug for promoting the expression of stem cell-like memory T cells.

[0030] The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0031] In some embodiments, the nucleotide sequence of the element containing the phase separation-free transcription factor is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0032] In some embodiments, the drug includes a drug for cell therapy, gene therapy, immune cell proliferation, cancer treatment, or treatment of aging-related diseases.

[0033] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0035] Figure 1 is the order of the components of O'SK circular RNA (FlexCirc system);

[0036] Figure 2 This is the result of in vitro transcription and circularization verification;

[0037] Figure 3 The results of HPLC purification and testing;

[0038] Figure 4 Validate cell-level results for circular RNA protein expression;

[0039] Figure 5 Characterization of the phase separation of Oct4 transcription factor Static phase separation observation results;

[0040] Figure 6 Characterization of the phase separation of Oct4 transcription factor Dynamic phase separation observation results;

[0041] Figure 7 The results of T cell expansion and viability treated with O'SK circular RNA according to one embodiment of the present application;

[0042] Figure 8The results of the changes in the ratio of Tscm in T cells after treatment with the O'SK circular RNA according to one embodiment of the present application are shown. DETAILED DESCRIPTION

[0043] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] the term

[0046] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0047] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0048] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0049] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0050] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0051] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0052] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0053] The terms "identity" and "homology" as used in this application refer to the use of amino acid sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% similarity and still have the same function as the original amino acid sequence or nucleotide sequence.

[0054] The term "variant" used in connection with a peptide or polypeptide refers to a peptide or polypeptide whose amino acid sequence differs due to insertion, deletion or conservative substitution of amino acids but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence substantially identical to a reference protein, wherein the reference protein has an amino acid sequence retaining at least one biological activity. Conservative substitution of amino acids, i.e., replacing amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered to involve minor changes in the art. As understood in the art, these minor changes can be identified in part by considering the hydropathic index of amino acids. (Kyte et al., 1982, J. Mol. Biology. 157: 105-132). The hydropathic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. On the one hand, amino acids have a hydropathic index of ±2 and are replaced. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins retaining biological function. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measurement reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Amino acids with hydrophilicity values ​​that differ from each other by ±2 can be substituted. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, especially the side chains of those amino acids, such as hydrophobicity, hydrophilicity, charge, size and other properties.

[0055] As used herein, the term "variant" with respect to nucleic acids refers to (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions. A variant may be a nucleic acid sequence that is substantially identical over the entire length of a gene sequence or a fragment thereof. A nucleic acid, protein sequence may be 66%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of a gene, protein sequence, or a fragment thereof.

[0056] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0057] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0059] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0060] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0061] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0062] The term "epigenetic reprogramming" refers to the process of changing cell fate through epigenetic modifications such as DNA methylation without changing the gene sequence. Epigenetic reprogramming is a key biological process that involves the erasure and re-establishment of epigenetic marks across the genome. The term "reprogramming factor" has the ability to regulate the epigenetic properties of cells, restore cells to early differentiation or even undifferentiated states, and can convert adult or differentiated cells into pluripotent stem cells. Specifically, reprogramming factors are factors from the Oct family, Sox family, Klf family, Myc family, Nanog family, Glis family or Lin family. Among them, "Yamanaka factors or Yamanaka factors" include but are not limited to a combination of four transcription factor genes, Oct-4, Sox4, Klf4 and c-Myc (collectively referred to as OSKM genes). In some aspects, they may also be Oct4, Sox-2 and Klf4 genes (OSK genes). In some aspects, they may also be Oct4, Klf4 and c-Myc genes (OKM genes). In some aspects, they may also be Sox-2, Klf4 and c-Myc genes (OKMS genes). Generally, people use viral vectors to deliver and overexpress multiple reprogramming factors without restriction, which is a conventional method for preparing induced pluripotent stem cells. T cells can also form induced pluripotent stem cells in this way. Therefore, reducing the number of factors, controlling the time of factor expression, and changing the phase separation characteristics of the factors can achieve dedifferentiation of T cells and generate memory T cells without producing induced pluripotent stem cells. Current studies have shown that the expression of exogenous Yamanaka factors (including Oct3 / 4, Sox2, cMyc and Klf4) can reshape the epigenetic profile of cells, thereby changing cell identity, that is, epigenetic reprogramming.

[0063] Initiation phase reprogramming refers to the process of engineering cell reprogramming factors to keep the reprogramming of old cells in the initiation phase, thereby providing a longer safe time window for rejuvenation and greatly reducing the risk of becoming iPSCs. The main difference between initiation phase reprogramming and transient reprogramming is the engineering of cell reprogramming factors, which allows the reprogramming phase to be artificially controlled at the molecular level.

