Cell programmable RNA translation switch regulated by mirna

Abstract

The present invention relates to the technical field of molecular biology, and specifically relates to a cell programmable RNA translation switch regulated by miRNA. Provided in the present invention is an RNA recombinant nucleic acid molecule. An RNA with cell-specific expression capability can be constructed only by means of engineering a single RNA molecule without needing to combine multiple RNA molecules nor needing to introduce an exogenous protein, and can respond various miRNA signals to implement logic operations. The strategy for engineering IRES provided in the present invention can be used for various IRESs, and has universality and transferability. In the present invention, a dynamic change in the structure of an engineered IRES element is realized by means of responding an endogenous miRNA signal of a cell, so that the translation activity of the IRES is dynamically regulated.

Description

A cellular programmable RNA translation switch regulated by miRNA Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a cell programmable RNA translation switch based on miRNA regulation. Background Art

[0002] RNA molecules have long been considered a promising new drug modality due to their ease of synthesis, modular coding region sequences that facilitate modification, and the fact that RNA functions exclusively in the cytoplasm, thus avoiding sequence integration into the genome. However, due to the rapid degradation of RNA molecules by ubiquitous RNases and the lack of effective RNA delivery technologies in the clinic, the application of RNA technology in medicine has been slow, with only a few oligonucleotides (siRNA) entering clinical trials. In recent years, breakthroughs in RNA delivery technology, RNA modifications to reduce immunogenicity, and machine learning-based RNA sequence design and optimization have enabled the rapid development of RNA technology. RNA-based therapies, such as antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and messenger RNA (mRNA) vaccines, have shown great potential in biomedical applications.

[0003] Breakthroughs in RNA technology have also brought new opportunities for the design and application of RNA-based gene circuits, making it possible to design RNA gene circuit devices with potential for clinical application. Researchers have previously designed RNA gene circuits that can sense miRNA signals, RNA-binding protein signals, and small chemical molecule signals, achieving proof-of-concept, but these designs still have limitations in practical application. Auslander et al. designed an RNA device containing the self-cleaving hammerhead ribozyme (HHR) and fused an RNA aptamer that can bind to small molecules or proteins to the sequence of the natural HHR. In the absence of signal input, the HHR undergoes self-cleavage, destroying the integrity of the RNA device. The RNA device, lacking end protection, is then degraded and cleared, thus failing to express the target protein. However, when the RNA device senses the small molecule or protein signal corresponding to the aptamer within the cell, the small molecule / protein binds to the HHR and alters the local spatial structure of the RNA, disrupting the self-cleavage activity of the HHR, allowing the protein encoded by the RNA to be expressed. However, a common drawback of this work and other efforts utilizing self-cleaving ribozymes is that this approach is only suitable for delivering DNA to cells, relying on the cell's own transcription and protection mechanisms to transport transcripts from the nucleus to the cytoplasm to function. This is because RNA can self-cleave after in vitro transcription, even before it is delivered to the cell. Therefore, although this approach achieves regulation at the RNA level, it still requires DNA delivery and is not a true RNA gene circuit, thus failing to leverage the advantages of RNA technology.

[0004] In 2017, Kawasaki et al. first proposed an RNA device capable of sensing endogenous RNA-binding proteins (RNAs) in cells. They placed an RNA aptamer in the 5' untranslated region (UTR) of a target gene. When the RNA-binding protein binds to the RNA aptamer, it blocks the ribosome's path from the mRNA's 5' cap region to the downstream, thereby inhibiting translation of the target gene. When the RNA-binding protein is absent or not bound to the aptamer, the target gene is expressed normally, thus achieving a sensing response to endogenous RNA-binding protein signals. This work, as well as other linear mRNA devices that place RNA aptamers in the 5' UTR, suffers from the following drawbacks: First, this approach has weak regulatory capabilities because it relies on the traditional 5' cap-initiated translation mechanism. During ribosome scanning from the 5' cap, if the RNA aptamer's structure is too stable, it hinders the ribosome's scanning passage. Consequently, target gene expression remains low in the absence of RNA-binding protein signals. However, if the RNA aptamer's structure is less stable, while ribosome scanning passage is facilitated, the aptamer's binding to the protein is weak in the presence of RNA-binding protein, leading to leaky expression of the target gene. On the other hand, this method turns off the expression of the target gene after sensing the signal of the endogenous RNA binding protein, that is, it plays the function of shutting down gene expression, but what is more practical is to turn on the expression of the target gene after sensing a specific signal, such as turning on the expression of the suicide gene after sensing the cell signal upregulated by the tumor.

[0005] In addition, there are other reported RNA device architectures, such as using RNA splicing mechanisms to regulate the expression of target genes: placing the binding sequence of RNA binding protein (RBP) near the RNA variable splicing site, thereby converting the RBP-RNA binding signal into different transcripts with RNA variable splicing. However, since RNA splicing must be completed in the cell nucleus, the disadvantage of this method is that it requires the introduction of additional auxiliary factors to help the RNA device shuttle from the cytoplasm into the nucleus to complete the splicing, and then enter the cytoplasm to function.

[0006] Compared to linear mRNA, circular RNA (circRNA) exhibits superior stability and extended persistence in mammalian cells due to its greater resistance to degradation by RNases and lower immunogenicity. Therefore, circRNA is considered a potential alternative to linear mRNA. To date, synthetic biologists have developed strategies for regulating gene expression in both linear mRNA and circRNA. As described above, these strategies typically require the use of RBP aptamers and miRNA binding sites (miRBS) to shut down translation in response to RBPs and miRNAs, and the tandem use of these shut-off modules to activate translation. Consequently, these strategies require multiple RNA molecules to construct functional devices such as cell classifiers and logic gates, increasing the size of the entire RNA device and introducing potentially immunogenic exogenous proteins. Previously reported strategies for activating RNA translation by miRNA rely on miRNA cleavage to release the polyadenosine tail (polyA tail) or excise the degradation domain of linear mRNA and are therefore not applicable to circRNA.

[0007] In summary, there is still no convenient and simple way to achieve translational activation of circRNA by miRNA.

[0008] Summary of the Invention

[0009] The present invention aims to address, at least to some extent, one of the technical problems in the related art. To this end, one objective of the present invention is to propose a novel strategy that allows miRNAs to activate and inhibit the translation of mRNA / circRNA by modifying only a single RNA molecule, thereby enabling the construction of RNA devices capable of logical operations and cell classification.

[0010] To this end, the first aspect of the present invention provides an RNA recombinant nucleic acid molecule. According to an embodiment of the present invention, the RNA recombinant nucleic acid molecule comprises:

[0011] (1) RNA translation switch element;

[0012] (2) IRES elements or elements with similar functions to IRES elements;

[0013] (3) target polypeptide encoding element,

[0014] Wherein, the IRES element or an element having similar functions to the IRES element includes a structure that plays a key role in translation activity

[0015] The RNA translation switch element comprises a first RNA sequence and a second RNA sequence, wherein the first RNA sequence is located upstream of the second RNA sequence, the first RNA sequence contains an RS sequence, the second RNA sequence contains a DS sequence, and a first spacer sequence is present between the RS sequence and the DS sequence.

[0016] (a) the RS sequence and at least a portion of the DS sequence are complementary and paired to form a stem-loop structure;

[0017] (b) the DS sequence is complementary to at least a portion of the structure that plays a key role in translation activity;

[0018] In the RNA recombinant nucleic acid molecule, (a) and (b) do not occur simultaneously, and the initiation of translation of the target polypeptide encoding element is achieved through changes in the two structures of (a) and (b).

[0019] According to one embodiment of the present invention, the first RNA sequence further comprises at least one miRBS sequence, which is complementary and incompletely complementary to at least a portion of a miRNA specifically expressed in a target cell, wherein the target cell is a cell into which the RNA recombinant nucleic acid molecule is to be introduced.

[0020] The RNA recombinant nucleic acid molecule provided by the present invention does not require the combination of multiple RNA molecules, nor the introduction of exogenous proteins. The present invention can construct RNA with cell-specific expression ability by engineering a single RNA molecule, and can sense multiple miRNA signals to achieve logical operations. The engineered IRES element proposed in the present invention can be used for both linear mRNA and circular RNA expression regulation. The strategy for engineering IRES proposed in the present invention can be applied to a variety of IRESs, and has universality and portability. The present invention achieves dynamic structural changes of the engineered IRES element by sensing endogenous miRNA signals in the cell, thereby dynamically regulating the translation activity of IRES. The present invention is the first that can simultaneously achieve upregulation or downregulation of the translation activity of the target protein by relying on a single circular RNA molecule, and can be used for logical operations of cell signals and cell classification.

[0021] The second aspect of the present invention provides an isolated DNA sequence, comprising a DNA sequence capable of being transcribed into the RNA recombinant nucleic acid molecule of the first aspect.

