WO2025035296A1 - 基于miRNA调控的细胞可编程RNA翻译开关 - Google Patents

基于miRNA调控的细胞可编程RNA翻译开关 Download PDF

Info

Publication number
WO2025035296A1
WO2025035296A1 PCT/CN2023/112676 CN2023112676W WO2025035296A1 WO 2025035296 A1 WO2025035296 A1 WO 2025035296A1 CN 2023112676 W CN2023112676 W CN 2023112676W WO 2025035296 A1 WO2025035296 A1 WO 2025035296A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
rna
mirbs
nucleic acid
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/112676
Other languages
English (en)
French (fr)
Inventor
谢震
宁辉
刘淦
黄慧雅
刘强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Syngentech Co Ltd
Tsinghua University
Original Assignee
Beijing Syngentech Co Ltd
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Syngentech Co Ltd, Tsinghua University filed Critical Beijing Syngentech Co Ltd
Priority to PCT/CN2023/112676 priority Critical patent/WO2025035296A1/zh
Publication of WO2025035296A1 publication Critical patent/WO2025035296A1/zh
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • 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.
  • RNA molecules have been regarded as a new drug form with great potential due to their easy synthesis and preparation, modular coding region sequences that are easy to modify, and the fact that RNA functions only in the cytoplasm and thus avoids sequence integration into the genome.
  • RNA-based therapies such as antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and messenger RNA (mRNA) vaccines, have shown great potential in biomedical applications.
  • ASOs antisense oligonucleotides
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • RNA gene circuits that can sense miRNA signals, RNA-binding protein signals, and chemical small molecule signals to complete concept verification, but these designs still have defects in practical applications.
  • Auslander et al. designed an RNA device containing a hammerhead ribozyme (HHR) with self-cleavage function, and fused RNA aptamers that can bind to small molecules or proteins to the sequence of the natural hammerhead ribozyme.
  • HHR hammerhead ribozyme
  • the hammerhead ribozyme undergoes self-cleavage, destroying the integrity of the RNA device, and the RNA device lacking end protection is then degraded and cleared, so the target protein is not expressed.
  • the RNA device senses the small molecule signal or protein signal corresponding to the aptamer in the cell, the small molecule/protein binds to the hammerhead ribozyme and changes the local spatial structure of the RNA, destroying the self-cleavage activity of the hammerhead ribozyme, so that the protein encoded by the RNA can be expressed.
  • RNA binding protein binds to the RNA aptamer, it blocks the path of the ribosome scanning downstream from the 5' cap region of the mRNA, thereby inhibiting the translation of the target gene; when the RNA binding protein does not exist or does not bind to the aptamer, the target gene is expressed normally, thus achieving the sensing response of the endogenous RNA binding protein signal.
  • this method achieves the goal of shutting down the expression of the target gene after sensing the signal of the endogenous RNA binding protein, that is, it has 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 of tumor upregulation.
  • RNA shearing mechanisms to regulate the expression of target genes: placing the binding sequence of RNA binding protein (RBP) near the RNA variable shearing site, thereby converting the binding signal of RBP to RNA into different transcripts of RNA variable shearing.
  • RBP RNA binding protein
  • 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 shearing, and then enter the cytoplasm to function.
  • circRNA circular RNA
  • synthetic biologists have developed strategies to regulate gene expression in linear mRNA and in circRNA.
  • these strategies usually require the use of RBP aptamers and miRNA binding sites (miRBS) to shut down translation in response to RBP and miRNA, and use these shutdown modules in tandem to activate translation. Therefore, these strategies require multiple RNA molecules to construct functional devices such as cell classifiers and logic gates, which increases the size of the entire RNA device and introduces foreign proteins with potential immunogenicity.
  • the strategies reported by previous researchers to activate 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.
  • the present invention aims to solve at least one of the technical problems in the related art to a certain extent.
  • one purpose of the present invention is to propose a new strategy that can achieve the translation activation and inhibition of mRNA/circRNA by miRNA by only modifying a single RNA molecule, and thereby construct an RNA device with logical operations and cell classification.
  • the first aspect of the present invention provides an RNA recombinant nucleic acid molecule.
  • the RNA recombinant nucleic acid molecule comprises:
  • IRES elements or elements with similar functions to IRES elements
  • 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.
  • the DS sequence is complementary to at least a portion of the structure that plays a key role in translation activity
  • RNA recombinant nucleic acid molecule 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).
  • 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.
  • the RNA recombinant nucleic acid molecule provided by the present invention does not need to combine multiple RNA molecules, nor does it need to introduce 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 of engineering IRES proposed in the present invention can be applied to a variety of IRES, 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 one that can simultaneously achieve upregulation or downregulation of the translation activity of the target protein with only a single circular RNA molecule, and can be used for logical operations of cell signals and cell classification.
  • the second aspect of the present invention provides an isolated DNA sequence, comprising a DNA sequence that can be transcribed into the RNA recombinant nucleic acid molecule described in the first aspect.
  • the third aspect of the present invention provides a vector comprising the isolated DNA sequence described in the second aspect.
  • 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.
  • 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.
  • 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.
  • a seventh aspect of the present invention provides a method for expressing a target polypeptide in a cell, the method comprising:
  • 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.
  • An eighth aspect of the present invention provides a method for treating a tumor, the method comprising:
  • 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,
  • the target cells are tumor cells, and the non-target cells are healthy cells.
  • RNA recombinant nucleic acid molecule designed by the present invention realizes the on-off regulation of the translation activity of the internal ribosome entry site (IRES); Design RNA secondary structure to realize engineered IRES elements activated/inhibited by specific microRNA (miRNA); construct engineered IRES elements activated/inhibited by specific miRNA into circular RNA (circRNA) vectors, deliver circRNA molecules through in vitro transcription and lipid nanoparticle (LNP) packaging, and realize specific expression of circRNA in tumor cells.
  • IRISPR internal ribosome entry site
  • the beneficial effects brought by 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 treatment method based on RNA translation regulation. Since the RNA encapsulated by LNP can be delivered to the tumor lesion by intravenous injection without intratumoral injection, it can give full play to the various 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.
  • 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;
  • FIG2 shows a schematic diagram (A) and effect diagram (B) of restoring the translation activity of HCV-IRES by designing RS sequence in HEK293 cell line;
  • FIG3 shows the inhibitory effect of DS and the restorative effect of RS tested in the Huh7 cell line
  • FIG4 shows a schematic diagram of designing RNA secondary structures containing miRBS and RS sequences to construct three engineered IRES elements (MITA) activated by miRNA in HEK293 cell lines;
  • FIG5 shows the response of three MITA to exogenous miRNA analogs tested in HEK293 cell lines
  • Figure 6 shows the response of three MITAs to endogenous miRNAs tested in HEK293 and Huh7 cell lines
  • FIG7 shows the design of RNA secondary structures containing different miRBS and RS sequences to construct engineered IRES elements with logical “OR” operation capabilities
  • FIG8 shows the design of RNA secondary structures containing different miRBS and RS sequences to construct engineered IRES elements with logical “AND” operation capabilities
  • FIG9 shows the design of miRBS binding sites containing fully complementary pairs to construct an engineered IRES element with logical “NOT” operation capability
  • 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 are connected in series;
  • FIG11 shows the plasmid vector designed and constructed by combining MITA and MITR to test the classification effect in HEK293 and Huh7 cell lines;
  • FIG12 shows a schematic diagram of a template for in vitro transcription of circular RNA containing a MITA element
  • FIG13 shows the expression inhibition and recovery of circular RNA containing dIRES and rIRES in HEK293 and Huh7 cell lines
  • FIG14 shows the expression differences of circular RNA containing three different MITAs in HEK293 and Huh7 cell lines
  • FIG15 shows the classification effect of circular RNA designed and constructed by combining MITA and MITR in HEK293 and Huh7 cell lines;
  • FIG16 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;
  • FIG17 shows the effect of designing RS sequence to restore the translation activity of tIRES in HEK293 cell line
  • FIG18 shows the effect of designing a 20-nt DS sequence to inhibit the translation activity of other IRES in HEK293 cell lines
  • FIG19A shows a schematic diagram of the construction of a cancer cell-specific killing circular RNA molecule without miRBS
  • FIG19B shows the results of a cancer cell specific killing experiment
  • FIG20A shows a schematic diagram of the construction of a cancer cell-specific killing circular RNA molecule containing 4 ⁇ miRBS
  • FIG20B shows the results of a cancer cell specific killing experiment
  • FIG. 20C shows micrographs of cancer cells and normal cells after transfection with killer circular RNA molecules.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
  • 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.
  • the term “and/or” encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein.
  • “A and/or B” encompasses “A,” “A and B,” and “B.”
  • “A, B, and/or C” encompasses “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”
  • IRES refers to the Internal Ribosomal Entry Site (IRES).
  • IRES Internal Ribosomal Entry Site
  • ITAF trans-acting Factor
  • 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 coding elements through structural changes by complementary or non-complementary pairing with the structure contained in IRES that plays a key role in translation activity.
  • the "DS sequence" used in this article is a part of the RNA sequence in the RNA translation switch element of the RNA recombinant nucleic acid molecule, which is complementary to at least a part of the structure contained in IRES that plays a key role in translation activity (such as the 3' pseudoknot structure).
  • 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 complementarily pairs with at least a portion of the DS sequence and forms a stem-loop structure.
  • the "3' pseudoknot structure" used in this article is a part 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.
  • target polypeptide encoding element refers to the mRNA sequence of the target polypeptide.
  • a "spacer sequence” is a non-functional mRNA sequence.
  • “Stem-loop structure” refers to the reverse repeat sequence present in single-stranded RNA molecules. Due to the hydrogen bond pairing between complementary bases, the long chain segment can be folded back to form a secondary structure. Definition: The double-stranded region between the paired bases forms the "stem", while the single-stranded region that cannot pair protrudes to form a "loop”.
  • Y-shaped double stem-loop structure used in this article refers to a single-stranded RNA molecule that has two inverted repeat sequences and forms two stem-loops. Structure, wherein the two stem-loop structures and the complementary paired sequences at both ends of the single-stranded RNA molecule together constitute a Y-shaped double stem-loop structure as used herein.
  • 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.
  • single-stranded loop portion refers to the "loop" portion formed by the protruding unpaired single-stranded region in the inverted repeat sequence formed by the single-stranded RNA molecule.
  • “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 relationship between A and T, A and U, and G and C.
  • the present invention specifically refers to the complementary pairing of bases in RNA molecules.
  • miRBS sequence refers to a sequence that complementarily pairs with a miRNA.
  • MicroRNA also known as MicroRNA, is a class of single-stranded non-coding RNA molecules with a length of about 20nt and evolutionarily conserved. 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.
  • mRNA target messenger RNA
  • RNA translation switch element of the present invention independently means that the multiple miRBS sequences contained in the RNA translation switch element of the present invention are the same or different, and can bind to the same or different miRNA types.
  • tandem miRBS sequences refers to multiple miRBS sequences directly linked at the first position.