[0064] The term "liquid-liquid phase separation" (LLPS), also known as phase separation, refers to the fact that certain proteins or nucleic acid molecules in cells can produce another phase with different physical and chemical properties in the surrounding originally uniform liquid environment through multivalent interactions, usually in the form of micron-scale droplets (similar to oil droplets in water). These droplets form a relatively independent space, selectively enriching certain protein molecules, making them have higher protein density and weakened molecular microscopic movement, which can promote certain biochemical reaction results, such as gene transcription regulation, changes in chromosome spatial structure, etc. There are many mechanisms for protein phase separation, one of which depends on the intrinsically disordered domain (IDR) of the protein. The acidic polar amino acids in this domain carry negative charges and can interact to form phase separation. "No phase separation" refers to the mutation of the IRD sequence or key amino acids that form phase separation, resulting in the inability of the variant protein to form LLPS, thereby affecting (reducing) its biological function in the cell.

[0065] The term "O'SK" refers to the Oct4mut-Sox2-Klf4 tandem sequence, and OSK refers to the Oct4-Sox2-Klf4 tandem sequence.

[0066] The term "LNP" lipid nanoparticles are nanoparticles composed of ionizable lipids, cholesterol, phospholipids and PEG lipids in a certain proportion. They are often used to encapsulate nucleic acids. Their components are similar to cell membranes and can achieve delivery to specific cells.

[0067] The terms "T cell" and "T lymphocyte" are interchangeable and used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1), T helper 2 (Th2), T helper 17 (Th17), or regulatory T (Treg) cells. T cells can be T helper cells (Th; CD4 + T cells), CD4 + T cells, CD8 + T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, stem cell-like memory T cells (Tscm cells), central memory T cells (T cm ), effector memory T cells (Tem), terminal effector T cells (T eff) or any other subset of T cells. Illustrative T cell populations suitable for use in specific embodiments include stem central memory T cells (T SCM ).

[0068] Naive T cells may have the following cell surface marker expression patterns: CCR7 + 、CD62L + 、CD45RA + 、CD45RO - 、CD95 - . Stem cell-like memory T cells (Tscm) may have the following cell surface marker expression patterns: CCR7 + 、CD62L + 、CD45RA + 、CD45RO - 、CD95 + Central memory T cells (Tcm) may have the following cell surface marker expression patterns: CCR7 + 、CD62L + 、CD45RA - 、CD45RO + 、CD95 + Effector memory T cells (T EM ) may have the following cell surface marker expression pattern: CCR7 - 、CD62L - 、CD45RA - ,CD45RO + 、CD95 + Terminal effector T cells (T eff ) may have the following cell surface marker expression pattern: CCR7 - 、CD62L - 、CD45RO - 、CD95 + See, e.g., Gattinoni et al. Nat. Med. 17 (2011): 1290-7; and Flynn et al. Clin. Translat. Immunol. 3 (2014): e20, which are incorporated herein by reference in their entirety for all purposes.

[0069] The term "Tn (naive T cells)" refers to T cells that have been released from the thymus but have not yet encountered the corresponding antigen.

[0070] The term "Tscm (stem cell memory T cells)" refers to memory stem cells, which are the smallest differentiated cells at the top of the memory T lymphocyte hierarchy system.

[0071] The term "Tcm (central memory T cells)" refers to central memory T cells, which are T cells with long-term memory produced after initial T cells are activated by antigens and can home to lymph nodes to receive antigen restimulation.

[0072] The term "Teff (effector T cells)" refers to effector T cells. Effector T cells are cells formed by proliferation and differentiation after T cells are stimulated by antigens.

[0073] The present application utilizes engineered transcription factors that remove the phase separation ability (hereinafter referred to as phase-free transcription factors) to participate in multi-factor partial reprogramming. The present application finds that phase-free transcription factors cause partial reprogramming to remain in the initial stage, maintaining cell identity characteristics while rejuvenating cells, so the present application refers to this as the initial stage reprogramming technology. This technology greatly reduces the risk of iPSC generation while improving the efficiency of cell rejuvenation, and is more likely to be used in clinical practice.

[0074] On the one hand, the present application provides a method for promoting the expression of stem cell-like memory T cells, comprising:

[0075] Phase-separation-free transcription factors were prepared by adopting the method of phase-separation-free engineered reprogramming factors.

[0076] The non-phase separation transcription factors are introduced into the target cells to perform initial phase reprogramming on the target cells, which include T cells.