[0022] The third aspect of the present invention provides a vector comprising the isolated DNA sequence described in the second aspect.

[0023] The fourth aspect of the present invention provides a cell comprising the RNA recombinant nucleic acid molecule described in the first aspect and / or the isolated DNA sequence described in the second aspect and / or the vector described in the third aspect.

[0024] The fifth aspect of the present invention provides a composition comprising the RNA recombinant nucleic acid molecule described in the first aspect and / or the isolated DNA sequence described in the second aspect and / or the vector described in the third aspect.

[0025] The sixth aspect of the present invention provides the use of the RNA recombinant nucleic acid molecule described in the first aspect, the isolated DNA sequence described in the second aspect, the vector described in the third aspect, and the composition described in the fifth aspect in the preparation of a pharmaceutical composition for treating or preventing a disease, wherein the disease is related to the expression of the target polypeptide, and the expression of the target polypeptide can treat or prevent the disease.

[0026] A seventh aspect of the present invention provides a method for expressing a target polypeptide in a cell, the method comprising:

[0027] The RNA recombinant nucleic acid molecule described in the first aspect and / or the isolated DNA sequence described in the second aspect and / or the vector described in the third aspect and / or the composition described in the fifth aspect are introduced into cells to express the target polypeptide.

[0028] An eighth aspect of the present invention provides a method for treating tumors, comprising:

[0029] administering the RNA recombinant nucleic acid molecule of the first aspect and / or the isolated DNA sequence of the second aspect and / or the vector of the third aspect and / or the composition of the fifth aspect to a subject, wherein the target polypeptide is expressed in tumor cells and not in healthy cells,

[0030] Wherein, the target cells are tumor cells, and the non-target cells are healthy cells.

[0031] The RNA recombinant nucleic acid molecule designed in the present invention realizes on-off regulation of the translation activity of the internal ribosome entry site (IRES); by designing the RNA secondary structure, an engineered IRES element activated / inhibited by a specific microRNA (miRNA) is realized; the engineered IRES element activated / inhibited by a specific miRNA is constructed into a circular RNA (circRNA) vector, and the circRNA molecule is delivered through in vitro transcription and lipid nanoparticle (LNP) packaging to achieve specific expression of the circRNA in tumor cells.

[0032] The beneficial effects of the present invention include: the application of the circular RNA molecules specifically expressed by tumor cells constructed by the present invention can achieve specific expression of tumor cells and can be used as a new tumor targeted therapy based on RNA translation regulation. Because the RNA encapsulated by LNP can be delivered to tumor lesions by intravenous injection without the need for intratumoral injection, it can fully utilize the multiple advantages of RNA technology: low cost, easy preparation and packaging delivery, rapid expression of target genes, and no integration into the cell genome. At the same time, the device can also be packaged into viruses to prepare new oncolytic viruses.

[0033] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0035] Figure 1 shows a schematic diagram (A) and effect diagram (B) of inhibiting the translation activity of HCV-IRES by designing DS sequence in HEK293 cell line;

[0036] Figure 2 shows a schematic diagram (A) and effect diagram (B) of restoring the translation activity of HCV-IRES by designing the RS sequence in the HEK293 cell line;

[0037] Figure 3 shows the inhibitory effect of DS and the restorative effect of RS tested in the Huh7 cell line;

[0038] FIG4 shows a schematic diagram of designing RNA secondary structures containing miRBS and RS sequences to construct three miRNA-activated engineered IRES elements (MITA) in HEK293 cell lines;

[0039] Figure 5 shows the responses of three MITA to exogenous miRNA analogs tested in HEK293 cell lines;

[0040] Figure 6 shows the responses of three MITAs to endogenous miRNAs tested in HEK293 and Huh7 cell lines;

[0041] FIG7 shows the design of RNA secondary structures containing different miRBS and RS sequences to construct engineered IRES elements with logical “OR” operation capabilities;

[0042] FIG8 shows the design of RNA secondary structures containing different miRBS and RS sequences to construct engineered IRES elements with logical “AND” operation capabilities;

[0043] FIG9 shows an engineered IRES element designed to contain fully complementary paired miRBS binding sites to construct a logical NOT operation capability;

[0044] Figure 10 shows the inhibitory activity of multiple miRNAs tested in HEK293 and Huh7 cell lines, where 4×miRNA binding sites means four miRNA binding sites connected in series;

[0045] Figure 11 shows the plasmid vector designed and constructed by combining MITA and MITR to test the classification effect in HEK293 and Huh7 cell lines;

[0046] Figure 12 shows a schematic diagram of a template for in vitro transcription of circular RNA containing a MITA element;

[0047] FIG13 shows the expression inhibition and restoration of circular RNA containing dIRES and rIRES in HEK293 and Huh7 cell lines;

[0048] FIG14 shows the expression differences of circular RNAs containing three different MITAs in HEK293 and Huh7 cell lines;

[0049] Figure 15 shows the classification effect of circular RNA designed and constructed by combining MITA and MITR in HEK293 and Huh7 cell lines;

[0050] Figure 16 shows a schematic diagram and effect diagram of designing a DS sequence to inhibit the translation activity of a truncated version of HCV-IRES (tIRES) in a HEK293 cell line;

[0051] FIG17 shows the effect of designing RS sequence to restore the translation activity of tIRES in HEK293 cell line;

[0052] FIG18 shows the effect of designing a 20-nt DS sequence to inhibit the translation activity of other IRES in HEK293 cell lines;

[0053] FIG19A shows a schematic diagram of the construction of a cancer cell-specific killing circular RNA molecule without miRBS;

[0054] FIG19B shows the results of a cancer cell-specific killing experiment;

[0055] FIG20A shows a schematic diagram of the construction of a cancer cell-specific killing circular RNA molecule containing 4×miRBS;

[0056] FIG20B shows the results of a cancer cell-specific killing experiment;

[0057] FIG20C shows micrographs of cancer cells and normal cells after transfection with killer circular RNA molecules.

[0058] Detailed Description of the Invention

[0059] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0060] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0061] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0062] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, protein and nucleic acid chemistry, molecular biology, related terms and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0063] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0064] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0065] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0066] the term

[0067] "IRES" refers to the Internal Ribosomal Entry Site (IRES). The mechanism by which IRES initiates RNA translation differs from the traditional 5' cap-dependent translation mechanism in eukaryotic cells. IRES is an RNA sequence with a specific spatial structure, originally discovered in the genomes of RNA viruses. Due to its unique higher-order structure, it can directly recruit eukaryotic ribosomes, or further recruit translation initiation factors and ribosomes by recruiting trans-acting factors (IRES Trans-Acting Factor, ITAF), thereby enabling translation to initiate directly from within the RNA sequence without relying on the 5' cap. Based on the sequence characteristics of IRES and the conservation of its secondary structure, internal ribosome entry sites can be divided into three categories.

[0068] The "RNA translation switch element" used in this article refers to an RNA sequence that regulates the initiation of translation of mRNA. The RNA translation switch element regulates the expression of downstream target polypeptide encoding elements through structural changes by complementary or non-complementary pairing with the structure contained in IRES that plays a key role in translation activity.

[0069] The "DS sequence" used herein is a portion of the RNA sequence in the RNA translation switch element of the RNA recombinant nucleic acid molecule, which complementarily pairs with at least a portion of the structure (such as the 3' pseudoknot structure) contained in IRES that plays a key role in translation activity.

[0070] The "RS sequence" used herein is a portion of the RNA sequence in the RNA translation switch element of the RNA recombinant nucleic acid molecule, which is complementary to at least a portion of the DS sequence and forms a stem-loop structure.

[0071] The "3' pseudoknot structure" used in this article is a portion of the sequence at the 3' end of IRES, which is in a single-stranded state or a complementary paired state, and determines the entry of the ribosome; when the 3' pseudoknot structure is in a single-stranded state, the ribosome can bind to the mRNA and initiate the translation of the downstream sequence connected to it; when the 3' pseudoknot structure is complementary paired with other sequences, the ribosome cannot bind to the mRNA and cannot initiate the translation of the downstream sequence.

[0072] As used herein, the "target polypeptide encoding element" refers to the mRNA sequence of the target polypeptide.

[0073] A "spacer sequence" is a non-functional mRNA sequence.

[0074] A "stem-loop" structure refers to an inverted repeat sequence found in single-stranded RNA molecules. Due to hydrogen bonding between complementary bases, the long strand can fold back, forming a secondary structure. The double-stranded region between the paired bases forms the "stem," while the unpaired single-stranded region protrudes to form the "loop."

[0075] The "Y-shaped double stem-loop structure" used in this article refers to a single-stranded RNA molecule that forms two stem-loop structures due to the presence of two inverted repeat sequences, wherein the two stem-loop structures and the complementary paired sequences at both ends of the single-stranded RNA molecule together constitute the Y-shaped double stem-loop structure used in this article.