  • 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.
  • Lipid nanoparticles are usually nanoparticles composed of ionized cationic lipids, phospholipids, cholesterol and polyethylene glycol lipids, with a solid or oily core in the center, while the smaller traditional unilamellar liposomes are typically characterized by a lipid bilayer on the surface and an aqueous "pool” in the center of the particle. Because liposomes have long been approved by regulatory agencies and used for clinical small molecule drugs, they were quickly identified by gene therapy pioneers as a potential delivery vehicle, especially for RNA small molecule drugs.
  • Liposomes are liposomes made of phosphatidylcholine and ceramide, and have a bilayer structure similar to the cell membrane structure. Liposomes are widely used as drug carriers for small molecule drugs, proteins, nucleic acids and imaging agents. In order to improve the therapeutic effect and improve patient compliance, a variety of drug delivery methods for liposomes have been developed, such as parenteral administration, pulmonary administration, oral administration, transdermal administration, ophthalmic administration and nasal administration.
  • composition is in a form that permits the biological activity of the active ingredients to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • the terms “subject” and “patient” are used interchangeably, regardless of whether the subject has been or is currently receiving any form of treatment.
  • 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.
  • the subject is a human.
  • the subject is a person suspected of having cancer, an autoimmune disease or condition, and/or an infection.
  • apoptosis-inducing polypeptide refers to a polypeptide fragment that is capable of initiating programmed cell death.
  • 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.
  • GSDMD Gasdermin D
  • HSV-TK Herpes Simplex Virus Thymidine Kinase
  • 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 antimetabolites such as ganciclovir, HSV-TK can convert the drug into its toxic form, thereby inducing cell death.
  • the present invention provides an RNA recombinant nucleic acid molecule, comprising:
  • IRES elements or elements with similar functions to IRES elements
  • 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.
  • the DS sequence is complementary to at least a portion of the structure that plays a key role in translation activity
  • RNA recombinant nucleic acid molecule 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).
  • RNA is modified after transcription, and the ribosome and other proteins can recognize the modification.
  • mRNA initiates translation through the cap structure of the head.
  • the cap structure is a necessary structure for the initiation of mRNA translation, providing a signal for the ribosome to recognize mRNA, assisting the ribosome to bind to mRNA, and starting translation from AUG.
  • the cap structure can increase the stability of mRNA and protect mRNA from the attack of 5' ⁇ 3' nuclease.
  • An IRES element or an element with similar functions to the IRES element is constructed in the RNA recombinant nucleic acid molecule, and the ribosome can recognize the element and initiate internal translation.
  • the inventors have proposed a new strategy that only requires the modification of the RNA molecule structure to achieve the translation activation and inhibition of mRNA/circRNA by miRNA, and can thereby construct an RNA device with logical operations and cell classification.
  • the translation of enhanced yellow fluorescent protein depends on the cap structure of RNA.
  • 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 part of the RS sequence and the DS sequence are complementary to each other and form a stem-loop structure, and the DS sequence can be complementary to at least a part of the pseudoknot structure.
  • the RS sequence, the DS sequence and the pseudoknot structure are used to complement each other.
  • the translation of the target polypeptide encoding element downstream of IRES is realized by using different complementary pairing modes. Specifically, in Figure 1, by inserting an upstream sequence (called DS) complementary to the pseudoknot region, the original structure of IRES will be destroyed, thereby inhibiting the translation activity mediated by IRES.
  • DS upstream sequence
  • the present invention can realize the dynamic opening and closing of the gene downstream of IRES.
  • the DS sequence is complementary to the pseudoknot structure
  • the translation of the gene downstream of IRES is in a closed state
  • the RS sequence is complementary to the DS sequence to form a stem-loop structure
  • the translation of the gene downstream of IRES is in an open state.
  • 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.
  • the miRBS sequence in the first RNA sequence 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 having a similar function 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 having a similar function to the IRES element is in a closed state and cannot recruit ribosomes to enter.
  • the miRBS sequence when the miRBS sequence is designed, 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.
  • Designing the bases of the two nucleotides at the 10th and 11th positions 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.
  • each miRBS sequence binds to the same or different miRNA types. It should be noted that multiple miRBS sequences can be designed on an RNA translation switch element, each miRBS sequence is the same or different, and each miRBS sequence binds to a miRNA specifically expressed in a complementary target cell.
  • 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,
  • the DS sequence is complementary to at least a portion of the structure contained in the IRES element that is critical for 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.
  • the RNA translation switch element comprises at least one of the first stem-loop structures, wherein each The miRBS sequences contained in the first stem-loop structure independently bind to the same or different miRNA types.
  • the balance can be maintained by regulating the length of the complementary pairing of miRBS in the stem-loop, so that when miRBS is not bound to miRNA, the stem-loop structure is stable, and when miRBS is bound to miRNA, the stem-loop is easily opened. If the RNA translation switch element closed structure is too stable, even if the miRBS is bound to miRNA, it is not easy to open, and the IRES translation switch that responds to miRNA cannot be played. If the RNA translation switch element closed structure is too unstable, the RNA translation switch element may be opened before miRNA is bound to miRBS, resulting in the inability to achieve the purpose of responding to miRNA signals to control the IRES translation switch.
  • 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.
  • the partial complementary pairing of the third spacer sequence and the RS sequence includes 2-3 non-complementary pairing regions.
  • the DS sequence 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.
  • 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.
  • a MITA IRES translation activation element responsive to miRNA
  • SL stem-loop
  • the closure will be too stable and the stem-loop will not be easy to open, so it is preferred to design several non-complementary paired bases to make it easier to open.
  • 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.
  • 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.
  • the RS sequence is located at the two double-stranded stems in the "Y"-shaped double stem-loop structure.
  • the two double-stranded stems in the "Y"-shaped double stem-loop structure respectively have 1-2 non-complementary pairing regions.
  • 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.
  • 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 non-target cells, and the RNA recombinant nucleic acid molecule has at least one miRBS sequence for non-target cells.
  • each of the miRBS sequences targeting 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.
  • 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.
  • the miRBS sequence targeting non-target cells is 3-5 tandem miRBS sequences.
  • the miRBS sequence for non-target cells is completely complementary to the miRNA specifically expressed in the non-target cells.
  • the length of the RS sequence is 9-25 nt, preferably 9-13 nt.
  • the length of the DS sequence is 10-25 nt, preferably 10-22 nt.
  • 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.
  • the element having a function similar to that of the IRES element is an element having a translation initiation function.
  • the element having similar functions to the IRES element comprises at least A deletion, insertion, or substitution of a nucleic acid element.
  • the IRES element is derived from a hepatitis C-like virus.
  • the hepatitis C-like virus includes at least one selected from the group consisting of hepatitis C virus (HCV), avian encephalomyelitis virus (AEV), classical swine fever virus (CSFV), and porcine enterovirus (PTV).
  • HCV hepatitis C virus
  • AEV avian encephalomyelitis virus
  • CSFV classical swine fever virus
  • PTV porcine enterovirus
  • IRES sequences derived from hepatitis C virus are not completely the same, their sequence identity is relatively high, and they all have a key pseudoknot structure.
  • the present invention mainly exemplifies the IRES sequence derived from HCV in the embodiments, in theory, other IRES sequences derived from viruses that contain structures (such as pseudoknot structures) that play a key role in translation activity are also covered within the protection scope of the present invention.
  • the structure contained in the IRES element that plays a key role in translation activity is a 3'-terminal pseudoknot structure.
  • the element having a similar function to the IRES element is a truncated IRES element.
  • 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.
  • the nucleic acid sequence of the truncated IRES element is a sequence obtained by removing 40 bases from the 5' end of the wild-type HCV IRES.
  • the RNA recombinant nucleic acid molecule is a linear RNA molecule or a circular RNA molecule, preferably a circular RNA molecule to enhance stability.
  • the miRBS sequence is determined based on the activity of the miRNA specifically expressed in the cell. For example, when the target polypeptide coding element is required to initiate translation in tumor cells, while the target polypeptide coding element in normal cells is not translated, the miRNA activity in normal cells and tumor cells is required to be different. If the miRNA screened is highly active in tumor cells and low 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 the translation of the target polypeptide coding element; at the same time, another miRNA with low activity in tumor cells and high activity in normal cells is selected, and this signal is designed as a shutdown signal.
  • the RNA recombinant nucleic acid molecule In normal cells, the RNA recombinant nucleic acid molecule binds to the miRNA in normal cells, and the RNA recombinant nucleic acid molecule is cut off in normal cells. In tumor cells, because there is no miRNA signal in normal cells, the RNA recombinant nucleic acid molecule is not cut.
  • the highly expressed miRNA in tumor cells can activate the IRES to initiate translation, and the highly expressed miRNA in normal cells will inhibit (cut) the RNA recombinant nucleic acid molecule of the present invention. Using these two methods at the same time, the target polypeptide coding element is only translated in tumor cells to ensure the safety of the RNA recombinant nucleic acid molecule.
  • the RNA recombinant nucleic acid molecule provided by the present invention does not need to combine multiple RNA molecules, nor does it need to introduce 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 of engineering IRES proposed in the present invention can be applied to a variety of IRES, 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 one that can simultaneously achieve upregulation or downregulation of the translation activity of the target protein with only a single circular RNA molecule, and can be used for logical operations of cell signals and cell classification.
  • the present invention provides an isolated DNA sequence, comprising a DNA sequence that can be transcribed into the aforementioned RNA recombinant nucleic acid molecule.
  • the present invention provides a vector comprising the isolated DNA sequence described above.
  • 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.
  • 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.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • 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.
  • the RNA recombinant nucleic acid molecule or the composition containing the RNA recombinant nucleic acid molecule provided by the present invention 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 the composition can also be used to treat tumor-related diseases, and can also achieve the 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.
  • 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.
  • the target polypeptide when the disease is a tumor, is selected from at least one of a polypeptide that induces cell apoptosis and a polypeptide that is toxic or lethal to tumor cells.
  • the present invention provides a method for expressing a target polypeptide in a cell, the method comprising:
  • 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.
  • 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.
  • the present invention provides a method for treating a tumor, the method comprising:
  • 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 and not expressed in healthy cells,
  • the target cells are tumor cells, and the non-target cells are healthy cells.
  • 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.
  • the method for treating tumors aims to highly express at least one of a polypeptide that induces apoptosis and a polypeptide that is toxic or lethal to tumor cells in tumor cells, while being lowly expressed or not expressed in normal cells.
  • the translation of the target polypeptide encoding element initiated by the IRES element is in an open state in tumor cells and in a closed state in normal cells.
  • the IRES system described above is used to achieve the expression of lethal genes.
  • the miRNA cleavage signal that is highly expressed in normal tissues is combined to improve safety.
  • RNA recombinant nucleic acid molecule of the present invention has a double guarantee of safety in treatment.
  • the miRBS for normal cells and the IRES for tumor cells are on the same RNA recombinant nucleic acid molecule.
  • 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.