[0077] The approach of engineering reprogramming factors without phase separation includes mutating the regions related to the phase separation function of transcription factors so that the transcription factor loss triggers the phase separation function.

[0078] In some embodiments, the non-phase separation transcription factor is introduced into the target cell to make CCR7 + CD45RA + The proportion of T cell population increased.

[0079] In some embodiments, the relevant regions of the phase separation function of the transcription factor include IDR regions. IDR regions, i.e., intrinsically disordered regions, are a special structural domain in proteins. IDR regions refer to protein fragments that do not have a fixed three-dimensional structure but can be deformed. These regions lack stable secondary or tertiary structures but have specific biological functions.

[0080] In some embodiments, the mutation treatment includes mutating polar charged amino acids or acidic amino acids in the IDR region.

[0081] In some of these embodiments, a polar charged amino acid or an acidic amino acid is replaced with a neutral amino acid.

[0082] In some of these embodiments, the acidic amino acids are mutated to neutral amino acids.

[0083] In some of the embodiments, the amino acid sequence shown in SEQ ID NO.1 is mutated at positions 1 to 140 and / or positions 288 to 360.

[0084] In some of these embodiments, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

[0085] In some embodiments, the invention comprises: mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.1.

[0086] In some embodiments, the amino acid sequence of the non-phase separation transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0087] In some of the embodiments, before the phase-separation-free transcription factor is introduced into the cell, the method further comprises the step of preparing the phase-separation-free transcription factor into an element comprising the phase-separation-free transcription factor.

[0088] In some embodiments, the element comprises one or more of DNA, RNA, a recombinant vector, a recombinant protein, and a recombinant cell.

[0089] In some embodiments, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector, and a lentivirus vector.

[0090] In some of these embodiments, the RNA includes one or more of linear mRNA, siRNA, circular RNA, circular mRNA, and tRNA.

[0091] In one embodiment, the circular RNA comprises multiple transcription factors in series.

[0092] In some of these embodiments, the transcription factors are linked via a linker peptide.

[0093] The elements in the present application can be further used to construct vectors, which can be monocistronic, bicistronic or polycistronic. In a specific embodiment of the present application, the coding regions of the bicistronic or polycistronic genes can be linked by at least one connecting peptide, and the connecting peptide can be an internal ribosome entry site (IRES) or a self-cleaving 2A peptide (2A).

[0094] IRES sequences that can be used include, but are not limited to, small RNA viruses (e.g., FMDV), pestiviruses (e.g., CFFV), polioviruses (e.g., PV), encephalomyocarditis viruses (e.g., ECMV), foot-and-mouth disease viruses (e.g., FMDV), hepatitis C viruses (e.g., HCV), classical swine fever viruses (e.g., CSFV), mouse corneal white eye viruses (e.g., MLV), simian immunodeficiency viruses (e.g., SIV), or cricket paralysis viruses (e.g., CrPV), etc. The coding region can also be divided by a "2A linker", which is a short peptide (18-25 amino acids) derived from a virus, also known as a "self-cleaving" peptide, which can make a single transcription product produce multiple proteins, including but not limited to one or more of P2A / T2A / E2A / F2A.

[0095] Optionally, the nucleotide sequence of the connecting peptide is shown in SEQ ID NO.3 to SEQ ID NO.4.

[0096] In some embodiments, the element comprises one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

[0097] In some of the embodiments, the nucleotide sequence of the element is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0098] In some embodiments, introducing the phase separation-free transcription factor into the target cell comprises using a delivery system;

[0099] Optionally, the delivery system includes one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

[0100] On the other hand, the present application provides the use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug for promoting the expression of stem cell-like memory T cells.

[0101] The amino acid sequence of the non-phase separation transcription factor is shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0102] In some embodiments, the nucleotide sequence comprising the element of the phase separation-free transcription factor is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0103] In some of these embodiments, the drug includes a drug for cell therapy, gene therapy, immune cell proliferation, cancer treatment, or treatment of aging-related diseases.

[0104] The present application performs amino acid modification on a specific site of Oct4 protein to weaken its phase separation ability, including the steps of selecting the most important transcription factor, performing amino acid modification on a specific site to weaken its phase separation ability, converting the amino acid sequence into a DNA sequence capable of expressing circular RNA through codon optimization, adding necessary auxiliary sequences, and preparing circular RNA.