[0076] The "double-stranded stem" used herein refers to the "stem" formed by the double-stranded region between the paired bases in the inverted repeat sequence formed by the single-stranded RNA molecule.

[0077] The "single-stranded loop portion" used herein refers to the "loop" portion formed by the protruding unpaired single-stranded region in the inverted repeat sequence formed by a single-stranded RNA molecule.

[0078] "Complementary pairing" refers to the phenomenon that the bases of each nucleotide residue in a nucleic acid molecule are connected to each other by hydrogen bonds according to the corresponding relationships of A and T, A and U, and G and C. The present invention specifically refers to the complementary pairing of bases in RNA molecules.

[0079] As used herein, "miRBS sequence" refers to a sequence that complementarily pairs with a miRNA.

[0080] "miRNA", also known as MicroRNA, is a class of evolutionarily conserved single-stranded non-coding RNA molecules with a length of about 20nt. It binds to target messenger RNA (mRNA) through base complementary pairing and prevents the production of target protein by guiding the degradation of target mRNA or inhibiting the translation of target mRNA.

[0081] As used herein, "independently" means that the multiple miRBS sequences contained in the RNA translation switch element of the present invention are identical or different, and can bind to the same or different miRNA types.

[0082] As used herein, "tandem miRBS sequences" refers to multiple miRBS sequences directly linked at the first position.

[0083] As used herein, a "truncated IRES element" refers to an IRES element with a partial sequence deleted, which may be a partial sequence deleted at both ends or in the middle.

[0084] "Lipid nanoparticles" are typically composed of ionized cationic lipids, phospholipids, cholesterol, and polyethylene glycol lipids, with a solid or oily core at the center. Traditional unilamellar liposomes, which are smaller in size, are typically characterized by a lipid bilayer on the surface and an aqueous "pool" at the center of the particle. Because liposomes have long been approved by regulatory agencies and used in clinical small molecule drugs, they were quickly identified by gene therapy pioneers as a potential delivery vehicle, particularly for RNA small molecule drugs.

[0085] Liposomes are lipids made from phosphatidylcholine and ceramide, with a bilayer structure similar to that of cell membranes. Liposomes are widely used as drug carriers for small molecule drugs, proteins, nucleic acids, and imaging agents. To enhance therapeutic efficacy and improve patient compliance, various drug delivery methods for liposomes have been developed, including parenteral, pulmonary, oral, transdermal, ophthalmic, and nasal administration.

[0086] As used herein, the term "composition" is in a form that permits the biological activity of the active ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0087] As used herein, the term "treat," ...

[0088] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has been or is currently receiving any form of treatment. As used herein, the terms "subject" or "patient" refer to a mammalian subject or patient. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0089] As used herein, "apoptosis-inducing polypeptide" refers to a polypeptide fragment that is capable of initiating programmed cell death.

[0090] As used herein, "polypeptides that are toxic or lethal to tumor cells" refer to protein fragments that can specifically kill or inhibit the growth of cancer cells while being relatively safe for normal cells. In the present invention, two representative polypeptides were used: the N-terminal portion of Gasdermin D (GSDMD) and Herpes Simplex Virus Thymidine Kinase (HSV-TK). GSDMD is a pore-forming protein involved in pyroptosis (an inflammatory cell death pathway), and its N-terminal portion plays a key role in inducing cell death. HSV-TK is an enzyme commonly used in gene therapy strategies. When combined with antimetabolite drugs such as ganciclovir, HSV-TK can convert the drug into its toxic form, thereby inducing cell death.

[0091] RNA recombinant nucleic acid molecules

[0092] According to one embodiment of the present invention, the present invention provides an RNA recombinant nucleic acid molecule, comprising:

[0093] (1) RNA translation switch element;

[0094] (2) IRES elements or elements with similar functions to IRES elements;

[0095] (3) target polypeptide encoding element,

[0096] Wherein, the IRES element or an element having similar functions to the IRES element includes a structure that plays a key role in translation activity

[0097] The RNA translation switch element comprises a first RNA sequence and a second RNA sequence, wherein the first RNA sequence is located upstream of the second RNA sequence, the first RNA sequence contains an RS sequence, the second RNA sequence contains a DS sequence, and a first spacer sequence is present between the RS sequence and the DS sequence.

[0098] (a) the RS sequence and at least a portion of the DS sequence are complementary and paired to form a stem-loop structure;

[0099] (b) the DS sequence is complementary to at least a portion of the structure that plays a key role in translation activity;

[0100] In the RNA recombinant nucleic acid molecule, (a) and (b) do not occur simultaneously, and the initiation of translation of the target polypeptide encoding element is achieved through changes in the two structures of (a) and (b).

[0101] In eukaryotes, RNA undergoes post-transcriptional modifications within its head, which are recognized by ribosomes and other proteins. mRNA translation is initiated through a cap structure within the head. The cap structure is essential for mRNA translation initiation, providing a signal for ribosome recognition, assisting ribosome binding and initiating translation from the AUG. The cap structure also increases mRNA stability, protecting it from 5'→3' exonucleases. By constructing an IRES element or an element with similar functions within an RNA recombinant nucleic acid molecule, the ribosome recognizes this element and initiates internal translation. The inventors have proposed a novel strategy that allows miRNAs to activate and inhibit mRNA / circRNA translation simply by modifying the RNA molecule's structure. This strategy also enables the construction of RNA devices capable of logical operations and cell classification.

[0102] According to a specific embodiment of the present invention, as shown in Figure 1, the translation of enhanced yellow fluorescent protein (EYFP) depends on the cap structure of RNA. As an internal reference gene, the translation of blue fluorescent protein (tagBFP) is initiated by IRES. The pseudoknot structure (Pseudoknot) must be in a single-stranded state. IRES has the function of initiating translation. Once the pseudoknot structure is destroyed (other sequences are complementary to it and are in a non-single-stranded state), the ribosome cannot enter and the downstream protein cannot be translated. The inventors designed RS sequence and DS sequence, wherein at least a portion of the RS sequence and the DS sequence are complementary to each other and form a stem-loop structure. The DS sequence can be complementary to at least a portion of the pseudoknot structure. The RS sequence, DS sequence and the different complementary pairing modes of the pseudoknot structure are utilized to achieve the translation of the target polypeptide encoding element downstream of IRES. Specifically, in Figure 1, by inserting an upstream sequence (called DS) that is complementary to the pseudoknot region, the original structure of IRES is destroyed, thereby inhibiting the translation activity mediated by IRES. This results in the downstream tagBFP translation being blocked, making it impossible to recruit ribosomes to initiate translation. The RS sequence can complement the DS sequence to form a stem-loop structure, achieving a pseudoknot structure in a single-stranded state, initiating downstream tagBFP translation. The present invention can dynamically switch the gene downstream of the IRES on and off. When the DS sequence complements the pseudoknot structure, the translation of the gene downstream of the IRES is in the off state; when the RS sequence complements the DS sequence to form a stem-loop structure, the translation of the gene downstream of the IRES is in the on state.

[0103] According to one embodiment of the present invention, the first RNA sequence further comprises at least one miRBS sequence, which is complementary and incompletely complementary to at least a portion of a miRNA specifically expressed in a target cell, wherein the target cell is a cell into which the RNA recombinant nucleic acid molecule is to be introduced.

[0104] When the miRBS sequence in the first RNA sequence is complementary to at least a portion of the miRNA specifically expressed in the target cell and is not completely complementary, it can initiate the translation of a polypeptide downstream of the IRES element or an element with similar functions to the IRES element. However, when the miRBS sequence is not complementary to the miRNA specifically expressed in the target cell, the IRES element or an element with similar functions to the IRES element is in a closed state and cannot recruit ribosomes to enter.

[0105] According to one embodiment of the present invention, when designing the miRBS sequence, it is necessary to ensure that when the miRBS sequence is complementary to the miRNA specifically expressed in the target cell, the bases of the two nucleotides at positions 10 and 11 in the complementary pairing region from the 5' to 3' direction are designed to be mismatched.

[0106] Designing the bases of the two nucleotides at positions 10 and 11 in the complementary pairing region as mismatches can ensure that the stem-loop structure is opened but will not be cut by the complex in the cell.

[0107] According to one embodiment of the present invention, each miRBS sequence binds to the same or different miRNA types. It should be noted that multiple miRBS sequences can be designed on a single RNA translation switch element, each miRBS sequence being the same or different, and each miRBS sequence binds to a complementary miRNA specifically expressed in target cells.