  • exogenous RNA RNA recombinant nucleic acid molecule or composition provided by the present invention
  • a solid tumor mouse is injected (such as by intravenous injection) into a solid tumor mouse to observe whether it is expressed in the tumor (with fluorescence) and there is no fluorescence in the non-planted tumor area (healthy area).
  • 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 and polypeptides that are toxic or lethal to tumor cells in tumor cells but not in healthy cells.
  • Restriction enzymes 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.
  • oligonucleotides were heated to 95°C in 1 ⁇ PNK buffer, gradually cooled (-1°C per minute) to 37°C, annealed, and 1 ⁇ M of the annealed product was phosphorylated with 0.5 U/ ⁇ L PNK in the presence of 0.5 mM ATP.
  • Golden Gate Assembly was performed according to the New England Biolabs protocol.
  • 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.
  • plasmid transfection 0.6 ⁇ g of plasmid was mixed with 0.6ug Lipofectamine 8000 and then transfected into each well of cells.
  • miRNA mimics or miRNA inhibitors were mixed with 0.6 ⁇ g plasmid and 0.6 ug Lipofectamine 8000 to a final transfection concentration of 50 nM and then transfected into each well of cells.
  • circRNA transfection circRNA was encapsulated with lipid nanoparticles as described below and transfected into cells at 0.6 ⁇ g per well.
  • CircRNA circular RNA
  • DNA templates for circRNA precursors were first generated by linearizing plasmid constructs using Xba I endonuclease.
  • CircRNA precursors were generated by in vitro transcription (IVT) from DNA templates using T7 Polymerase (Novoprotein).
  • IVT in vitro transcription
  • RNA products were treated with DNase I (Thermo Fisher Scientific) for 30 min to digest the DNA template.
  • GTP Novoprotein
  • GTP Novoprotein
  • precursor RNA was removed by treatment with RNase R (Novoprotein) at 37 °C for 15 min. CircRNAs treated with RNase R were purified using LiCl (Novoprotein).
  • Lipid nanoparticles encapsulate circRNA.
  • 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).
  • SM-102 ionizable lipid
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • cholesterol 1,2-distearoyl-sn-glycero-3-phosphocholine
  • 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 circRNA in a ratio of 1:3 through a Lipid Mixer (Micronano INano L).
  • the preparations were then dialyzed through an ultrafiltration centrifugal filter unit (Millipore) with a 100 kD molecular weight cutoff (Sartorius Stedim Biotech) and dialyzed against PBS (pH 7.4) for 30 times the volume to concentrate the preparations to the desired concentration and stored at -20°C. All preparations were tested for particle size, distribution, RNA concentration, and encapsulation efficiency.
  • the cell culture medium was removed and 100 ⁇ L of Bright-Light Luciferase Assay System (Vazyme) was added, mixed and incubated for 3 min. 100 ⁇ L of the culture mixture was transferred to a flat-bottom white wall plate (Corning) and the luminescence intensity was measured using a microplate luminometer (Thermo Fisher Scientific).
  • Vazyme Bright-Light Luciferase Assay System
  • LDH analysis was performed according to the manufacturer's protocol. Briefly, 3 ⁇ 10 5 cells were seeded in each well of each 96-well culture plate (Corning) one day before transfection, and 1 mL of DMEM complete medium was added. The next day, 0.2-0.5 ug of LNP-encapsulated circRNA was transfected into each well.
  • the cell culture plate was centrifuged at 400 g for 5 min, and the supernatant was carefully aspirated, and 150 ⁇ L of 10-fold diluted lactate dehydrogenase (LDH) release reagent (the volume ratio of reagent to phosphate buffered saline (PBS) was 1:10) was added and mixed. After one hour of incubation, the cell culture plate was centrifuged again at 400 g for 5 min, and the resulting supernatant (120 ⁇ L per well) was transferred to a new 96-well plate for subsequent sample quantification.
  • LDH lactate dehydrogenase
  • EYFP or mKate when fully complementary miRBS is included in the construct
  • fluorescent protein is used as an internal control.
  • tagBFP fluorescence intensity mean value (tagBFP MFI)
  • EYFP or mKate MFI of the positive gated cells were calculated.
  • the relative IRES-mediated tagBFP fluorescence intensity is defined as the ratio of tagBFP/EYFP (or tagBFP/mKate when fully complementary miRBS is included in the construct) MFI.
  • the normalized IRES-mediated tagBFP fluorescence intensity is defined as the relative IRES-mediated tagBFP fluorescence intensity divided by the positive control (nIRES or rIRES) tested in the same cell line.
  • the luminous intensity mean value of each sample was calculated.
  • Normalized luciferase activity is defined as luminous intensity divided by the positive control (nIRES or rIRES) tested in the same cell line.
  • HCV-IRES internal entry site
  • HCV-IRES hepatitis C virus
  • HCV-IRES hepatitis C virus
  • DS upstream sequence
  • a bicistronic fluorescent reporter system was constructed, and the translation efficiency of engineered IRES with different DS lengths in human embryonic kidney cells (HEK293) was evaluated using flow cytometry (B in Fig. 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, so the ratio of the mean fluorescence intensity (MFI) of tagBFP and EYFP can characterize the translation efficiency mediated by IRES.
  • MFI mean fluorescence intensity
  • the MFI of tagBFP Compared with the unmodified natural IRES (nIRES), the MFI of tagBFP began to decrease when the upstream DS length exceeded 12 nucleotides (12-nt), and when the DS length reached 20-nt, the MFI of tagBFP decreased by more than 10 times compared with nIRES ( Figure 1, the IRES containing 20-nt DS is called dIRES).
  • RS recovery sequence
  • Figure 2A the pairing of the pseudoknot region with DS (translation closed state) changes to the pairing of RS with DS (translation initiation state), the pseudoknot region is released, and the initiation of translation is restored.
  • a programmable IRES translation activation element that responds to miRNA
  • the inventors attempted to design a secondary structure with a miRNA binding site (miRBS) so that the RS is fixed in a relatively stable structure.
  • miRBS miRNA binding site
  • the miRBS is designed as a miRNA sponge, that is, there are 2 nucleotide mismatches with the miRNA at positions 10 and 11.
  • Three types of MITA were constructed, namely the shoe last (TL), stem loop (SL) and three-arm (AJ) structures (corresponding to A-C, respectively).
  • the 13-nt RS In the absence of 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 off state).
  • the complementary miRNA 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 to restore the IRES structure and translation activity (i.e., the device is in the on state).
  • FIG. 5 The left column of each group in Figure 5 is the control group (addition of miRNA non-complementary to miRBS equal to miRNA-FF4), and the right column is the experimental group with addition of miRNA-FF4 (FF4mimics).
  • the shoe last structure with 16 nucleotide base pairings (TL16), the stem-loop structure with 3 nucleotide mismatches (SL3), and the 3-arm structure with 2 nucleotide mismatches (AJ2) were designed.
  • miR-21-5p is a miRNA that is highly expressed in most cancer cells but lowly expressed in HEK293.
  • 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).
  • Figure 10 mKATE expression is not regulated by miRNA, while EYFP is regulated by miRNA.
  • the mKATE plasmid is used 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.
  • miR-21-5p was finally selected as the Huh7 high-activity miRNA to construct MITA module
  • miR-106a-5p and miR-18a were selected as highly active miRNAs in HEK293 to realize the logic "NOT” gate.
  • MITA IRES-related sequence
  • TL16, SL3 and AJ2 responsive to miR-21-5p was constructed, and four miR-106a-5p and miR-18a binding sites were inserted in series upstream of MITA and downstream of tagBFP reporter gene ( Figure 11). These plasmid vectors were transfected into Huh7 and HEK293 cells, respectively, and mKate fluorescent protein was used as an internal reference control.
  • plasmid templates containing replaced intron-exon were constructed for in vitro transcription (IVT) of circRNA ( Figure 12).
  • circRNAs encoding nIRES, dIRES, and rIRES were first transfected into HEK293 and Huh7 cells, respectively.
  • the same dIRES inhibition and rIRES restoration effects as plasmid transfection were observed ( Figure 13, the left column of each group is HEK293, and the right column is Huh7 cells).
  • 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 design showed more than a 3-fold increase in fluorescence signal in Huh7 compared with HEK293, while AJ2 showed more than a 6-fold increase in fluorescence signal. These results show that the MITA module of the present invention can also be activated by miRNA in circRNA. Finally, the cell type classifier of the circRNA version was tested ( Figure 15, each group of left columns 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 shows that the cell type classifier circRNA of the present invention can accurately identify the desired cancer cells and may be used as a potential cancer targeted therapy.
  • truncated HCV-IRES truncated HCV-IRES
  • the inventors found that the truncated IRES structure could also function with the first neck loop removed in the wild-type IRES structure, thus achieving the goal of shortening the length of DS and RS when designing the IRES system.
  • the results showed that the insertion of only 10-nt DS significantly reduced the translation activity of tIRES by more than 10 times (the structure is called dtIRES, Figure 16).
  • the inventors also tested RS that restored the translation activity of dtIRES.
  • Simply inserting a 9-nt RS can restore the translation activity of dtIRES to more than 70% of that of tIRES ( Figure 17). Therefore, the present invention can also be used to develop MITA and MITR in a truncated version of HCV-IRES.
  • DS is designed to be 20nt and RS is designed to be 13nt. After truncation, DS is designed to be 10nt and RS is designed to be 9nt to achieve the opening and closing of translation.
  • the inventors also designed a 20-nt DS to destroy the pseudoknot structure of other IRES to demonstrate the universality and scalability of the strategy used in the present invention. Similar inhibitory effects were also observed in the IRES 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 IRES (Figure 18).