[0105] The amino acid sequence of the Oct4 protein before modification is as shown in SEQ ID NO.1:

[0106] MAGHLASDFAFSPPPGGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFS QTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN

[0107] The amino acid sequence of the modified Oct4mut protein is shown in SEQ ID NO.2:

[0108] MAGHLAS A FAFSPPPGGGG A GPGGP A PGW A PRTWLSFQGPPGGPGIGPGVGPGS A VWGIPPCPPPY A FCGGMAYCGPQVGVGLVPQGGL A TSQP A G A AGVGV A SNS A GASP A PCTVTPGAVKL A K A KL A QNP AA SQ A IKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSS A YQ AA F A AAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFP A G A AFPPVSVTTLGSPMHSN

[0109] Design linker sequences, such as P2A and T2A sequences, as shown in SEQ ID NO.3 to SEQ ID NO.4.

[0110]

[0111] Oct4 or Oct4mut is connected to Sox2 and Klf4 through the above elements to form a tandem sequence, which is only an example and can be shown in SEQ ID NO.5 to SEQ ID NO.6, and the names are Oct4-P2A-Sox2-T2A-Klf4 tandem sequence (abbreviated as OSK, corresponding to SEQ ID NO.5) and Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence (abbreviated as O'SK, corresponding to SEQ ID NO.6).

[0112]

[0113]

[0114] In some of these embodiments, the Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence further comprises one or more of an internal ribosome entry site of the target fragment, a 5' homology arm, a ribozyme, a spacer sequence, a 3' homology arm, and a regulatory element.

[0115] In some of the embodiments, the nucleotide sequence of the internal ribosome entry site of the target fragment is shown as SEQ ID NO.7, and the internal ribosome entry site can allow the ribosome to directly bind and start translation in the absence of a 5' cap.

[0116] Wherein, the nucleotide sequence of SEQ ID NO.7 is:

[0117] (SEQ ID NO.7)

[0118] In some embodiments, the auxiliary looping fragment includes one or more of: a 5' homology arm, a ribozyme, a spacer sequence, a 3' homology arm, and a regulatory element.

[0119] In some of these embodiments, the spacer sequence includes a 5' spacer sequence and a 3' spacer sequence.

[0120] In some embodiments, the regulatory element includes one or more of substrate E1, IG3, IG5 and a promoter.

[0121] In some embodiments, the Oct4mut-P2A-Sox2-T2A-Klf4 tandem sequence can be, for example, the nucleotide sequence of the circular RNA is shown in SEQ ID NO.8.

[0122]

[0123] On the other hand, the present application provides the use of the above circular RNA in the preparation of a drug for promoting the expression of stem cell-like memory T cells.

[0124] On the one hand, the present application provides a nucleic acid molecule, which includes a fragment encoding a circular RNA.

[0125] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0126] Another aspect of the present application provides a vector, which includes a nucleic acid molecule.

[0127] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can direct the expression of the genes to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play equivalent functions. Engineered mRNA, including linear mRNA and circular mRNA, is a new type of gene vector suitable for non-viral gene delivery systems and can be used for in vivo gene therapy. Circular mRNA is preferred in this application, which has lower immunogenicity and higher stability than linear mRNA, and is suitable for long-term expression of therapeutic proteins in vivo. Therefore, it is increasingly used in gene therapy. As long as the vector can be used for carrying, it is within the scope of protection of this application.

[0128] In some embodiments, the vector comprises an adeno-associated virus or a plasmid.

[0129] On the other hand, the present application provides a cell, which includes the above-mentioned circular RNA, the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0130] The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0131] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0132] On the other hand, the present application provides a method for preparing cells, which comprises the step of introducing a nucleic acid molecule or a vector into a target cell.

[0133] In one example, the introduction was carried out by lipofectamine transfection.

[0134] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197. The introduction methods include, but are not limited to, liposome transfection, microvesicle transfection, exosome transfection, electroporation, nanocarrier transfection, chemical transfection using a transfection reagent; the nanocarrier includes a lipid, a polymer, or a lipid-polymer hybrid.

[0135] The present application provides a delivery system on the other hand for delivering the above-mentioned nucleic acid molecules, recombinant vectors comprising nucleic acid molecules, recombinant DNA molecules, mRNA, recombinant proteins, etc. In some embodiments, lipid nanoparticles or "LNPs" are used to deliver nucleic acids to cells. As described above, LNPs may include natural lipids or synthetic lipids, including conjugated lipids or polymers (e.g., pegylated lipids). LNPs may include any one or more of neutral lipids, zwitterionic lipids, ionizable lipids, cationic lipids, and anionic lipids. In some embodiments, LNPs include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA) in the form of natural or synthetic monoacyl or diacyl groups, or monoacyl, diacyl, triacyl, or tetraacyl forms of cardiolipin. In some embodiments, LNPs are micelles or reverse micelles (reverse micelles). In other embodiments, LNPs are unilamellar liposomes or multilamellar liposomes.