[0108] According to one embodiment of the present invention, as shown in FIG4A , when the first RNA sequence includes a miRBS sequence, the miRBS sequence is located upstream of the RS sequence;

[0109] The miRBS sequence is connected to the RS sequence via a second spacer sequence, and the second spacer sequence is partially complementary to the RS sequence.

[0110] When the DS sequence complementarily pairs with at least a portion of the structure contained in the IRES element that plays a key role in translation activity, at least a portion of the miRBS sequence, the second spacer sequence, the RS sequence, and at least a portion of the first spacer sequence form a first stem-loop structure.

[0111] According to one embodiment of the present invention, the RNA translation switch element comprises at least one of the first stem-loop structures, wherein the miRBS sequences contained in each of the first stem-loop structures independently bind to the same or different miRNA types.

[0112] According to a preferred embodiment of the present invention, as shown in Figure 4A, the balance can be maintained by regulating the length of the complementary pairing of the miRBS in the stem-loop, so that when the miRBS is not bound to the miRNA, the stem-loop structure is stable, and when the miRBS is bound to the miRNA, the stem-loop is easily opened. If the closed structure of the RNA translation switch element is too stable, then even if the miRBS is bound to the miRNA, it is not easy to open, and the function of the IRES translation switch in response to the miRNA cannot be achieved. If the closed structure of the RNA translation switch element is too unstable, the RNA translation switch element may open before the miRNA binds to the miRBS, resulting in the inability to achieve the purpose of controlling the IRES translation switch in response to the miRNA signal.

[0113] According to another embodiment of the present invention, as shown in FIG4B , when the first RNA sequence includes two miRBS sequences, the two miRBS sequences are located upstream of the RS sequence;

[0114] The two miRBS sequences are connected by a third spacer sequence, and the third spacer sequence is partially complementary to the RS sequence.

[0115] According to one embodiment of the present invention, the partial complementary pairing of the third spacer sequence and the RS sequence includes 2-3 non-complementary pairing regions.

[0116] According to one embodiment of the present invention, when the DS sequence is complementary to at least a portion of the structure contained in the IRES element that plays a key role in translation activity, at least a portion of the miRBS sequence located upstream, the third spacer sequence, the miRBS sequence located downstream, the RS sequence, and at least a portion of the first spacer sequence form a second stem-loop structure.

[0117] According to one embodiment of the present invention, the RNA translation switch element comprises at least one of the second stem-loop structures, wherein the miRBS sequences contained in each of the second stem-loop structures independently bind to the same or different miRNA types.

[0118] According to a preferred embodiment of the present invention, as shown in B in FIG4 , in a MITA (miRNA-responsive IRES translation activation element) having a stem-loop (SL) structure, if the RS sequence is fully complementary to the third spacer sequence (the connecting sequence between the two miRBS sequences), the closure will be too stable and the stem-loop will not be easy to open. Therefore, it is preferred to design several non-complementary paired bases to make it easier to open. Preferably, when designing the RS sequence, it is ensured that when the RS sequence is complementary to the third spacer sequence region, there are 2-3 non-complementary paired bases. According to another embodiment of the present invention, as shown in C in FIG4 , when the first RNA sequence includes three miRBS sequences, two of the three miRBS sequences are located upstream of the RS sequence, and another one of the three miRBS sequences is located between the RS sequence and the DS sequence.

[0119] According to one embodiment of the present invention, when the DS sequence is complementary to at least a portion of the structure contained in the IRES element that plays a key role in translation activity, a "Y"-shaped double stem-loop structure is formed between the sequences upstream of the DS sequence in the RNA translation switch element, and at least a portion of each of the three miRBS sequences is a double-stranded stem portion and / or a single-stranded loop portion in the "Y"-shaped double stem-loop structure.

[0120] According to one embodiment of the present invention, the RS sequence is located in the two double-stranded stems in the "Y"-shaped double stem-loop structure.

[0121] According to one embodiment of the present invention, the two double-stranded stems in the "Y"-shaped double stem-loop structure respectively have 1-2 non-complementary pairing regions.

[0122] According to one embodiment of the present invention, the RNA translation switch element comprises at least one "Y"-shaped double stem-loop structure, wherein the miRBS sequences contained in each of the "Y"-shaped double stem-loop structures independently bind to the same or different miRNA types. According to one embodiment of the present invention, the RNA recombinant nucleic acid molecule further comprises a non-target cell-specific miRBS sequence that is complementary to at least a portion of a miRNA specifically expressed in non-target cells, and the RNA recombinant nucleic acid molecule comprises at least one non-target cell-specific miRBS sequence.

[0123] According to one embodiment of the present invention, each of the miRBS sequences for non-target cells is located upstream of (1), and / or between (1) and (2), and / or between (2) and (3), and / or between (3) and the stop codon.

[0124] According to one embodiment of the present invention, the miRBS sequence targeting non-target cells is a plurality of tandem miRBS sequences, and each independently binds to the same or different miRNA types specifically expressed in non-target cells.

[0125] According to one embodiment of the present invention, the miRBS sequence targeting non-target cells is 3-5 tandem miRBS sequences.

[0126] According to one embodiment of the present invention, the miRBS sequence targeting non-target cells is completely complementary to the miRNA specifically expressed in the non-target cells.

[0127] According to one embodiment of the present invention, the length of the RS sequence is 9-25 nt, preferably 9-13 nt.

[0128] According to one embodiment of the present invention, the length of the DS sequence is 10-25 nt, preferably 10-22 nt.

[0129] According to one embodiment of the present invention, the length of the miRBS sequence that binds to the miRNA specifically expressed in target cells or non-target cells is 18-25 nt, preferably 20-24 nt.

[0130] According to one embodiment of the present invention, the element having a function similar to that of the IRES element is an element having a translation initiation function.

[0131] According to one embodiment of the present invention, the element having a function similar to that of the IRES element includes an element having at least one nucleic acid deletion, insertion or substitution compared to the IRES element.

[0132] According to one embodiment of the present invention, the IRES element is derived from a hepatitis C-like virus.

[0133] According to one embodiment of the present invention, the hepatitis C-like virus includes at least one selected from hepatitis C virus (HCV), avian encephalomyelitis virus (AEV), classical swine fever virus (CSFV), and porcine enterovirus (PTV).

[0134] Although these hepatitis C virus-derived IRES sequences are not identical, they share a high degree of sequence identity and all contain the key pseudoknot structure. While the present invention primarily cites HCV-derived IRES sequences in the examples, theoretically, IRES sequences derived from other viruses that contain structures critical for translational activity (e.g., pseudoknot structures) are also encompassed within the scope of the present invention.

[0135] According to one embodiment of the present invention, the structure contained in the IRES element that plays a key role in translation activity is a 3'-end pseudoknot structure.

[0136] According to one embodiment of the present invention, the element having a similar function to the IRES element is a truncated IRES element.

[0137] According to one embodiment of the present invention, the nucleic acid sequence of the truncated IRES element is a sequence obtained by removing the 5'-end 30-40 bases of the wild-type IRES. For example, the nucleic acid sequence of the truncated IRES element is a sequence obtained by removing the 5'-end 40 bases of the wild-type HCV IRES.

[0138] According to one embodiment of the present invention, the RNA recombinant nucleic acid molecule is a linear RNA molecule or a circular RNA molecule, preferably a circular RNA molecule to enhance stability.

[0139] In the present invention, the miRBS sequence is determined based on the activity of the miRNA specifically expressed in the cell. For example, when a target polypeptide encoding element is required to initiate translation in tumor cells, but not in normal cells, the miRNA activity in normal and tumor cells must differ. If the miRNA screened has high activity in tumor cells but low activity in normal cells, the RNA recombinant nucleic acid molecule binds to the miRNA in the tumor cells, and the IRES in the tumor cells initiates translation of the target polypeptide encoding element. Simultaneously, another miRNA with low activity in tumor cells but high activity in normal cells is selected, and this signal is designed to be an off signal. In normal cells, the RNA recombinant nucleic acid molecule binds to the miRNA in normal cells, and the RNA recombinant nucleic acid molecule is cleaved in normal cells. In tumor cells, due to the lack of the normal miRNA signal, the RNA recombinant nucleic acid molecule is not cleaved. The miRNA highly expressed in tumor cells can activate the IRES to initiate translation, while the miRNA highly expressed in normal cells will inhibit (cleave) the RNA recombinant nucleic acid molecule of the present invention. Utilizing these two approaches simultaneously, the target polypeptide encoding element is translated only in tumor cells, thereby ensuring the safety of the RNA recombinant nucleic acid molecule.