  • AEV avian encephalomyelitis virus
  • CSFV classical swine fever virus
  • PTV porcine enterovirus
  • Example 7 The killing effect of RNA molecules of the present invention on cancer cells
  • FIG. 20C is a micrograph corresponding to FIG. 20B, which intuitively reflects the situation of cell killing.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明涉及分子生物学技术领域，具体涉及基于miRNA调控的细胞可编程RNA翻译开关。本发明提供一种RNA重组核酸分子，无需组合多个RNA分子，也无需引入外源蛋白，仅凭工程化改造单独一个RNA分子即可构建具有细胞特异性表达能力的RNA，并且能够感应多种miRNA信号实现逻辑运算。本发明提出的工程化改造IRES的策略，可以应用到多种IRES中，具有普适性和可迁移性。本发明通过感应细胞内源miRNA信号，实现工程化改造后的IRES元件的结构动态变化，从而动态调控IRES的翻译活性。

Description

基于miRNA调控的细胞可编程RNA翻译开关 技术领域
本发明涉及分子生物学技术领域，具体涉及基于miRNA调控的细胞可编程RNA翻译开关。
背景技术
长期以来，RNA分子由于其易于合成制备，编码区序列模块化从而易于改造，且RNA只在细胞质发挥功能因此避免序列整合至基因组等特点，一直被视作有巨大潜力的新型药物形式，但是由于无处不在的RNA酶对RNA分子的快速降解，且临床上缺乏有效的RNA递送技术，RNA技术在医药领域的应用进展缓慢，只有少数寡聚核苷酸(siRNA)进入临床试验。近年来，RNA递送技术的突破，RNA修饰降低免疫原性，以及基于机器学习的RNA序列设计优化，使RNA技术得以迅速发展。基于RNA的治疗方法，如反义寡核苷酸(ASO)、小干扰RNA(siRNA)和信使RNA(mRNA)疫苗，在生物医学应用中显示出巨大的潜力。
RNA技术的突破也为基于RNA的基因线路设计与应用带来了新机遇，使得设计具有临床应用潜力的RNA基因线路装置成为可能。研究者们曾设计能感知miRNA信号、RNA结合蛋白信号、化学小分子信号的RNA基因线路完成了概念验证，但是这些设计在实际应用上仍有缺陷。Auslander等人设计了含有自剪切功能的锤状核酶(Hammerhead ribozyme,HHR)的RNA装置，并且在天然锤状核酶的序列中融合了可与小分子或蛋白结合的RNA适配体。在没有信号输入时，锤状核酶发生自剪切，破坏RNA装置的完整性，缺乏末端保护的RNA装置随即被降解清除，因此不表达目标蛋白。而当该RNA装置在细胞内感应到适配体对应的小分子信号或蛋白信号时，由于小分子/蛋白结合至锤状核酶上并且改变RNA局部的空间结构，破坏了锤状核酶的自剪切活性，从而该RNA编码的蛋白得以表达。然而，这一工作以及其他利用自剪切核酶的工作的共同缺点在于，该方式只适用于将DNA递送至细胞，凭借细胞自身的转录和保护机制将转录本从细胞核运输至细胞质发挥功能。这是因为RNA在体外转录后还未被递送至细胞就有可能发生自剪切，因此该方案虽然是在RNA水平实现调控，但仍然需要递送DNA，并不是真正意义上的RNA基因线路，因此无法发挥RNA技术的优势。
Kawasaki等人在2017年首次提出能感应细胞内源RNA结合蛋白的RNA装置，他们将RNA适配体置于目标基因的5’非翻译区，当RNA结合蛋白结合至RNA适配体上时，阻遏核糖体从mRNA的5’帽子区域往下游扫描的路径，从而抑制目标基因的翻译；当RNA结合蛋白不存在或未结合至适配体上时，目标基因正常表达，因此实现了内源RNA结合蛋白信号的传感响应。这一工作，以及其他将RNA适配体置于5’非翻译区的线性mRNA装置的共同缺点在于：一方面该方案的调控功能较弱，因为该方法依赖于传统的5’帽起始翻译机制，核糖体从5’帽向下扫描过程中，如果RNA适配体的结构过于稳定，则不利于核糖体的扫描通过，因此在没有RNA结合蛋白信号时目标基因的表达仍然很低，然而如果RNA适配体的结构不够稳定，虽然有利于核糖体的扫描通过，但是在RNA结合蛋白存在时适配体与蛋白的结合能力较弱，因此存在目标基因泄露表达。另一方面，该方法实现的是感应到内源RNA结合蛋白的信号后关闭目标基因的表达，即起到关闭基因表达的功能，但更有实际应用价值的是在感应到特定信号后开启目标基因的表达，例如感应到肿瘤上调表达的细胞信号后开启自杀基因的表达。
此外，还有其他已经报道的RNA装置架构，例如利用RNA剪切机制来调控目的基因的表达：将RNA结合蛋白(RBP)的结合序列置于RNA可变剪切位点附近，从而将RBP与RNA的结合信号转化为RNA可变剪切的不同转录本。然而，由于RNA的剪切需要在细胞核内完成，因此该方法的缺点在于需要引入额外的辅助因素帮助RNA装置从细胞质穿梭进入核中完成剪切，再进入细胞质中发挥功能。
与线性mRNA相比，环状RNA(circRNA)在哺乳动物细胞中表现出优异的稳定性和延长的持续时间，因为circRNA对RNA酶的降解的抵抗能力更强，且免疫原性较低。因此，circRNA被认为是线性mRNA的一种潜在的替代物。迄今为止，合成生物学家已经开发了在线性mRNA和在circRNA中调控基因表达的策略。如上所述，这些策略通常需要用到RBP适配体和miRNA结合位点(miRBS)，以响应RBP和miRNA来关闭翻译，并串联使用这些关闭模块来激活翻译。因此，这些策略都需要多个RNA分子来构建细胞分类器、逻辑门等功能器件，这便增加了整个RNA装置的大小，并且引入了具有潜在免疫原性的外源蛋白。而前人报道的由miRNA激活RNA翻译的策略依赖于miRNA切割来释放聚腺苷尾部(polyA尾)或切除线性mRNA的降解域，因此不适用于circRNA。
综上所述，目前仍未有一种便捷简单的方式来实现miRNA对于circRNA的翻译激活。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出了一种全新的策略，只需要改造单独一个RNA分子即可实现miRNA对于mRNA/circRNA的翻译激活和抑制，并且能够由此构建具有逻辑运算和细胞分类的RNA装置。
为此，本发明第一方面提供一种RNA重组核酸分子。根据本发明的实施方案，所述RNA重组核酸分子包括：
(1)RNA翻译开关元件；
(2)IRES元件或与IRES元件具有类似功能的元件；
(3)目标多肽编码元件，
其中，所述IRES元件或与IRES元件具有类似功能的元件包括对翻译活性起关键作用的结构
所述RNA翻译开关元件包括第一RNA序列和第二RNA序列，所述第一RNA序列位于所述第二RNA序列的上游，所述第一RNA序列含有RS序列，所述第二RNA序列含有DS序列，所述RS序列与所述DS序列之间具有第一间隔序列，
(a)所述RS序列和所述DS序列的至少一部分互补配对并形成茎环结构；
(b)所述DS序列与所述对翻译活性起关键作用的结构的至少一部分互补配对；
在所述RNA重组核酸分子中，(a)和(b)不同时发生，通过(a)和(b)两种结构的变化，实现所述目标多肽编码元件的起始翻译与否。
根据本发明一个实施方案，所述第一RNA序列进一步包括至少一个miRBS序列，所述miRBS序列与靶细胞中特异性表达的miRNA的至少一部分互补且不完全互补，所述靶细胞为所述RNA重组核酸分子待导入的细胞。
本发明提供的RNA重组核酸分子，无需组合多个RNA分子，也无需引入外源蛋白，本发明仅凭工程化改造单独一个RNA分子即可构建具有细胞特异性表达能力的RNA，并且能够感应多种miRNA信号实现逻辑运算。本发明提出的工程化改造IRES元件既可用于线性mRNA也可用于环状RNA的表达调控。本发明提出的工程化改造IRES的策略，可以应用到多种IRES中，具有普适性和可迁移性。本发明通过感应细胞内源miRNA信号，实现工程化改造后的IRES元件的结构动态变化，从而动态调控IRES的翻译活性。本发明是首个能够仅凭单独一个环状RNA分子即可同时实现目标蛋白翻译活性的上调或下调，并且能够用于细胞信号的逻辑运算和细胞分类。
本发明第二方面提供一种分离的DNA序列，包括能够转录为第一方面所述的RNA重组核酸分子的DNA序列。
本发明第三方面提供一种载体，包括第二方面所述的分离的DNA序列。
本发明第四方面提供一种细胞，包括第一方面所述的RNA重组核酸分子和/或第二方面所述的分离的DNA序列和/或第三方面所述的载体。
本发明第五方面提供一种组合物，包括第一方面所述的RNA重组核酸分子和/或第二方面所述的分离的DNA序列和/或第三方面所述的载体。
本发明第六方面提供第一方面所述的RNA重组核酸分子、第二方面所述的分离的DNA序列、第三方面所述的载体、第五方面所述的组合物在制备治疗或预防疾病的药物组合物中的应用，所述疾病与所述目标多肽的表达相关，所述目标多肽的表达能够治疗或者预防所述疾病。
本发明第七方面提供一种在细胞内表达目标多肽的方法，所述方法包括：
将第一方面所述的RNA重组核酸分子和/或第二方面所述的分离的DNA序列和/或第三方面所述的载体和/或第五方面所述的组合物导入细胞中，表达目标多肽。
本发明第八方面提供一种治疗肿瘤的方法，所述方法包括：
向受试者施用第一方面所述的RNA重组核酸分子和/或第二方面所述的分离的DNA序列和/或第三方面所述的载体和/或第五方面所述的组合物，所述目标多肽在肿瘤细胞中表达且在健康细胞中不表达，
其中，所述靶细胞为肿瘤细胞，所述非靶细胞为健康细胞。
本发明设计的RNA重组核酸分子，实现核糖体内部进入位点(IRES)翻译活性的开-关调控；通过 设计RNA二级结构，实现由特定微小RNA(miRNA)激活/抑制的工程化IRES元件；将由特定miRNA激活/抑制的工程化IRES元件构建至环状RNA(circRNA)载体中，通过体外转录和脂质纳米颗粒(LNP)包装递送circRNA分子，实现circRNA在肿瘤细胞的特异性表达。
本发明所带来的有益效果包括：应用本发明所构建的肿瘤细胞特异性表达的环状RNA分子，能够实现肿瘤细胞的特异性表达，可作为一种基于RNA翻译调控的新型肿瘤靶向治疗手段。由于LNP包裹的RNA可以通过静脉注射递送至肿瘤病灶，而无需瘤内注射，能够充分发挥RNA技术的多种优势：成本较低，易于制备和包装递送，目标基因表达迅速，不会整合至细胞基因组等。与此同时，该装置也可以包装成病毒，制备新型溶瘤病毒。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了在HEK293细胞系中通过设计DS序列来抑制HCV-IRES的翻译活性示意图(A)和效果图(B)；
图2显示了在HEK293细胞系中通过设计RS序列来恢复HCV-IRES的翻译活性示意图(A)和效果图(B)；
图3显示了在Huh7细胞系中测试DS的抑制效果和RS的恢复效果；