[0136] It is understandable that the present application also relates to other delivery systems, such as GalNAc conjugated delivery system, liposome LNP nano delivery system (small molecule ligands, antibodies and other molecules) and virus-like particle VLP delivery, protamine, high molecular polymers, inorganic nanoparticles, exosomes, polymer matrices, viral transfection, etc.

[0137] On the other hand, the present application provides the use of the above-mentioned circular RNA, the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned cell in the preparation of T cell therapy products.

[0138] On the other hand, the present application provides the use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug for promoting the expression of stem cell-like memory T cells.

[0139] In some embodiments, the amino acid sequence of the non-phase separation transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.2.

[0140] In some embodiments, the nucleotide sequence comprising the element of the phase separation-free transcription factor is as shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.8.

[0141] On the other hand, the present application provides a method for constructing a circular RNA, comprising:

[0142] A tandem sequence of Oct4mut-P2A-Sox2-T2A-Klf4 was constructed to prepare a vector. The template of the vector was linearized, and then an in vitro transcription reaction and a cyclization catalytic reaction were performed. An exoribonuclease was used for digestion reaction to construct a circular RNA.

[0143] In some embodiments, the method comprises linearizing the template of the vector using restriction endonuclease EcoR I for linearization.

[0144] In some embodiments, the exonuclease described in the above method includes RNase R enzyme. RNase R (Ribonuclease R) is a 3'-5' exoribonuclease derived from the Escherichia coli RNR superfamily. RNase R can cut and degrade RNA from the 3'-5' direction, digesting almost all linear RNA molecules, but it is not easy to digest circular RNA, lasso structure or double-stranded RNA molecules with less than 7 nucleotides at the 3' end protruding end. RNase R is mainly used for the identification and enrichment experiments of circRNA (circular RNA). It enriches circRNA by digesting linear RNA, thereby playing a role in gene expression and variable splicing research.

[0145] On the other hand, the present application provides a method for treating or a method for promoting Tscm expression, which comprises administering an effective dose of the above circular RNA to a subject.

[0146] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0147] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0148] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing risk, reducing severity or delaying the onset of a condition, including biochemical, histological and / or behavioral symptoms of the condition, its complications and intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present application or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the patient's target antigen-related condition of the present disclosure. Diseases treated include diseases associated with insufficient production or secretion of memory T cells.

[0149] The present application provides an application of a circular RNA in the preparation of a drug for promoting the expression of stem cell-like memory T cells, wherein the circular RNA comprises a target fragment encoding a target protein, a ribosome entry fragment, and an auxiliary circularization fragment, wherein the target fragment encoding the target protein is specifically designed in the present application, and its specific site is modified with amino acids to weaken its phase separation ability, and then the corresponding codon optimization is performed, and the purity of the prepared circular RNA is>85%, and the three target proteins contained therein can be expressed after transfection into cells, and after transfection into human primary T cells, the proportion of Tscm in the T cell population can be effectively increased.

[0150] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0151] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0152] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0153] Example 1

[0154] 1. Design, production and validation of O'SK circular RNA

[0155] 1. Vector construction

[0156] The order of each element of O'SK circular RNA (FlexCirc system) is as follows Figure 1 As shown: 5' homology arm, IG5, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, IG3, 3' homology arm, the above-mentioned FlexCirc system ring system sequence, including the 5' end T7 promoter sequence, and the DNA template linearization restriction site EcoR I sequence (see SEQ ID NO.8 for details) were recombined into the pBlue Script plasmid, and the required plasmid was obtained by direct synthesis by a third-party sequence synthesis company, and the DNA sequence was confirmed to be correct by sequencing.

[0157] 2. In vitro transcription and circularization verification

[0158] (1) Preparation of DNA linearized template: The mutation vector plasmid synthesized by a third party is linearized using restriction endonuclease EcoRI to obtain a transcription template.

[0159] Specifically, EcoR I restriction endonuclease was used to linearize and digest the plasmid qualified by PCR to prepare the IVT transcription template. The enzyme digestion system is shown in Table 1.

[0160] The purity of the linearized template was tested by HPLC to ensure that the prepared linearized template met the requirements of subsequent IVT transcription.