[0140] The RNA recombinant nucleic acid molecule provided by the present invention does not require the combination of multiple RNA molecules, nor the introduction of exogenous proteins. The present invention can construct RNA with cell-specific expression ability by engineering a single RNA molecule, and can sense multiple miRNA signals to achieve logical operations. The engineered IRES element proposed in the present invention can be used for both linear mRNA and circular RNA expression regulation. The strategy for engineering IRES proposed in the present invention can be applied to a variety of IRESs, and has universality and portability. The present invention achieves dynamic structural changes of the engineered IRES element by sensing endogenous miRNA signals in the cell, thereby dynamically regulating the translation activity of IRES. The present invention is the first that can simultaneously achieve upregulation or downregulation of the translation activity of the target protein by relying on a single circular RNA molecule, and can be used for logical operations of cell signals and cell classification.

[0141] Isolated DNA sequences, vectors, cells, compositions

[0142] According to one embodiment of the present invention, the present invention provides an isolated DNA sequence, comprising a DNA sequence capable of being transcribed into the aforementioned RNA recombinant nucleic acid molecule.

[0143] According to another embodiment of the present invention, the present invention provides a vector comprising the isolated DNA sequence described above.

[0144] According to another embodiment of the present invention, the present invention provides a cell comprising the aforementioned RNA recombinant nucleic acid molecule and / or the aforementioned isolated DNA sequence and / or the aforementioned vector.

[0145] According to one embodiment of the present invention, the present invention provides a composition comprising the aforementioned RNA recombinant nucleic acid molecule and / or the aforementioned isolated DNA sequence and / or the aforementioned vector.

[0146] According to one embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier.

[0147] According to one embodiment of the present invention, the present invention provides that the carrier is a lipid nanoparticle or a liposome, and the lipid nanoparticle or the liposome encapsulates the aforementioned RNA recombinant nucleic acid molecule and / or the aforementioned isolated DNA sequence and / or the aforementioned carrier.

[0148] It should be noted that when the RNA recombinant nucleic acid molecule provided by the present invention or a composition containing the RNA recombinant nucleic acid molecule is used to treat tumor-related diseases, the RNA recombinant nucleic acid molecule does not contain the miRBS binding site of normal cells. The RNA recombinant nucleic acid molecule or composition can also be used to treat tumor-related diseases and can also achieve differentiated expression of the target polypeptide in tumors and normal cells. However, if it contains the miRBS binding site of normal cells, the RNA recombinant nucleic acid molecule expresses the target polypeptide with greater differentiation between tumors and normal cells, and has better protection of safety switch.

[0149] Application in preparing pharmaceutical compositions for treating or preventing diseases

[0150] The present invention provides the use of the aforementioned RNA recombinant nucleic acid molecule, the aforementioned isolated DNA sequence, the aforementioned vector, and the aforementioned composition in the preparation of a pharmaceutical composition for treating or preventing a disease, wherein the disease is related to the expression of the target polypeptide, and the expression of the target polypeptide can treat or prevent the disease.

[0151] According to one embodiment of the present invention, when the disease is a tumor, the target polypeptide is selected from at least one of a polypeptide that induces cell apoptosis and a polypeptide that is toxic or lethal to tumor cells.

[0152] Method for expressing target polypeptide in cells and method for treating tumors

[0153] The present invention provides a method for expressing a target polypeptide in a cell, comprising:

[0154] The aforementioned RNA recombinant nucleic acid molecule and / or the aforementioned isolated DNA sequence and / or the aforementioned vector and / or the aforementioned composition are introduced into cells to express the target polypeptide.

[0155] According to one embodiment of the present invention, at least a portion of the miRNA specifically expressed in the cell is complementary and not completely complementary to the miRBS sequence in the first RNA sequence in the RNA recombinant nucleic acid molecule expressed in the cell after the introduction of the exogenous molecule.

[0156] According to one embodiment of the present invention, the present invention provides a method for treating tumors, comprising:

[0157] administering the aforementioned RNA recombinant nucleic acid molecule and / or the aforementioned isolated DNA sequence and / or the aforementioned vector and / or the aforementioned composition to a subject, wherein the target polypeptide is expressed in tumor cells but not in healthy cells,

[0158] Wherein, the target cells are tumor cells, and the non-target cells are healthy cells.

[0159] According to one embodiment of the present invention, the target polypeptide is selected from at least one of a polypeptide that induces cell apoptosis and a polypeptide that is toxic or lethal to tumor cells.

[0160] The method for treating tumors provided by the present invention aims to achieve high expression of at least one of a polypeptide that induces apoptosis and a polypeptide that is toxic or lethal to tumor cells in tumor cells, while low expression or no expression in normal cells. The IRES element initiates translation of the target polypeptide encoding element in tumor cells, which is in an open state and in a closed state in normal cells. In tumor cells, the IRES system described above is used to achieve expression of lethal genes. On this basis, in order to increase safety, expression in normal tissues is minimized, and miRNA cleavage signals that are highly expressed in normal tissues are combined to enhance safety. This is equivalent to the situation in which, in normal cells, the IRES structure is damaged and cannot be expressed because there is no tumor signal (miRNA specific to tumor cells), while on the other hand, the miRNA signal highly expressed in normal cells is sensed. The RNA recombinant nucleic acid molecule of the present invention has a double guarantee of safety in treatment. According to a preferred embodiment of the present invention, the miRBS for normal cells and the IRES for tumor cells are on the same RNA recombinant nucleic acid molecule.

[0161] According to a specific embodiment of the present invention, regarding the method for treating tumors, the control of the translation of the target polypeptide encoding element (taking fluorescent protein as an example) by the RNA recombinant nucleic acid molecule or composition of the present invention can be verified by taking tumor model mice (solid tumors) as an example. For example, exogenous RNA (the RNA recombinant nucleic acid molecule or composition provided by the present invention) is injected (such as by intravenous injection) into a mouse with a solid tumor to observe whether it is expressed in the tumor (fluorescent) and whether there is no fluorescence in the non-tumor-planted area (healthy area). This can verify whether the RNA recombinant nucleic acid molecule or composition provided by the present invention can be used to control the translation of polypeptides that induce cell apoptosis, polypeptides that are toxic or lethal to tumor cells in tumor cells but not in healthy cells.

[0162] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0163] Experimental Materials and Methods

[0164] 1. Sources of reagents and enzymes.

[0165] Restriction endonucleases, ATP, polynucleotide kinase (PNK), T4 DNA ligase, and Q5 high-fidelity DNA polymerase were purchased from New England Biolabs. Oligonucleotides were synthesized by Qingke Biotechnology Co., Ltd. miRNA mimics and inhibitors were purchased from Hesheng Biotechnology Co., Ltd.

[0166] 2. Plasmid construction.

[0167] As needed, equimolar amounts of oligonucleotides were heated to 95°C in 1× PNK buffer and gradually cooled (-1°C per minute) to 37°C for annealing. 1 μM of the annealed product was then phosphorylated with 0.5 U / μL PNK in the presence of 0.5 mM ATP. Golden Gate Assembly was performed according to the New England Biolabs protocol.

[0168] 3. Cell Culture and Transfection.

[0169] HEK293 (HEK293FT) cell line was purchased from Life Technologies. Huh7 cell line was purchased from BeNa culture collection Co., Ltd. Hela cell line was purchased from ATCC. All cells were cultured in high glucose DMEM (Invitrogen) containing 10% FBS (DiNing) and 1% penicillin-streptomycin mixture (Invitrogen) at 37°C, 100% humidity and 5% CO2. Lipofectamine 8000 (Beyond Time Co., Ltd.) was used for plasmid and miRNA mimic transfection. Transfection was performed according to the manufacturer's protocol. Briefly, one day before transfection, approximately 3 × 10 cells were seeded in each well of each 24-well plate (Corning). 5 For each cell, 1 mL of DMEM complete medium was added. For plasmid transfection, 0.6 μg of plasmid was mixed with 0.6 μg of Lipofectamine 8000 and then transfected into each well. For co-transfection of miRNA mimics or miRNA inhibitors, the miRNA mimics or miRNA inhibitors were mixed with 0.6 μg of plasmid and 0.6 μg of Lipofectamine 8000 to a final transfection concentration of 50 nM and then transfected into each well. For circRNA transfection, the circRNA was encapsulated with lipid nanoparticles as described below and transfected into the cells at 0.6 μg per well.

[0170] 4. Preparation and purification of circular RNA.

[0171] Preparation and purification of circular RNA (circRNA) were performed as follows. First, the DNA template of the circRNA precursor was generated by linearizing the plasmid construct using Xba I endonuclease. The circRNA precursor was generated by in vitro transcription (IVT) from the DNA template using T7 Polymerase (Novoprotein). After IVT, the RNA product was treated with DNase I (Thermo Fisher Scientific) for 30 minutes to digest the DNA template. After DNase I digestion, GTP (Novoprotein) was added to the RNA product to a final concentration of 2mM and incubated at 55°C for 15 minutes to catalyze the circularization of circRNAs. Then, the precursor RNA was removed by treatment with RNase R (Novoprotein) at 37°C for 15 minutes. The circRNA after RNase R treatment was purified using LiCl (Novoprotein).