图4显示了在HEK293细胞系中设计含有miRBS和RS序列的RNA二级结构构建三种由miRNA激活的工程化IRES元件(MITA)的示意图；
图5显示了在HEK293细胞系中测试三种MITA对外源miRNA类似物的响应；
图6显示了在HEK293、Huh7细胞系中测试三种MITA对内源miRNA的响应；
图7显示了设计含有不同miRBS和RS序列的RNA二级结构构建具有逻辑“或”运算能力的工程化IRES元件；
图8显示了设计含有不同miRBS和RS序列的RNA二级结构构建具有逻辑“与”运算能力的工程化IRES元件；
图9显示了设计含有完全互补配对的miRBS结合位点来构建具有逻辑“非”运算能力的工程化IRES元件；
图10显示了在HEK293、Huh7细胞系中测试多种miRNA的抑制活性，其中，4×miRNA binding sites表示四个miRNA结合位点串联连接；
图11显示了结合MITA和MITR设计构建细胞类型分类器的质粒载体在HEK293、Huh7细胞系中测试分类效果；
图12显示了含有MITA元件的环状RNA体外转录的模版示意图；
图13显示了测试含有dIRES和rIRES的环状RNA在HEK293、Huh7细胞系中的表达抑制和恢复情况；
图14显示了测试含有三种不同MITA的环状RNA在HEK293、Huh7细胞系中的表达差异情况；
图15显示了结合MITA和MITR设计构建细胞类型分类器的环状RNA在HEK293、Huh7细胞系中测试分类效果；
图16显示了在HEK293细胞系中设计DS序列来抑制截短版HCV-IRES(tIRES)的翻译活性示意图和效果图；
图17显示了在HEK293细胞系中设计RS序列来恢复tIRES的翻译活性效果图；
图18显示了在HEK293细胞系中设计20-nt的DS序列来抑制其他IRES的翻译活性的效果图；
图19A显示了不含miRBS的癌细胞特异性杀伤环状RNA分子构建示意图；
图19B显示了癌细胞特异性杀伤实验结果图；
图20A显示了含有4×miRBS的癌细胞特异性杀伤环状RNA分子构建示意图；
图20B显示了癌细胞特异性杀伤实验结果图；
图20C显示了癌细胞与正常细胞在转染杀伤性环状RNA分子后的显微照片图。
发明详细描述
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
术语
“IRES”是指核糖体内部进入位点(Internal Ribosomal Entry Site,IRES)，IRES起始RNA翻译的机制不同于真核细胞中传统的5’帽依赖的翻译机制，IRES是一段具有特定空间结构的RNA序列，最初发现于RNA病毒的基因组中。由于其特殊的高级结构能够直接招募真核细胞的核糖体，或者通过招募反式作用因子(IRES Trans-Acting Factor,ITAF)进一步招募翻译起始因子和核糖体，从而能够不依赖于5’帽，直接从RNA序列内部起始翻译。根据IRES序列特征和二级结构的保守性，可以将核糖体内部进入位点分为三类。
本文使用的“RNA翻译开关元件”是指调控mRNA起始翻译的一段RNA序列，RNA翻译开关元件通过与IRES中含有的对翻译活性起关键作用的结构之间进行互补配对或者非互补配对，通过结构变化来调控下游目标多肽编码元件的表达。
本文使用的“DS序列”是RNA重组核酸分子的RNA翻译开关元件中的一部分RNA序列，其与IRES中含有的对翻译活性起关键作用的结构(如3’端假结结构)的至少一部分互补配对。
本文使用的“RS序列”是RNA重组核酸分子的RNA翻译开关元件中的一部分RNA序列，其与DS序列的至少一部分互补配对并形成茎环结构
本文使用的“3’端假结结构”是IRES的3’末端的一部分序列，其处于单链状态或者互补配对状态，决定着核糖体的进入；当3’端假结结构处于单链状态，核糖体可以与mRNA结合，起始与之相连的下游序列的翻译；当3’端假结结构与其他序列互补配对，则核糖体无法与mRNA结合，无法起始下游序列的翻译。
本文使用的“目标多肽编码元件”是指目标多肽的mRNA序列。
“间隔序列”是一段无功能的mRNA序列。
“茎环结构”是指单链RNA分子中存在的反向重复序列，由于互补碱基间的氢键配对，长链区段可以回折形成的一种二级结构。定义配对碱基间的双链区形成“茎”，而不能配对的单链区部分则突出形成“环”。
本文使用的“Y型双茎环结构”是指单链RNA分子由于存在两个反向重复序列，形成了两个茎环 结构，其中，两个茎环结构以及单链RNA分子两端的互补配对序列共同本文使用的组成了Y型双茎环结构。
本文使用的“双链茎部”是指单链RNA分子形成的反向重复序列中，配对碱基间的双链区形成的“茎”部。
本文使用的“单链环部”是指单链RNA分子形成的反向重复序列中，不配对的单链区部分突出形成的“环”部。
“互补配对”是指核酸分子中各核苷酸残基的碱基按A与T、A与U和G与C的对应关系互相以氢键相连的现象，本发明特指RNA分子中的碱基互补配对。
本文使用的“miRBS序列”是指与miRNA互补配对的序列。
“miRNA”也称为MicroRNA，是一类长度约20nt，进化上保守的单链非编码RNA分子，它通过碱基互补配对作用，结合目标信使RNA(mRNA)，并通过引导目标mRNA的降解或抑制目标mRNA的翻译，从而阻止目标蛋白质的产生。
本文使用的“各自独立地”是指本发明中的RNA翻译开关元件含有的多个miRBS序列相同或者不相同，可以结合相同或者不相同的miRNA类型。
本文使用的“串联的miRBS序列”是指多个miRBS序列首位直接相连接。
本文使用的“截短的IRES元件”是指缺失部分序列的IRES元件，可以是两端缺失部分序列，也可以是中间缺失部分序列。
“脂质纳米颗粒”通常是由离子化阳离子脂质、磷脂、胆固醇和聚乙二醇脂质组成的纳米颗粒，中央为一个固体或油性内核，而粒径较小的传统单层脂质体的典型特征是表面有脂质双层，颗粒中央为一个水性“池”。由于脂质体在很早之前已被监管机构批准并用于临床小分子药物，它们很快被基因治疗先驱确定为一种潜在的递送载体，特别应用于作为RNA小分子药物的递送载体。
“脂质体”是由卵磷脂和神经酰胺等制得的脂质体，具有的双分子层结构与细胞膜结构类似。脂质体作为药物载体被广泛应用于小分子药物、蛋白质、核酸和显像剂。为提高治疗效果和改善患者依从性，针对脂质体的多种给药已被开发出来，例如肠外给药、肺部给药、口服给药、经皮给药、眼科给药和鼻腔给药等。
如本文所用，术语“组合物”是以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
如本文所用，术语“治疗”是指暂时或永久地、部分或完全地消除、减少、抑制或改善事件、疾病或病状的临床症状、表现或进展。
在许多实施例中，术语“受试者”和“患者”可互换使用，而不管受试者是否已经或当前正在接受任何形式的治疗。如本文所用，术语“受试者”或“患者”是指哺乳动物受试者或患者。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。示例性受试者包括但不限于人、猴、犬、猫、小鼠、大鼠、牛、马、骆驼、禽、山羊和绵羊。在某些实施方案，所述受试者是人。在一些实施方案，所述受试者是疑似患有癌症、自体免疫性疾病或病况、和/或感染的人。
如本文所用，“诱导细胞凋亡的多肽”指的是能够启动细胞程序性死亡的多肽片段。
如本文所用，“对肿瘤细胞有毒性或杀伤性的多肽”是指那些能够特异性地杀死或抑制癌细胞生长的蛋白质片段，而对正常细胞相对安全。在本发明中，使用了两个代表性的多肽：Gasdermin D(GSDMD)的N-terminal部分和Herpes Simplex Virus Thymidine Kinase(HSV-TK)。GSDMD是一种参与pyroptosis(一种炎症性细胞死亡途径)的孔形成蛋白，其N-terminal部分在引发细胞死亡中具有关键作用。而HSV-TK则是一种常用于基因治疗策略的酶，当与抗代谢药物如ganciclovir配合时，HSV-TK可以转化该药物为其有毒形式，从而诱导细胞死亡。
RNA重组核酸分子
根据本发明一个实施方案，本发明提供一种RNA重组核酸分子，包括：
(1)RNA翻译开关元件；
(2)IRES元件或与IRES元件具有类似功能的元件；
(3)目标多肽编码元件，
其中，所述IRES元件或与IRES元件具有类似功能的元件包括对翻译活性起关键作用的结构
所述RNA翻译开关元件包括第一RNA序列和第二RNA序列，所述第一RNA序列位于所述第二RNA序列的上游，所述第一RNA序列含有RS序列，所述第二RNA序列含有DS序列，所述RS序列与所述DS序列之间具有第一间隔序列，
(a)所述RS序列和所述DS序列的至少一部分互补配对并形成茎环结构；
(b)所述DS序列与所述对翻译活性起关键作用的结构的至少一部分互补配对；
在所述RNA重组核酸分子中，(a)和(b)不同时发生，通过(a)和(b)两种结构的变化，实现所述目标多肽编码元件的起始翻译与否。
在真核生物中，RNA转录后头部发生了修饰，核糖体以及其他蛋白质能够识别该修饰。mRNA通过头部的帽子结构起始翻译，帽子结构是mRNA翻译起始的必要结构，为核糖体识别mRNA提供了信号，协助核糖体与mRNA结合，使翻译从AUG开始。同时帽子结构可增加mRNA的稳定性，保护mRNA免遭5’→3’核酸外切酶的攻击。在RNA重组核酸分子中构建IRES元件或与IRES元件具有类似功能的元件，核糖体可识别该元件，起始内部翻译。发明人提出了一种全新的策略，只需要改造RNA分子结构即可实现miRNA对于mRNA/circRNA的翻译激活和抑制，并且能够由此构建具有逻辑运算和细胞分类的RNA装置。
根据本发明一个具体的实施方案，如图1所示，增强型黄色荧光蛋白(EYFP)的翻译依赖于RNA的帽结构，作为内参基因，蓝色荧光蛋白(tagBFP)的翻译是由IRES起始的，假结结构(Pseudoknot)必须是单链状态IRES才具有起始翻译的功能，一旦破坏假结结构(其他序列与之互补配对，处于非单链状态)，则核糖体无法进入，无法翻译下游蛋白。发明人通过设计RS序列、DS序列，其中，RS序列和DS序列的至少一部分互补配对并形成茎环结构，DS序列可与假结结构的至少一部分互补配对，利用RS序列、DS序列与假结结构的不同互补配对方式，实现IRES下游目标多肽编码元件的翻译。具体地，图1中通过插入与假结区域互补配对的上游序列(称为DS)，使得IRES的原有结构将被破坏，从而抑制IRES介导的翻译活性。导致下游tagBFP翻译受阻，无法招募核糖体起始翻译，而RS序列又能够与DS序列互补形成茎环结构，实现假结结构出于单链状态，起始下游tagBFP翻译。本发明可以实现IRES下游基因动态的开和关的作用，当DS序列与假结结构互补配对，则IRES下游基因翻译处于关闭状态；当RS序列与DS序列互补形成茎环结构，IRES下游基因翻译处于开启状态。
根据本发明一个实施方案，所述第一RNA序列进一步包括至少一个miRBS序列，所述miRBS序列与靶细胞中特异性表达的miRNA的至少一部分互补且不完全互补，所述靶细胞为所述RNA重组核酸分子待导入的细胞。