[0161] Table 1: EcoR I enzyme 500μL digestion system formula

[0162] Components Volume (μL) Plasmids 50μg EcoRI (10U / μL) 50 Tingo Buffer 50 Enzyme-free water Up to 500

[0163] (2) In vitro transcription and circularization verification

[0164] Prepare 20 μL of reaction system according to Table 2, place the transcription system in the PCR instrument, and react at 37°C for 2 hours. After the reaction is completed, add 1 μL of DNase I and 2 μL of 10×DNase I buffer to each reaction system, and react at 37°C for 20 minutes. Transfer each transcription system to a 1.5 mL centrifuge tube, add RNase R enzyme and incubate at 37°C for 30 minutes, then add 0.5 times the volume of 7.5M lithium chloride and 50mM EDTA RNA precipitation solution, mix well and precipitate in a -20°C refrigerator for 1 hour. After precipitation is completed, centrifuge at 18360×g for 15 minutes at 4°C, remove the supernatant as much as possible, add 500 μL of 70% ethanol solution, blow and wash the precipitate, centrifuge again under the same conditions, remove the supernatant as much as possible, evaporate the ethanol, and resuspend each experimental group with 100 μL of enzyme-free water. Samples were taken to detect RNA concentration and purity, and 2% E-Gel electrophoresis was used to determine the circRNA synthesis effect.

[0165] The results are as follows Figure 2 As shown, in 2% E-gel electrophoresis, lanes 5 (OSK circular RNA) and 6 (O'SK circular RNA) can form a single band with a slower migration rate, and its size is higher than 6000 nt, which is consistent with the theory.

[0166] Table 2 In vitro transcription system

[0167]

[0168] (3) HPLC purification and testing

[0169] This part of the work was completed by Shenzhen XingGene Technology Co., Ltd. using its internal methods, such as Figure 3 As shown, the main peak of O'SK circular RNA is obvious, and the impurity peak is relatively low. The main peak is integrated to obtain its purity. The concentration and purity of the prepared O'SK circular RNA are shown in Table 3 below:

[0170] Table 3: In vitro transcription system

[0171]

[0172] Verification results:

[0173] 1. Circular RNA protein expression verification

[0174] 1. Cell culture: HEK293T cells were cultured in a 37°C 5% CO2 biological incubator using DMEM (Gibco) complete medium containing 10% fetal bovine serum and 1% double antibody. When the cells grew to 90%, 0.05% Trypsin-EDTA was used to digest the cells and count them according to 1.0×10 5 The cells were seeded at a density of 1.54 × 10 / well in a 24-well plate and mRNA transfection was performed the next day.

[0175] 2. Cell transfection: According to the instructions, use lipofectamine 3000 (Thermo Fisher) to transfect O'SK circular RNA in HEK293T cells. Add 1.0 μg of circular RNA to 50 μL Opti-MEM and mix thoroughly; take another tube, add 50 μL Opti-MEM and 1.0 μL lipofectamine 3000, mix the two tubes thoroughly, incubate at room temperature for 15 minutes, and add to the corresponding cells. After 48 hours of transfection, add 50 μL RIPA lysis buffer (Biyuntian) to each well, place on ice for 30 minutes, fully lyse, and collect protein lysate for WB (protein blotting) experiments. Among them, Oct4mut antibody (Wuhan Proleger) and Sox2 antibody (Jingjie Bio, PTM-6930) were used for WB experiments.

[0176] The results are as follows Figure 4 As shown, the internal reference gene ( Figure 4 Group A in), Oct4mut( Figure 4 in Group B) and Sox2( Figure 4 Group C in the sample was only detected in sample 1.

[0177] 2. Phase separation characterization of Oct4 transcription factor

[0178] 1. Plasmid synthesis: GFP-Oct4 plasmid and GFP-Oct4mut (O'SK) plasmid were synthesized at GENEWISE (Suzhou GENEWISE Biotechnology Co., Ltd.) and cloned into pCDNA3.4 vector. Subsequent experiments were performed after the DNA sequence was confirmed to be correct.

[0179] The nucleotide sequence of GFP-Oct4 plasmid is shown in SEQ ID NO.9:

[0180]

[0181] The nucleotide sequence of GFP-O'SK plasmid is shown in SEQ ID NO.10:

[0182]

[0183] 2. Cell culture: HEK293T cells were cultured in a 37°C 5% CO2 biological incubator using DMEM (Gibco) complete medium containing 10% fetal bovine serum and 1% double antibody. When the cells grew to 90%, 0.05% Trypsin-EDTA was used to digest the cells and count them according to 1.0×10 5 The cells were seeded at a density of 1.54 × 10 / well in a 24-well plate and mRNA transfection was performed the next day.