[0172] 5. Lipid nanoparticles encapsulate circRNA.

[0173] CircRNAs were encapsulated in lipid nanoparticles (LNPs) dissolved in ethanol containing an ionizable lipid (SM-102), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid (molar ratio of 50:10:38.5:1.5). The lipid mixture was mixed with 50 mM citrate buffer (pH 4.0) containing the circRNA at a ratio of 1:3 using a Lipid Mixer (Micronano INano L). The formulation was then dialyzed against an ultrafiltration centrifugal filter unit (Millipore) with a 100 kD molecular weight cutoff (Sartorius Stedim Biotech) and 30 volumes of PBS (pH 7.4) to concentrate to the desired concentration and store at −20°C. All formulations were tested for particle size, distribution, RNA concentration, and encapsulation efficiency.

[0174] 6. Flow cytometry.

[0175] Three days after transfection, cells were trypsinized and centrifuged at 300 g for 5 min at 4°C. The supernatant was removed and the cells were resuspended in 1× PBS (Invitrogen) without calcium or magnesium. Fluorescence activated flow cytometry analysis was performed using a Fortessa flow cytometer (BD Biosciences) with the following settings: EYFP fluorescence was excited at 514 nm and emitted in the range of 519-568 nm. TagBFP fluorescence was excited at 405 nm and emitted in the range of 410-507 nm. mKate fluorescence was excited at 543 nm and emitted in the range of 571-651 nm. For each sample, approximately 1×10 5 to about 5×10 5 Cell events.

[0176] 7. Luciferase assay.

[0177] 24 hours after transfection, the cell culture medium was removed and 100 μL of Bright-Light Luciferase Assay System (Vazyme) was added, mixed and incubated for 3 min. 100 μL of the culture mixture was transferred to a flat-bottom white-walled plate (Corning) and the luminescence intensity was measured using a microplate luminometer (Thermo Fisher Scientific).

[0178] 8. Lactate dehydrogenase cytotoxicity assay.

[0179] Cell death was assessed using a lactate dehydrogenase cytotoxicity assay kit (Beyotime, Shanghai, China). LDH analysis was performed according to the manufacturer's protocol. Briefly, 3 × 10 cells / well of each 96-well culture plate (Corning) were seeded one day before transfection. 5 cells and added 1 mL of DMEM complete medium. The next day, 0.2-0.5 μg of LNP-encapsulated circRNA was transfected into each well. 24 hours later, the cell culture plate was centrifuged at 400 g for 5 minutes, and the supernatant was carefully aspirated. 150 μL of 10-fold diluted lactate dehydrogenase (LDH) release reagent (the volume ratio of the reagent to phosphate buffered saline (PBS) was 1:10) was added and mixed. After incubation for one hour, the cell culture plate was centrifuged again at 400 g for 5 minutes, and the resulting supernatant (120 μL per well) was transferred to a new 96-well plate for subsequent sample quantification.

[0180] 9. Data analysis.

[0181] For all studies, the data presented are representative results of three independent biological replicates. For flow cytometry measurements, EYFP or mKate (when the construct includes a fully complementary miRBS) fluorescent protein was used as an internal control. For each sample, the tagBFP fluorescence intensity mean (tagBFP MFI) and EYFP or mKate MFI of the positive gated cells were calculated. The relative IRES-mediated tagBFP fluorescence intensity was defined as the ratio of tagBFP / EYFP (or tagBFP / mKate when the construct includes a fully complementary miRBS) MFI. The normalized IRES-mediated tagBFP fluorescence intensity was defined as the relative IRES-mediated tagBFP fluorescence intensity divided by the positive control (nIRES or rIRES) tested in the same cell line. For luciferase assays, the luminescence intensity mean of each sample was calculated. Normalized luciferase activity was defined as the luminescence intensity divided by the positive control (nIRES or rIRES) tested in the same cell line.

[0182] Example 1 Inhibition and restoration of HCV-IRES translation activity

[0183] The present embodiment is described with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) as an example.HCV-IRES is composed of four domains, and the special pseudoknot structure in HCV-IRES domain 4 plays a vital role in the translation initiation mediated by HCV-IRES, and the pseudoknot is spatially adjacent to the upstream end of HCV-IRES. The inventors make the original structure of HCV-IRES be destroyed by inserting the upstream sequence (called DS) complementary to the pseudoknot region, thereby inhibiting the translation activity mediated by IRES (A in FIG1 ). A bicistronic fluorescent reporter system was constructed, and the translation efficiency (B in FIG1 ) of the engineered IRES with different DS lengths in human embryonic kidney cells (HEK293) was evaluated by flow cytometry. The translation of enhanced yellow fluorescent protein (EYFP) depends on the cap structure of RNA as an internal reference gene. The translation of blue fluorescent protein (tagBFP) is initiated by IRES, so the mean fluorescence intensity (MFI) ratio of tagBFP to EYFP can characterize the translation efficiency mediated by IRES. Compared with the unmodified natural IRES (nIRES), the MFI of tagBFP began to decrease when the upstream DS length exceeded 12 nucleotides (12-nt). When the DS length reached 20-nt, the MFI of tagBFP decreased by more than 10-fold compared with nIRES ( Figure 1 , the IRES containing a 20-nt DS is called dIRES).

[0184] Next, a recovery sequence (RS) complementary to the 20-nt DS was designed and inserted, because the base pairing between RS and DS will restore the original structure and translation initiation activity of HCV-IRES (A in Figure 2), wherein the pairing of the pseudoknot region with DS (translation closed state) is changed to the pairing of RS with DS (translation initiation state), the pseudoknot region is released, and translation is restored. By introducing RS upstream of DS, when the RS length exceeds 8-nt, the MFI of tagBFP increases. When the RS length reaches 13-nt, the MFI of tagBFP is restored to more than 70% of nIRES (the dIRES containing 13-nt RS is called rIRES) (B in Figure 2). In addition, the inventors also observed tagBFP translation inhibition of dIRES similar to that in HEK293 cells and tagBFP translation recovery of rIRES in human hepatocellular carcinoma cells (Huh7) (Figure 3).

[0185] The nucleic acid sequences involved in this embodiment are shown in Table 1 below:

[0186] Table 1

[0187] Example 2 Construction of three types of MITA

[0188] To further design a programmable miRNA-responsive IRES translation activation element (MITA), the inventors attempted to design a secondary structure with a miRNA binding site (miRBS) to fix the RS in a relatively stable structure. To ensure that the miRNA only binds to the miRBS and is not cleaved, the miRBS was designed as a miRNA sponge, that is, there were two nucleotide mismatches with the miRNA at positions 10 and 11. Three types of MITA were constructed (Figure 4), namely the shoe last (TL), stem-loop (SL), and three-arm (AJ) structures (corresponding to AC, respectively). In the absence of a complementary miRNA, the 13-nt RS is fixed in the designed structure, so that the IRES-mediated tagBFP translation is inhibited by the 20-nt DS (i.e., the device is in the closed state). When the complementary miRNA is present, the miRNA binds to the designed miRBS and destroys the designed RNA secondary structure, releasing the 13-nt RS and thus restoring the IRES structure and translation activity (i.e., the device is in the open state).

[0189] First, miRBSs complementary to the artificial miRNA FF4 were designed and co-transfected with miRNA mimics to test all three MITA types in HEK293 cells (Figure 5). The left column in each group in Figure 5 represents a control group (addition of an equal amount of non-complementary miRNA to the miRBS as miRNA FF4), while the right column represents an experimental group in which miRNA FF4 was added (FF4 mimics). The designed structures included a shoe-last structure with 16 nucleotide base pairs (TL16), a stem-loop structure with three nucleotide mismatches (SL3), and a three-arm structure with two nucleotide mismatches (AJ2). In the absence of the corresponding miRNA mimics, all three MITA types showed reduced tagBFP translation, indicating that in the absence of the required miRNA, MITA is in an off state. Following co-transfection with the miRNA FF4 mimic, tagBFP translation was largely restored, indicating that the presence of miRNA FF4 triggers MITA to switch to an on state.

[0190] Next, an attempt was made to use MITA to sense endogenous miRNA. A miRBS complementary to miR-21-5p was designed (miR-21-5p is a miRNA that is highly expressed in most cancer cells but lowly expressed in HEK293). The tagBFP translation levels of all three types of MITA in Huh7 and HEK293 cells were detected (Figure 6, the left column of each group in the figure is HEK293 cells, and the right column is Huh7 cells). Since the initial structure of the design will stabilize the closed state if there are more base pairings, and more base mispairings will be detrimental to the closed state, the inventors changed the number of base pairings and mispairings to optimize the MITA structure. The results showed that all MITA showed higher tagBFP translation activity in Huh7 compared to HEK293, and TL16, SL3 and AJ2 showed the highest fold change (more than 3 times).