当第一RNA序列中的miRBS序列与靶细胞中特异性表达的miRNA的至少一部分互补且不完全互补时，能够启动IRES元件或与IRES元件具有类似功能的元件下游多肽的翻译，而当miRBS序列未与靶细胞中特异性表达的miRNA互补时，IRES元件或与IRES元件具有类似功能的元件处于关闭状态，无法招募核糖体进入。
根据本发明一个实施方案，所述miRBS序列进行序列设计时，需要保证当所述miRBS序列与靶细胞中特异性表达的miRNA互补配对时，自5’至3’方向，在互补配对区的第10位和第11位的两个核苷酸的碱基设计为错配。
将互补配对区的第10位和第11位的两个核苷酸的碱基设计为错配，可以保证茎环结构被打开，但不会被细胞中的复合物剪切。
根据本发明一个实施方案，每一个miRBS序列结合相同或者不同的miRNA类型。需要说明的是，一个RNA翻译开关元件上可以设计多个miRBS序列，各个miRBS序列相同或不相同，每一个miRBS序列结合与之互补的靶细胞中特异性表达的miRNA。
根据本发明一个实施方案，如图4中A所示，当所述第一RNA序列包括一个miRBS序列时，所述miRBS序列位于所述RS序列的上游；
所述miRBS序列与所述RS序列通过第二间隔序列连接，且所述第二间隔序列与所述RS序列部分互补，
当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构的至少一部分互补配对时，所述miRBS序列的至少一部分、所述第二间隔序列、所述RS序列、所述第一间隔序列的至少一部分形成第一茎环结构。
根据本发明一个实施方案，所述RNA翻译开关元件包括至少一个所述第一茎环结构，其中，各个 所述第一茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。
根据本发明一个优选的实施方案，图4中A所示，可以通过调控miRBS在茎环中互补配对的长度来维持平衡，使得miRBS未与miRNA结合时，茎环结构稳定存在，当miRBS与miRNA结合时茎环容易打开。如果RNA翻译开关元件关闭结构太稳定，则即便miRBS与miRNA结合也不容易打开，起不到响应miRNA的IRES翻译开关的作用，若RNA翻译开关元件关闭结构太不稳定，可能miRNA还未与miRBS结合，RNA翻译开关元件就打开了，导致无法实现响应miRNA信号来控制IRES翻译开关的目的。
根据本发明另一个实施方案，如图4中B所示，当所述第一RNA序列包括两个miRBS序列时，两个所述miRBS序列位于所述RS序列的上游；
两个所述miRBS序列之间通过第三间隔序列连接，且所述第三间隔序列与所述RS序列部分互补配对。
根据本发明一个实施方案，所述第三间隔序列与所述RS序列部分互补配对包括具有2-3个非互补配对区域。
根据本发明一个实施方案，当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构中的至少一部分互补配对时，位于上游的所述miRBS序列的至少一部分、所述第三间隔序列、位于下游的所述miRBS序列、所述RS序列、所述第一间隔序列的至少一部分形成第二茎环结构。
根据本发明一个实施方案，所述RNA翻译开关元件包括至少一个所述第二茎环结构，其中，各个所述第二茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。
根据本发明一个优选的实施方案，如图4中B所示，在具有茎环(SL)结构的MITA(响应miRNA的IRES翻译激活元件)中，如果RS序列与第三间隔序列(两个miRBS序列之间的连接序列)完全互补，会导致关闭太稳定，茎环不易打开，因此优选设计几个非互补配对碱基，更容易打开。优选地，在设计RS序列时，保证RS序列与第三间隔序列区互补时，存在2-3个不互补配对碱基。根据本发明另一个实施方案，如图4中C所示，当所述第一RNA序列包括三个miRBS序列时，三个所述miRBS序列其中的两个位于所述RS序列的上游，三个所述miRBS序列其中的另外一个位于所述RS序列和所述DS序列之间。
根据本发明一个实施方案，当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构中的至少一部分互补配对时，所述RNA翻译开关元件中的DS序列上游的序列之间形成“Y”型双茎环结构，三个所述miRBS序列中的每一个的至少一部分在所述“Y”型双茎环结构中的双链茎部和/或单链环部。
根据本发明一个实施方案，所述RS序列位于所述“Y”型双茎环结构中的两个双链茎部。
根据本发明一个实施方案，所述“Y”型双茎环结构中的两个双链茎部分别具有1-2个非互补配对区域。
根据本发明一个实施方案，所述RNA翻译开关元件包括至少一个所述“Y”型双茎环结构，其中，各个所述“Y”型双茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。根据本发明一个实施方案，所述RNA重组核酸分子进一步包括与非靶细胞中特异性表达的miRNA的至少一部分互补的针对非靶细胞的miRBS序列，所述RNA重组核酸分子具有至少一个针对非靶细胞的miRBS序列。
根据本发明一个实施方案，每一个所述针对非靶细胞的miRBS序列位于(1)的上游，和/或(1)和(2)之间，和/或(2)和(3)之间，和/或(3)与终止密码子之间。
根据本发明一个实施方案，所述针对非靶细胞的miRBS序列为多个串联的miRBS序列，且各自独立地结合相同或不同的非靶细胞中特异性表达的miRNA类型。
根据本发明一个实施方案，所述针对非靶细胞的miRBS序列为3-5个串联的miRBS序列。
根据本发明一个实施方案，所述针对非靶细胞的miRBS序列与非靶细胞中特异性表达的miRNA完全互补。
根据本发明一个实施方案，所述RS序列的长度为9-25nt，优选为9-13nt。
根据本发明一个实施方案，所述DS序列的长度为10-25nt，优选为10-22nt。
根据本发明一个实施方案，与靶细胞或非靶细胞中特异性表达的miRNA结合的miRBS序列的长度为18-25nt，优选20-24nt。
根据本发明一个实施方案，所述与IRES元件具有类似功能的元件为具有翻译起始功能的元件。
根据本发明一个实施方案，所述与IRES元件具有类似功能的元件包括与IRES元件相比，具有至少 一个核酸的缺失、插入或替换的元件。
根据本发明一个实施方案，所述IRES元件源自类丙型肝炎病毒。
根据本发明一个实施方案，所述类丙型肝炎病毒包括选自丙型肝炎病毒(HCV)、禽脑脊髓炎病毒(AEV)、猪瘟病毒(CSFV)、猪肠病毒(PTV)中的至少之一。
这些类丙型肝炎病毒来源的IRES序列虽然不完全相同，但序列同一性比较高，且均存在关键的假结结构。虽然本发明在实施例中主要举例了HCV来源的IRES序列，但理论上，其他含有对翻译活性起关键作用的结构(例如假结结构)的病毒来源的IRES序列，也都涵盖在本发明的保护范围之内。
根据本发明一个实施方案，所述IRES元件中含有的对翻译活性起关键作用的结构为3’端假结结构。
根据本发明一个实施方案，所述与IRES元件具有类似功能的元件为截短的IRES元件。
根据本发明一个实施方案，所述截短的IRES元件的核酸序列为去除野生型IRES的5’端30-40个碱基的序列。例如，所述截短的IRES元件的核酸序列为去除野生型HCV IRES的5’端40个碱基的序列。
根据本发明一个实施方案，所述RNA重组核酸分子为线性RNA分子或者环状RNA分子。优选环状RNA分子，以增强稳定性。
在本发明中，所述miRBS序列是依据细胞中特异性表达的miRNA的活性确定的。例如，当在肿瘤细胞需要目标多肽编码元件起始翻译，而正常细胞中目标多肽编码元件不翻译时，这时要求miRNA在正常细胞和肿瘤细胞中活性有差异。如果筛选的是在肿瘤细胞活性高而在正常细胞活性低的miRNA，则RNA重组核酸分子与肿瘤细胞中miRNA结合，肿瘤细胞中IRES起始目标多肽编码元件翻译；同时选择在肿瘤细胞活性低在正常细胞中活性高的另一种miRNA，设计这个信号为关闭信号，在正常细胞中，RNA重组核酸分子与正常细胞中miRNA结合，RNA重组核酸分子在正常细胞中被切割掉，在肿瘤细胞中因为没有正常细胞中miRNA信号，RNA重组核酸分子就不被切割。在肿瘤细胞中高表达的miRNA能够激活IRES起始翻译，在正常细胞中高表达miRNA会抑制(切割)本发明的RNA重组核酸分子。同时运用这两种方式，使得目标多肽编码元件只在肿瘤细胞中翻译，用以保证RNA重组核酸分子的安全性。
本发明提供的RNA重组核酸分子，无需组合多个RNA分子，也无需引入外源蛋白，本发明仅凭工程化改造单独一个RNA分子即可构建具有细胞特异性表达能力的RNA，并且能够感应多种miRNA信号实现逻辑运算。本发明提出的工程化改造IRES元件既可用于线性mRNA也可用于环状RNA的表达调控。本发明提出的工程化改造IRES的策略，可以应用到多种IRES中，具有普适性和可迁移性。本发明通过感应细胞内源miRNA信号，实现工程化改造后的IRES元件的结构动态变化，从而动态调控IRES的翻译活性。本发明是首个能够仅凭单独一个环状RNA分子即可同时实现目标蛋白翻译活性的上调或下调，并且能够用于细胞信号的逻辑运算和细胞分类。
分离的DNA序列、载体、细胞、组合物
根据本发明一个实施方案，本发明提供一种分离的DNA序列，包括能够转录为前面所述的RNA重组核酸分子的DNA序列。
根据本发明另一个实施方案，本发明提供一种载体，包括前面所述的分离的DNA序列。
根据本发明另一个实施方案，本发明提供一种细胞，包括前面所述的RNA重组核酸分子和/或前面所述的分离的DNA序列和/或前面所述的载体。
根据本发明一个实施方案，本发明提供一种组合物，包括前面所述的RNA重组核酸分子和/或前面所述的分离的DNA序列和/或前面所述的载体。
根据本发明一个实施方案，所述组合物进一步包括药学上可接受的载体。
根据本发明一个实施方案，本发明提供所述载体为脂质纳米颗粒或脂质体，所述脂质纳米颗粒或脂质体将前面所述的RNA重组核酸分子和/或前面所述的分离的DNA序列和/或前面所述的载体包裹其中。
需要说明的是，当本发明提供的RNA重组核酸分子或含有所述RNA重组核酸分子的组合物用于治疗肿瘤相关疾病时，所述RNA重组核酸分子不含有正常细胞的miRBS结合位点，该RNA重组核酸分子或组合物也能够用于治疗肿瘤相关疾病，也能够实现目的多肽在肿瘤和正常细胞的区分表达，但是若含有正常细胞的miRBS结合位点，则RNA重组核酸分子表达目的多肽在肿瘤和正常细胞区分度更大，更具有安全开关的保障。
制备治疗或预防疾病的药物组合物中的应用
本发明提供前面所述的RNA重组核酸分子、前面所述的分离的DNA序列、前面所述的载体、前面所述的组合物在制备治疗或预防疾病的药物组合物中的应用，所述疾病与所述目标多肽的表达相关，所述目标多肽的表达能够治疗或者预防所述疾病。
根据本发明一个实施方案，当所述疾病为肿瘤时，所述目标多肽选自诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽中的至少之一。