[0184] 3. Cell transfection: According to the instructions, lipofectamine 3000 (Thermo Fisher) was used to transfect GFP-Oct4 plasmid and GFP-Oct4mut plasmid in HEK293T cells. 1.0 μg of plasmid was added to 50 μL Opti-MEM and mixed thoroughly; in another tube, 50 μL Opti-MEM and 1.0 μL lipofectamine 3000 were added, the two tubes were mixed thoroughly, incubated at room temperature for 15 min, and added to the corresponding cells.

[0185] 4. Static phase separation observation: 48 hours after transfection, cells were fixed with 4% paraformaldehyde (Biyuntian, P0099). After being fully washed with PBS, DAPI (Biyuntian, P0131) was used for nucleus staining and sealing. For cells in the 1,6-hexanediol treatment group, 1.5% 1,6-hexanediol (Sangong, A601513) was added for incubation for 5 minutes 48 hours after transfection. After washing with PBS, cells were fixed and stained. The morphology and aggregation degree of GFP in the cell nucleus were observed under a 100x oil lens using a Nikon A1 confocal fluorescence microscope. GFP-Oct4 exists in the cell nucleus in the form of highly condensed droplets. After treatment with 1,6-hexanediol, it becomes a uniformly distributed form, indicating that Oct4 has the characteristics of classic phase separation; while GFP-Oct4mut has always been in a diffusely distributed form, indicating that its phase separation characteristics can be destroyed by amino acid modification. The results are as follows: Figure 5 shown.

[0186] 5. Dynamic phase separation observation (fluorescence decolorization and fluorescence recovery)

[0187] The cells were transfected in the same way, and the GFP signal in the nucleus of living cells was dynamically observed under a Nikon A1 confocal fluorescence microscope. The GFP fluorescence spots were first quenched using a 2s laser (excitation light 488nm, fluorescence intensity 21%), and then recorded every 2s within a 10min observation window. It can be found that GFP-Oct4 has the ability to recover quickly, while the fluorescence spots of GFP-Oct4mut do not have the ability to recover after quenching. The results are shown in Figure 2. Figure 6 shown.

[0188] III. Characterization of memory T cell phenotype after T cell transfection

[0189] 1. Cell culture

[0190] Thaw the cryopreserved tube of primary human T cells (Miaoshun Biotechnology, PB03-N-1C) in a 37°C water bath. When a small amount of ice crystals remain in the tube, remove it from the water bath and transfer it to a biosafety cabinet. Add a small amount of RPMI-1640 complete medium containing 10% fetal bovine serum and 1% double antibody, transfer it to a centrifuge tube containing 8mL complete medium, rinse the cryopreserved tube and the tip of the gun, centrifuge at 400g for 10min at room temperature, and count. Add Dynabeads Human T-Activator CD3CD28 (Gibco 11161D) and 30IU / mL IL-2 (PeProtech, 200-02; 10μg) in a 1:1 ratio. After four days of stimulation, the experiment was carried out in groups.

[0191] 2. Cell processing and detection

[0192] After stimulation, the cells were collected and the magnetic beads were removed. 6 Cells were inoculated at a density of 100 μg / well. According to the instructions of ProteanFect Max (West Lake Aggregate, PT0201), Reagent A, Regent B, O'SK circular RNA and Regent C were prepared in proportion and order, incubated with T cells at 37°C for 30 min, added with 200 μL RPMI 1640 complete medium, centrifuged at 300 g for 5 min, discarded the supernatant, resuspended the cell pellet with 1 mL RPMI 1640 complete medium, added with 30 IU / mL IL-2, and cultured for three days. Cells were collected every three days and the total cell volume and viability were counted. The experiment was terminated on the tenth day. Some cells were lysed with RIPA lysis buffer and subjected to WB detection. Other cells were resuspended with flow cytometry washing buffer, centrifuged, and PE anti-human CCR7 (Biolegend, 353203), Brilliant Violet 605 anti-human CD45RA (Biolegend, 304134) and Brilliant Violet 421 anti-human CD95 (Biolegend, 305624) were added. After being resuspended twice with flow cytometry washing buffer, the expression of the above indicators was detected by the machine.

[0193] The results showed that the proliferation of T cells treated with O'SK circular RNA could be maintained at 3-4 times for a long time, and the survival rate was comparable to that of the group treated with IL-2 only. Figure 7In addition, after O'SK circular RNA treatment, the proportion of Tscm in cells increased significantly, as shown in Figure 8 shown.