[0191] The nucleic acid sequences involved in this embodiment are shown in Table 2 below:

[0192] Table 2

[0193] Example 3 Designing MITA to implement logical operations in cells

[0194] After demonstrating that sequence programming of MITA can respond to any miRNA, the inventors attempted to design MITA to implement logical operations in cells. The inventors tandemly connected two TL16 sequences of miRNA-FF4 and miRNA-199a-3p (neither of these miRNAs is expressed in HEK293) and tested them in HEK293 by co-transfection with miRNA mimics (Figure 7). When co-transfected with miRNA-FF4 or miRNA-199a-3p mimics, the IRES-mediated tagBFP translation activity was restored, indicating that the design of tandem TL16 can achieve a logical "or" function, that is, any one of the miRNAs, miRNA-FF4 or miRNA-199a-3p, can initiate translation of tagBFP mediated by IRES. The inventors then designed an SL3 motif with both miRNA-FF4 and miRNA-199a-3p binding sites (Figure 8). The results showed that the translation of tagBFP could only be restored when miRNA-FF4 and miRNA-199a-3p mimics were co-transfected at the same time, indicating that the dual-input SL3 design can be used as a logic "AND" gate. In order to achieve the logic "NOT" function, the inventors directly inserted the fully complementary binding sites of miRNA-FF4 upstream and downstream of HCV-IRES to construct a programmable miRNA-based IRES translation repressor (MITR) (Figure 9). When miRNA-FF4 was co-transfected with MITR, the translation of tagBFP was significantly inhibited, indicating that MITR can achieve a logic "NOT" gate. In the "NOT" gate, no miRNA signal can be expressed. After receiving the miRNA signal, the miRBS is fully complementary to the miRNA and will be recruited to the shearing complex in the cell, causing the mRNA to be sheared and IRES to be unable to initiate translation of downstream sequences. The nucleic acid sequences involved in this embodiment are shown in Table 3 below:

[0195] Table 3

[0196] Example 4 Construction of cell type classifier

[0197] The inventors attempted to use the aforementioned MITA and MITR design strategies to construct a cell type classifier. The inhibitory activity of several miRNAs in HEK293 and Huh7 was tested (Figure 10). In Figure 10, mKATE expression is not regulated by miRNA, while EYFP is regulated by miRNA. The mKATE plasmid serves as a control. When there is a miRNA signal in the cell, the pCMV-EYFP-4×miRNA binding sites plasmid will be cut, affecting the expression of YFP. Based on the inhibitory activity results, miR-21-5p was ultimately selected as the Huh7 highly active miRNA to construct the MITA module, and miR-106a-5p and miR-18a were selected as the HEK293 highly active miRNAs to implement the logical "NOT" gate. A MITA (IRES-related sequence) containing miR-21-5p-responsive TL16, SL3, and AJ2 was constructed. Four miR-106a-5p and miR-18a binding sites were inserted in series upstream of the MITA and downstream of the tagBFP reporter gene (Figure 11). These plasmid vectors were transfected into Huh7 and HEK293 cells, respectively, using mKate fluorescent protein as an internal control. In Huh7 cells, the 4xmiR-18a RBS and 4xmiR-106a RBS do not bind to miR-18a and miR-106a, and no cleavage occurs. At the same time, MITA senses the tumor cell miRNA signal, thereby promoting BFP expression. In normal cells, the opposite is true: MITA does not sense the miRNA signal, BFP is not expressed or is weakly expressed, while the 4xmiR-18a RBS and 4xmiR-106a RBS bind to the highly expressed miRNAs in normal cells, and the entire structure is cleaved. Figure 11 shows that tagBFP fluorescence intensity increased up to 7-fold in Huh7 cells compared to HEK293 cells (left column in each group is HEK293 cells, right column is Huh7 cells). Notably, when MITA or MITR were used alone, only a 3-fold or 4-fold increase in fluorescence signal was observed. Therefore, the results suggest that MITA and MITR have a synergistic effect in cell type classification.

[0198] The nucleic acid sequences involved in this embodiment are shown in Table 4 below:

[0199] Table 4

[0200] Example 5

[0201] To directly apply MITA and cell type classifiers to circRNAs, plasmid templates containing a replaced intron-exon (PIE) were constructed for in vitro transcription (IVT) of circRNAs (Figure 12). Using firefly luciferase as a reporter gene, circRNAs encoding nIRES, dIRES, and rIRES were first transfected into HEK293 and Huh7 cells, respectively. The same dIRES inhibition and rIRES restoration effects as those observed with plasmid transfection were observed (Figure 13, the left column of each group is HEK293, and the right column is Huh7 cells). Next, circRNAs encoding MITA of TL16, SL3, or AJ2 were transfected into Huh7 and HEK293 cells, respectively (Figure 14, the left column of each group is HEK293, and the right column is Huh7 cells). The results showed that the TL16 or SL3 designs showed a more than 3-fold increase in fluorescence signal in Huh7 compared to HEK293, while AJ2 showed a more than 6-fold increase in fluorescence signal. These results indicate that the MITA module of the present invention can also be activated by miRNA in circRNA. Finally, the circRNA version of the cell type classifier was tested (Figure 15, the left column of each group in the right figure is HEK293, and the right column is Huh7 cells). Compared with HEK293, an increase of up to 8 times in fluorescence signal was observed in Huh7, which indicates that the cell type classifier circRNA of the present invention can accurately identify the desired cancer cells and may serve as a potential cancer targeted therapy.

[0202] The nucleic acid sequences involved in this embodiment are shown in Table 5 below:

[0203] Table 5

[0204] Example 6 Improvement of MITA

[0205] To further refine the MITA design, the inhibitory effect of the DS in a truncated HCV-IRES (tIRES) domain 1 was tested, as the inventors believed that shortening the spatial distance between the DS and the key pseudoknot site of the IRES would promote complementary base pairing. The inventors found that the truncated IRES structure could also function even when the first neck loop of the wild-type IRES structure was removed, thus enabling the design of IRES systems with shortened DS and RS lengths. The results showed that the insertion of just a 10-nt DS significantly reduced the translational activity of tIRES by more than 10-fold (this structure is referred to as dtIRES, Figure 16).

[0206] The inventors also tested RSs that restore the translational activity of dtIRES. Simply inserting a 9-nt RS restored the translational activity of dtIRES to over 70% of that of tIRES (Figure 17). Therefore, the present invention can also be applied to the development of MITA and MITR in truncated versions of HCV-IRES. In a full-length IRES structure, the DS is typically designed to be 20 nt and the RS to be 13 nt. After truncation, a 10-nt DS and a 9-nt RS can achieve translational on / off switching.

[0207] In addition, the inventors also designed a 20-nt DS to disrupt the pseudoknot structure of other IRESs to demonstrate the universality and scalability of the strategy used in the present invention. Similar inhibitory effects were observed in the IRESs of avian encephalomyelitis virus (AEV), classical swine fever virus (CSFV), and porcine enterovirus (PTV), indicating that the strategy of the present invention can be extended to other IRESs (Figure 18).

[0208] The nucleic acid sequences involved in this embodiment are shown in Table 6 below:

[0209] Table 6

[0210] Example 7: Killing effect of RNA molecules of the present invention on cancer cells

[0211] To verify the specific toxicity of the circular RNA of the present invention to cancer cells, the luciferase reporter gene in the circular RNA was replaced with the N-terminal portion of Gasdermin D (GSDMD) (Figures 19A and 20A). Cytotoxicity was determined using lactate dehydrogenase (LDH). As shown in Figure 19B, the experimental results showed that for undesigned common circular RNA molecules (native IRES), LDH activity in Huh7 cells was significantly increased by 18 times compared to HEK293 cells (the left column of each group in Figure 19B is HEK293, and the right column is Huh7 cells. The circular RNA containing AJ2 MITA does not contain a miRBS sequence).

[0212] When the cell type classifier circular RNA of the present invention was transfected into Huh7 and HEK293 cells, the observed cytotoxicity difference was even more significant, reaching 89-fold (Figure 20B, the left column of each group is HEK293, and the right column is Huh7 cells. The circular RNA containing AJ2 MITA contains 4× miRNA binding site sequences). Figure 20C is a micrograph corresponding to Figure 20B, which visually reflects the cell killing.

[0213] These results demonstrate the specific killing effect of the circular RNA of the present invention on cancer cells and highlight the potential applicability of the MITA module and cell type classifier of the present invention in targeted cancer therapy applications.