细胞内表达目标多肽的方法以及治疗肿瘤的方法
本发明提供一种在细胞内表达目标多肽的方法，所述方法包括：
将前面所述的RNA重组核酸分子和/或前面所述的分离的DNA序列和/或前面所述的载体和/或前面所述的组合物导入细胞中，表达目标多肽。
根据本发明一个实施方案，所述细胞中特异性表达的miRNA的至少一部分与导入外源分子后细胞中表达的RNA重组核酸分子中的第一RNA序列中的miRBS序列互补且不完全互补。
根据本发明一个实施方案，本发明提供一种治疗肿瘤的方法，所述方法包括：
向受试者施用前面所述的RNA重组核酸分子和/或前面所述的分离的DNA序列和/或前面所述的载体和/或前面所述的组合物，所述目标多肽在肿瘤细胞中表达且在健康细胞中不表达，
其中，所述靶细胞为肿瘤细胞，所述非靶细胞为健康细胞。
根据本发明一个实施方案，所述目标多肽选自诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽中的至少之一。
本发明提供的治疗肿瘤的方法，目的是希望诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽中的至少之一在肿瘤细胞中高表达，而在正常细胞中低表达或不表达。IRES元件起始目标多肽编码元件的翻译在肿瘤细胞中是开启表状态，在正常细胞中是关闭状态。在肿瘤细胞中，采用前边所述的IRES系统，实现杀伤性的基因的表达，在此基础上，为了增加安全性，尽可能在正常组织中不表达，同时联合在正常组织中高表达的miRNA切割信号，以提升安全性。相当于在正常细胞中，一方面因为没有肿瘤信号(肿瘤细胞中特异性的miRNA)，IRES结构损坏，不能表达，另一方面，又感知到正常细胞中高表达的miRNA信号，本发明的RNA重组核酸分子在治疗方面，安全性有了双重保障。根据本发明一个优选的实施方案，针对正常细胞的miRBS与针对肿瘤细胞的IRES在同一个RNA重组核酸分子上。
根据本发明一个具体的实施方案，关于治疗肿瘤的方法，可以以肿瘤模型小鼠(实体瘤)为例验证本发明的RNA重组核酸分子或组合物对目标多肽编码元件(以荧光蛋白为例)翻译的控制。例如，将外源RNA(本发明提供的RNA重组核酸分子或组合物)注入(如通过静脉注射)实体瘤小鼠体内，观察是否在肿瘤处表达(有荧光的)，在非种植肿瘤区域(健康区域)无荧光，侧面可以印证本发明提供的RNA重组核酸分子或组合物是否能够用于控制诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽在肿瘤细胞中翻译而在健康细胞中不翻译。
下面将结合实施例对本公开的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实验材料和方法
1.试剂和酶来源。
限制性内切酶、ATP、多核苷酸激酶(PNK)、T4 DNA连接酶和Q5高保真DNA聚合酶均从New England Biolabs购买。寡核苷酸由擎科生物科技有限公司合成。miRNA模拟物和抑制剂均从合生生物有限公司购买。
2.质粒构建。
根据需要，将等摩尔量的寡核苷酸在1×PNK缓冲液中加热至95℃，逐渐冷却(每分钟-1℃)至37℃，进行退火，然后用0.5U/μL PNK在0.5mM ATP存在下将1μM的退火产物磷酸化。按照New England Biolabs的方案进行Golden Gate Assembly。
3.细胞培养和转染。
HEK293(HEK293FT)细胞系从Life Technologies购买。Huh7细胞系从BeNa culture collection Co.,Ltd.购买。Hela细胞系从ATCC购买。所有细胞都在含有10％FBS(DiNing)和1％青霉素-链霉素混合物(Invitrogen)的高葡萄糖DMEM(Invitrogen)中在37℃、100％湿度和5％CO2下培养。Lipofectamine 8000(Beyond Time Co.,Ltd.)用于质粒和miRNA模拟物转染。转染按照生产厂家的方案进行。简而言之，转染前一天，在每个24孔板(Corning)的每孔中接种约3×105个细胞，加入1mL DMEM完全培养基。对于质粒转染，将0.6μg质粒与0.6ug Lipofectamine 8000混合，然后转染到每孔细胞中。对于miRNA模拟物或miRNA抑制剂共转染，将miRNA模拟物或miRNA抑制剂与0.6μg质粒和0.6ug Lipofectamine 8000混合，最终转染浓度为50nM，然后转染到每孔细胞中。对于circRNA转染，将circRNA用脂质纳米颗粒包裹，如下所述，并以0.6μg每孔的量转染到细胞中。
4.环状RNA的制备和纯化。
环状RNA(circRNA)的制备和纯化按照以下方法进行。首先通过使用Xba I内切酶线性化质粒构建物来生成circRNA前体的DNA模板。利用T7 Polymerase(Novoprotein)从DNA模板进行体外转录(IVT)，产生circRNA前体。IVT后，用DNase I(Thermo Fisher Scientific)处理RNA产物30min，消化DNA模板。DNase I消化后，在RNA产物中加入GTP(Novoprotein)，最终浓度为2mM，并在55℃孵育15min，催化circRNAs的环化。然后，用RNase R(Novoprotein)在37℃处理15min，去除前体RNA。RNase R处理后的circRNA用LiCl(Novoprotein)纯化。
5.脂质纳米颗粒包裹circRNA。
circRNAs被包裹在脂质纳米颗粒(LNPs)中，脂质溶解在含有可电离性脂质(SM-102)、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、胆固醇和PEG-脂质(摩尔比为50:10:38.5:1.5)的乙醇中。将脂质混合物与含有circRNA的50mM柠檬酸缓冲液(pH4.0)以1:3的比例通过Lipid Mixer(Micronano INano L)混合。然后，通过超滤离心滤器单元(Millipore)对制剂进行透析，滤器具有100kD分子量截止值(Sartorius Stedim Biotech)，并对PBS(pH7.4)进行30倍体积的透析，将制剂浓缩到所需浓度，并在-20℃保存。所有制剂都经过粒径、分布、RNA浓度和包裹率的测试。
6.流式细胞术。
转染后3天，用胰蛋白酶消化细胞，并在4℃下以300g离心5min。去除上清液，用不含钙或镁的1×PBS(Invitrogen)重悬细胞。用Fortessa流式细胞仪(BD Biosciences)进行荧光活化流式分析，设置如下：EYFP荧光用514nm激发，519-568nm范围内发射。tagBFP荧光用405nm激发，410-507nm范围内发射。mKate荧光用543nm激发，571-651nm范围内发射。对于每个样品，收集约1×105到约5×105个细胞事件。
7.荧光素酶测定。
转染后24小时，去除细胞培养基，并加入100μL Bright-Light荧光素酶测定系统(Vazyme)，混匀并孵育3min。将100μL的培养基混合物转移到平底白壁板(Corning)中，并用微孔板发光仪(Thermo Fisher Scientific)测量发光强度。
8.乳酸脱氢酶细胞毒性测定。
用乳酸脱氢酶细胞毒性测定试剂盒(Beyotime，上海，中国)评估细胞死亡。LDH分析按照生产厂家的方案进行。简而言之，在转染前一天，在每个96孔培养板(Corning)的每孔中接种3×105个细胞，加入1mL DMEM完全培养基。次日，转染0.2-0.5ug LNP包裹的circRNA到每孔中。24小时后，将细胞培养板以400g离心5min，并小心吸去上清液，加入150μL经过10倍稀释的乳酸脱氢酶(LDH)释放试剂(试剂与磷酸盐缓冲液(PBS)的体积比为1:10)，混匀。孵育一小时后，将细胞培养板再次以400g离心5min，并将产生的上清液(每孔120μL)转移到新的96孔板中进行后续样品定量。
9.数据分析。
对于所有研究，所呈现的数据是三个独立生物重复实验的代表性结果。对于流式细胞术测量，EYFP或mKate(当构建物中包含完全互补miRBS时)荧光蛋白作为内部对照。对于每个样品，计算了阳性门限细胞的tagBFP荧光强度平均值(tagBFP MFI)和EYFP或mKate MFI。相对IRES介导的tagBFP荧光强度定义为tagBFP/EYFP(或当构建物中包含完全互补miRBS时为tagBFP/mKate)MFI之比。归一化IRES介导的tagBFP荧光强度定义为相对IRES介导的tagBFP荧光强度除以在同一细胞系中测试的正对照(nIRES或rIRES)。对于荧光素酶测定，计算了每个样品的发光强度平均值。归一化荧光素酶活性定义为发光强度除以在同一细胞系中测试的正对照(nIRES或rIRES)。
实施例1 HCV-IRES的翻译活性的抑制和恢复
本实施例以丙肝病毒(HCV)核糖体内部进入位点(IRES)为例进行阐述。HCV-IRES由四个结构域组成，HCV-IRES结构域4中的特殊假结结构在HCV-IRES介导的翻译起始中起着至关重要的作用，并且该假结在空间上与HCV-IRES的上游末端相邻。发明人通过插入与假结区域互补配对的上游序列(称为DS)，使得HCV-IRES的原有结构将被破坏，从而抑制IRES介导的翻译活性(图1中A)。构建了双顺反子荧光报告系统，使用流式细胞术评估具有不同DS长度的工程化IRES在人胚胎肾细胞(HEK293)中的翻译效率(图1中B)。增强型黄色荧光蛋白(EYFP)的翻译依赖于RNA的帽结构，作为内参基因。蓝色荧光蛋白(tagBFP)的翻译是由IRES起始的，因此tagBFP与EYFP的平均荧光强度(MFI)比值可以表征IRES介导的翻译效率。与未改造的天然IRES(nIRES)相比，当上游DS长度超过12个核苷酸(12-nt)时，tagBFP的MFI开始降低，当DS长度达到20-nt时，tagBFP的MFI相比于nIRES降低了超过10倍(图1，称含有20-nt的DS IRES为dIRES)。
接下来，设计并插入与20-nt的DS互补的恢复序列(RS)，因为RS和DS之间的碱基配对将恢复HCV-IRES的原始结构和翻译起始活性(图2中A)，其中由假结区域与DS的配对(翻译关闭状态)变为RS与DS的配对(翻译起始状态)，假结区域释放，回复起始翻译。通过在DS上游引入RS，当RS长度超过8-nt时，tagBFP的MFI增加，当RS长度达到13-nt时，tagBFP的MFI恢复到nIRES的70％以上(称含有13-nt RS的dIRES为rIRES)(图2中B)。此外，发明人还在人肝细胞癌细胞(Huh7)中也观察到与HEK293细胞中相似的dIRES的tagBFP翻译抑制和rIRES的tagBFP翻译恢复(图3)。
本实施例涉及的核酸序列如下表1所示：
表1






实施例2三种类型的MITA的构建