[0194] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for promoting the expression of stem cell-like memory T cells, characterized in that: include: Using a phase-separation-free engineered reprogramming factor approach to prepare a phase-separation-free transcription factor; Introducing the phase-separation-free transcription factor into target cells to perform initial phase reprogramming on the target cells; The method of engineering reprogramming factors without phase separation includes mutating the relevant regions of the phase separation function of the transcription factor so that the transcription factor lacks the function of inducing phase separation; The target cells include T cells.

2. The method for promoting the expression of stem cell-like memory T cells according to claim 1, characterized in that: The phase-separation-free transcription factor is introduced into the target cell, so that CCR7 + CD45RA + The proportion of T cell population increased.

3. The method for promoting the expression of stem cell-like memory T cells according to claim 1, characterized in that: The region related to the phase separation function of the transcription factor includes the IDR region.

4. The method for promoting the expression of stem cell-like memory T cells according to claim 3, characterized in that: The mutation treatment includes mutating the polar charged amino acid or acidic amino acid in the IDR region; Optionally, the polar charged amino acid or the acidic amino acid is replaced with a neutral amino acid; Optionally, the acidic amino acids are mutated to neutral amino acids.

5. The method for promoting the expression of stem cell-like memory T cells according to claim 4, characterized in that: The method comprises mutating positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence shown in SEQ ID NO.1, Optionally, positions 1 to 140 and / or positions 288 to 360 of the amino acid sequence further include 10 to 20 amino acids before and after.

6. The method for promoting the expression of stem cell-like memory T cells according to claim 5, characterized in that: include: The invention relates to a method for mutating at least one of positions 8, 20, 31, 108, 138, 291, 297, 26, 56, 68, 91, 96, 98, 104, 113, 125, 127, 130, 134, 135, 296, 299, 341, 343, 145, 147, 166, 188, 209, 210, 215, 224, 238, 246, 270 and 272 of the amino acid sequence shown in SEQ ID NO.

1.

7. The method for promoting the expression of stem cell-like memory T cells according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

2.

8. The method for promoting the expression of stem cell-like memory T cells according to claim 7, characterized in that: Before the phase-separation-free transcription factor is introduced into cells, the method further comprises the step of preparing the phase-separation-free transcription factor into an element containing the phase-separation-free transcription factor.

9. The method for promoting the expression of stem cell-like memory T cells according to claim 8, characterized in that: The elements include: one or more of DNA, RNA, recombinant vector, recombinant protein and recombinant cell; Optionally, the recombinant vector comprises one or more of an adeno-associated virus vector, a poxvirus vector and a lentivirus vector; Optionally, the RNA comprises one or more of linear mRNA, siRNA, circular RNA, circular mRNA and tRNA.

10. The method for promoting the expression of stem cell-like memory T cells according to claim 9, characterized in that: The circular RNA comprises a plurality of transcription factors connected in series; Optionally, the transcription factor is linked via a linker peptide; Optionally, the nucleotide sequence of the connecting peptide is shown as SEQ ID NO.3 to SEQ ID NO.

4.

11. The method for promoting the expression of stem cell-like memory T cells according to claim 10, characterized in that: The elements include one or more of Oct4, Sox2, Klf4, c-Myc, L-Myc, Lin28, Nanog and Glis1.

12. The method for promoting the expression of stem cell-like memory T cells according to claim 9, characterized in that: The nucleotide sequence of the element is shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

8.

13. The method for promoting the expression of stem cell-like memory T cells according to any one of claims 1 to 6 or 8 to 12, characterized in that: Introducing the phase separation-free transcription factor into target cells comprises using a delivery system; Optionally, the delivery system comprises one or more of liposome LNP, exosome delivery, GalNAc system, VLP delivery, protamine, high molecular polymer, inorganic nanoparticles, exosomes, polymer matrix and viral transfection.

14. Use of a phase-separation-free transcription factor or an element comprising a phase-separation-free transcription factor in the preparation of a drug for promoting the expression of stem cell-like memory T cells; The amino acid sequence of the phase-separation-free transcription factor is as shown in SEQ ID NO.2, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

2.

15. The use according to claim 14, characterized in that: The nucleotide sequence of the element containing the phase-separation-free transcription factor is shown in SEQ ID NO.8, or a sequence having at least 85%, 90% or more identity with SEQ ID NO.

8.

16. The use according to claim 14, characterized in that The drugs include drugs for cell therapy, gene therapy, immune cell proliferation, cancer treatment or treatment of aging-related diseases.

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