[0214] The nucleic acid sequences involved in this embodiment are shown in Table 7 below:

[0215] Table 7

[0216] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0217] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An RNA recombinant nucleic acid molecule, wherein: include: (1) RNA translation switch element; (2) IRES elements or elements with similar functions to IRES elements; (3) target polypeptide encoding element, Wherein, the IRES element or an element having a similar function to the IRES element includes a structure that plays a key role in translation activity, The RNA translation switch element comprises a first RNA sequence and a second RNA sequence, wherein the first RNA sequence is located upstream of the second RNA sequence, the first RNA sequence contains an RS sequence, the second RNA sequence contains a DS sequence, and a first spacer sequence is present between the RS sequence and the DS sequence. (a) the RS sequence and at least a portion of the DS sequence are complementary to each other and form a stem-loop structure; (b) the DS sequence is complementary to at least a portion of the structure that plays a key role in translation activity; In the RNA recombinant nucleic acid molecule, (a) and (b) do not occur at the same time, and the initiation of translation of the target polypeptide encoding element is achieved through the changes in the two structures of (a) and (b).

2. The RNA recombinant nucleic acid molecule according to claim 1, wherein The first RNA sequence further includes at least one miRBS sequence, which is complementary and incompletely complementary to at least a portion of a miRNA specifically expressed in a target cell, wherein the target cell is a cell into which the RNA recombinant nucleic acid molecule is to be introduced.

3. The RNA recombinant nucleic acid molecule according to claim 2, wherein: When designing the miRBS sequence, it is necessary to ensure that when the miRBS sequence is complementary to the miRNA specifically expressed in the target cell, the bases of the two nucleotides at the 10th and 11th positions in the complementary pairing region from the 5' to 3' direction are designed to be mismatched.

4. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 3, wherein Each miRBS sequence binds the same or different miRNA types.

5. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 4, wherein When the first RNA sequence includes a miRBS sequence, the miRBS sequence is located upstream of the RS sequence; The miRBS sequence is connected to the RS sequence via a second spacer sequence, and the second spacer sequence is partially complementary to the RS sequence, Optionally, when the DS sequence is complementary to at least a portion of the structure contained in the IRES element that plays a key role in translation activity, at least a portion of the miRBS sequence, the second spacer sequence, the RS sequence, and at least a portion of the first spacer sequence form a first stem-loop structure; Optionally, the RNA translation switch element comprises at least one of the first stem-loop structures, wherein the miRBS sequences contained in each of the first stem-loop structures independently bind to the same or different miRNA types.

6. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 4, wherein When the first RNA sequence includes two miRBS sequences, the two miRBS sequences are located upstream of the RS sequence; The two miRBS sequences are connected by a third spacer sequence, and the third spacer sequence is partially complementary to the RS sequence. Optionally, the third spacer sequence partially complementarily pairs with the RS sequence and comprises 2-3 non-complementary pairing regions; Optionally, when the DS sequence is complementary to at least a portion of the structure contained in the IRES element that plays a key role in translation activity, at least a portion of the miRBS sequence located upstream, the third spacer sequence, the miRBS sequence located downstream, the RS sequence, and at least a portion of the first spacer sequence form a second stem-loop structure; Optionally, the RNA translation switch element comprises at least one of the second stem-loop structures, wherein the miRBS sequences contained in each of the second stem-loop structures independently bind to the same or different miRNA types.

7. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 4, wherein When the first RNA sequence includes three miRBS sequences, two of the three miRBS sequences are located upstream of the RS sequence, and another one of the three miRBS sequences is located between the RS sequence and the DS sequence; Optionally, when the DS sequence is complementary to at least a portion of the structure contained in the IRES element that plays a key role in translation activity, a "Y"-shaped double stem-loop structure is formed between the sequences upstream of the DS sequence in the RNA translation switch element, and at least a portion of each of the three miRBS sequences is in the double-stranded stem portion and / or the single-stranded loop portion of the "Y"-shaped double stem-loop structure; Optionally, the RS sequence is located at the two double-stranded stems in the "Y"-shaped double stem-loop structure; Optionally, the two double-stranded stems in the "Y"-shaped double stem-loop structure each have 1-2 non-complementary pairing regions; Optionally, the RNA translation switch element comprises at least one of the "Y"-shaped double stem-loop structures, wherein the miRBS sequences contained in each of the "Y"-shaped double stem-loop structures independently bind to the same or different miRNA types.

8. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 7, wherein The RNA recombinant nucleic acid molecule further comprises a miRBS sequence for non-target cells that is complementary to at least a portion of the miRNA specifically expressed in the non-target cells, and the RNA recombinant nucleic acid molecule has at least one miRBS sequence for non-target cells; Optionally, each of the miRBS sequences for non-target cells is located upstream of (1), and / or between (1) and (2), and / or between (2) and (3), and / or between (3) and the stop codon; Optionally, the miRBS sequence for non-target cells is a plurality of tandem miRBS sequences, and each independently binds to the same or different miRNA types specifically expressed in non-target cells; Optionally, the miRBS sequence for non-target cells is 3-5 tandem miRBS sequences, Optionally, the miRBS sequence for non-target cells is completely complementary to the miRNA specifically expressed in the non-target cells.

9. The RNA recombinant nucleic acid molecule according to any one of claims 2 to 8, wherein The length of the RS sequence is 9-25 nt, preferably 9-13 nt; Optionally, the length of the DS sequence is 10-25 nt, preferably 10-22 nt; Optionally, the length of the miRBS sequence that binds to the miRNA specifically expressed in the target cell or non-target cell is 18-25 nt, preferably 20-24 nt.

10. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 9, wherein The element having a similar function to the IRES element is an element having a translation initiation function; Optionally, the element having a similar function to the IRES element includes an element having at least one nucleic acid deletion, insertion or substitution compared to the IRES element; Optionally, the IRES element is derived from a hepatitis C-like virus; Optionally, the hepatitis C-like virus includes at least one selected from the group consisting of hepatitis C virus, avian encephalomyelitis virus, classical swine fever virus, and porcine enterovirus; Optionally, the structure contained in the IRES element that plays a key role in translation activity is a 3'-terminal pseudoknot structure; Optionally, the element having a similar function to the IRES element is a truncated IRES element; Optionally, the nucleic acid sequence of the truncated IRES element is a sequence obtained by removing 30-40 bases from the 5' end of the wild-type IRES.

11. The RNA recombinant nucleic acid molecule according to any one of claims 1 to 10, wherein: The RNA recombinant nucleic acid molecule is a linear RNA molecule or a circular RNA molecule.

12. An isolated DNA sequence, wherein: The invention comprises a DNA sequence capable of being transcribed into the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11.

13. A vector, wherein Comprising the isolated DNA sequence of claim 12.

14. A cell, wherein: The invention comprises the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11 and / or the isolated DNA sequence according to claim 12 and / or the vector according to claim 13.

15. A composition, wherein The invention comprises the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11 and / or the isolated DNA sequence according to claim 12 and / or the vector according to claim 13.

16. The composition according to claim 15, wherein The composition further comprises a pharmaceutically acceptable carrier; Optionally, the carrier is a lipid nanoparticle or a liposome, which encapsulates the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11 and / or the isolated DNA sequence according to claim 12 and / or the carrier according to claim 13.

17. Use of the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11, the isolated DNA sequence according to claim 12, the vector according to claim 13, or the composition according to claim 15 or 16 in the preparation of a pharmaceutical composition for treating or preventing a disease, wherein: The disease is associated with the expression of the target polypeptide, and the expression of the target polypeptide can treat or prevent the disease.

18. The use according to claim 17, wherein: When the disease is a tumor, the target polypeptide is selected from at least one of a polypeptide that induces cell apoptosis and a polypeptide that is toxic or lethal to tumor cells.

19. A method for expressing a target polypeptide in a cell, wherein: The method comprises: Introducing the RNA recombinant nucleic acid molecule according to any one of claims 1 to 11 and / or the isolated DNA sequence according to claim 12 and / or the vector according to claim 13 and / or the composition according to claim 15 or 16 into a cell to express the target polypeptide; Optionally, at least a portion of the miRNA specifically expressed in the cell is complementary and not completely complementary to the miRBS sequence in the first RNA sequence in the RNA recombinant nucleic acid molecule expressed in the cell after the introduction of the exogenous molecule.

20. A method for treating a tumor, wherein: The method comprises: Administering the RNA recombinant nucleic acid molecule of any one of claims 1 to 11 and / or the isolated DNA sequence of claim 12 and / or the vector of claim 13 and / or the composition of claim 15 or 16 to a subject, wherein the target polypeptide is expressed in tumor cells and not in healthy cells, Wherein, the target cells are tumor cells, and the non-target cells are healthy cells.

21. The method according to claim 20, wherein: The target polypeptide is selected from at least one of a polypeptide that induces cell apoptosis and a polypeptide that is toxic or lethal to tumor cells.

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