为了进一步设计可编程的响应miRNA的IRES翻译激活元件(MITA)，发明人试图设计具有miRNA结合位点(miRBS)的二级结构，以便将RS固定在相对稳定的结构中。为了确保miRNA只与miRBS结合而不被切割，将miRBS设计为miRNA海绵，即在10和11位与miRNA有2个核苷酸错配。构建了三种类型的MITA(图4)，即鞋楦(TL)、茎环(SL)和三臂(AJ)结构(分别对应A-C)。在缺乏互补miRNA的情况下，13-nt的RS被固定在设计的结构中，使得IRES介导的tagBFP翻译被20-nt的DS抑制(即装置处于关闭状态)。当存在互补miRNA时，miRNA与设计的miRBS结合并破坏设计的RNA二级结构，释放13-nt的RS从而恢复IRES结构和翻译活性(即装置处于开启状态)。
首先设计了与人工miRNA-FF4互补的miRBS，并用miRNA类似物共转染在HEK293中测试了所有三种类型的MITA(图5)。图5中每一组左侧柱子为对照组(加入与miRNA-FF4等量的与miRBS非互补的miRNA)，右侧柱子为加入miRNA-FF4的实验组(FF4mimics)，设计了具有16个核苷酸碱基配对的鞋楦结构(TL16)、具有3个核苷酸错配的茎环结构(SL3)和具有2个核苷酸错配的3臂结构(AJ2)。在未加入对应的miRNA类似物时，所有三种类型的MITA都表现出tagBFP翻译减少，这表明在缺乏所需miRNA的情况下，MITA处于关闭状态。在miRNA-FF4模拟物共转染后，tagBFP翻译大部分恢复，表明miRNA-FF4的存在触发了MITA至开启状态。
接着，试图使用MITA来感知内源性miRNA。设计了与miR-21-5p互补的miRBS(miR-21-5p是一种在大多数癌细胞中高表达但在HEK293中低表达的miRNA)。检测了在Huh7和HEK293细胞中所有三种类型MITA的tagBFP翻译水平(图6，图中每一组的左侧柱子为HEK293细胞，右侧柱子为Huh7细胞)。由于设计的初始结构中如果存在更多的碱基配对将稳定关闭状态，而更多的碱基错配将不利于关闭状态，因此发明人改变碱基配对和错配的数量以优化MITA结构。结果显示，与HEK293相比，所有MITA均在Huh7中表现出更高的tagBFP翻译活性，并且TL16、SL3和AJ2表现出最高的倍数变化(超过3倍)。
本实施例涉及的核酸序列如下表2所示：
表2


实施例3设计MITA来实现细胞的逻辑运算
在证明了对MITA进行序列编程可以响应任意miRNA之后，发明人试图设计MITA来实现细胞的逻辑运算。发明人串联了miRNA-FF4和miRNA-199a-3p的两个TL16序列(这两种miRNA在HEK293中都不表达)，通过miRNA模拟物共转染的方式在HEK293中测试(图7)。当用miRNA-FF4或miRNA-199a-3p模拟物共转染时，IRES介导的tagBFP翻译活性均恢复，表明串联TL16的设计可以实现逻辑“或”功能，即miRNA-FF4或miRNA-199a-3p中的任意一种miRNA都能使得IRES介导的tagBFP起始翻译。然后发明人设计了同时具有miRNA-FF4和miRNA-199a-3p结合位点的SL3基序(图8)。结果显示，只有在miRNA-FF4和miRNA-199a-3p模拟物同时共转染的情况下，tagBFP的翻译才能恢复，这表明双输入的SL3设计可以作为逻辑“与”门。为了实现逻辑“非”功能，发明人在HCV-IRES的上游和下游直接插入miRNA-FF4的完全互补结合位点，构建可编程的基于miRNA的IRES翻译阻遏物(MITR)(图9)。当使用miRNA-FF4与MITR共转染时，tagBFP的翻译被显著抑制，表明MITR可以实现逻辑“非”门。“非”门，没有miRNA信号可以表达，接收到miRNA信号后，miRBS与miRNA完全互补配对，将招募到细胞中的剪切复合物，导致mRNA被剪切，IRES不能起始下游序列翻译。本实施例涉及的核酸序列如下表3所示：
表3
实施例4构建细胞类型分类器
发明人试图使用前述的MITA和MITR设计策略来构建细胞类型分类器。测试了若干种miRNA在HEK293和Huh7中的抑制活性(图10)。图10中mKATE表达不受miRNA调控，EYFP受到miRNA调控，mKATE质粒作为对照，当细胞中具有miRNA信号时，pCMV-EYFP-4×miRNA binding sites质粒会被切开，影响YFP的表达。根据抑制活性结果，最终选择miR-21-5p作为Huh7高活性miRNA来构建 MITA模块，选择miR-106a-5p和miR-18a作为HEK293高活性miRNA来实现逻辑“非”门。构建了含有miR-21-5p响应性的TL16、SL3和AJ2的MITA(IRES相关序列)，在MITA上游和tagBFP报告基因下游分别串联插入4个miR-106a-5p和miR-18a结合位点(图11)。将这些质粒载体分别转染到Huh7和HEK293细胞中，用mKate荧光蛋白作为内参对照。在Huh7细胞中，4xmiR-18a RBS和4x miR-106aRBS不会与miR-18a和miR-106a结合，不发生切割，与此同时，MITA感受到肿瘤细胞miRNA信号，从而促进BFP表达。而正常细胞中，恰恰相反，MITA未感知到miRNA信号，BFP不表达或者弱表达，而4xmiR-18a RBS和4x miR-106aRBS与正常细胞中高表达的miRNA结合，整个结构被切割。图11结果表明，与HEK293相比，Huh7中的tagBFP荧光强度至多增加了7倍(每组左侧柱子为HEK293，右侧柱子为Huh7细胞)。值得注意的是，当单独使用MITA或MITR时，仅观察到荧光信号的3倍或4倍增加。因此，结果表明MITA和MITR在细胞类型分类方面具有协同效应。
本实施例涉及的核酸序列如下表4所示：
表4
实施例5
为了直接在circRNA中应用MITA和细胞类型分类器，构建了含有置换的内含子-外显子(PIE)的质粒模板，用于circRNA的体外转录(IVT)(图12)。以萤火虫荧光素酶为报告基因，首先将编码nIRES、dIRES和rIRES的circRNA分别转染到HEK293和Huh7细胞中。观察到与质粒转染相同的dIRES抑制和rIRES恢复效果(图13，每组左侧柱子为HEK293，右侧柱子为Huh7细胞)。接下来，将编码TL16、SL3或AJ2的MITA的circRNA分别转染到Huh7和HEK293细胞中(图14，每组左侧柱子为HEK293，右侧柱子为Huh7细胞)。结果表明与HEK293相比，TL16或SL3设计在Huh7中表现出超过3倍的荧光信号增加，而AJ2则表现出超过6倍的荧光信号增加。这些结果表明本发明的MITA模块在circRNA中也同样能够受到miRNA的激活。最后，测试了circRNA版本的细胞类型分类器(图15，右图中每组左侧柱子为HEK293，右侧柱子为Huh7细胞)。与HEK293相比，在Huh7中观察到高达8倍的荧光信号增加，这表明本发明的细胞类型分类器circRNA可以准确地识别所需的癌细胞，并可能作为潜在的癌症靶向治疗手段。
本实施例涉及的核酸序列如下表5所示：
表5




实施例6 MITA的改进
为了进一步改进MITA设计，测试了在结构域1截短的HCV-IRES(tIRES)中DS的抑制作用，因为发明人认为缩短DS和IRES关键假结部位之间的空间距离将促进碱基互补配对。发明人发现，野生型IRES结构中，第一个颈环去掉，截短的IRES结构也能够发挥功能，从而实现了在设计IRES系统时，可以缩短DS和RS的长度。结果显示，只需10-nt的DS插入便显著降低了10倍以上的tIRES的翻译活性(称该结构为dtIRES，图16)。
发明人还测试了恢复dtIRES翻译活性的RS，只需插入9-nt的RS便能使dtIRES的翻译活性恢复到tIRES的70％以上(图17)。因此，在截短版本的HCV-IRES中也可应用本发明开发MITA和MITR。在全长的IRES结构中，一般DS要设计20nt，RS要设计13nt。在截短后，DS设计10nt，RS设计9nt就可以实现翻译的打开和关闭。
此外，发明人还设计了20-nt长度的DS来破坏其他IRES的假结结构以证明本发明中使用的策略的普适性和可扩展性。在禽脑脊髓炎病毒(AEV)、猪瘟病毒(CSFV)和猪肠病毒(PTV)的IRES中也观察到了类似的抑制效果，这表明本发明的策略可以扩展到其他IRES(图18)。
本实施例涉及的核酸序列如下表6所示：
表6


实施例7本发明RNA分子对癌细胞的杀伤作用
为验证本发明的环状RNA对癌细胞的特异性毒性，将环状RNA中的荧光素酶报告基因替换为Gasdermin D(GSDMD)的N-terminal部分(图19A和20A)。采用乳酸脱氢酶(Lactate Dehydrogenase Cytotoxicity，LDH)进行细胞毒性测定。如图19B所示，实验结果显示，对于未经设计的普通环状RNA分子(native IRES)，Huh7细胞中LDH活性与HEK293细胞相比显著增高18倍(图19B中每组左侧柱子为HEK293，右侧柱子为Huh7细胞，含有AJ2 MITA的环状RNA不含有miRBS序列)。
当将本发明的细胞类型分类器环状RNA转染到Huh7和HEK293细胞中时，观察到的细胞毒性差异更为显著，高达89倍(图20B中每组左侧柱子为HEK293，右侧柱子为Huh7细胞，含有AJ2 MITA的环状RNA含有4×miRNA binding sites序列)。图20C为显微照片图，与图20B相对应，直观反映了细胞被杀伤的情况。
这些结果证明了本发明的环状RNA对癌细胞的特异性杀伤作用，并突显了本发明的MITA模块和细胞类型分类器在靶向癌症治疗应用中的潜在应用性。
本实施例涉及的核酸序列如下表7所示：
表7

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、“一些实施方案”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (21)

  1. 一种RNA重组核酸分子，其中，包括：
    (1)RNA翻译开关元件；
    (2)IRES元件或与IRES元件具有类似功能的元件；
    (3)目标多肽编码元件，
    其中，所述IRES元件或与IRES元件具有类似功能的元件包括对翻译活性起关键作用的结构，
    所述RNA翻译开关元件包括第一RNA序列和第二RNA序列，所述第一RNA序列位于所述第二RNA序列的上游，所述第一RNA序列含有RS序列，所述第二RNA序列含有DS序列，所述RS序列与所述DS序列之间具有第一间隔序列，
    (a)所述RS序列和所述DS序列的至少一部分互补配对并形成茎环结构；
    (b)所述DS序列与所述对翻译活性起关键作用的结构的至少一部分互补配对；
    在所述RNA重组核酸分子中，(a)和(b)不同时发生，通过(a)和(b)两种结构的变化，实现所述目标多肽编码元件的起始翻译与否。
  2. 根据权利要求1所述的RNA重组核酸分子，其中，所述第一RNA序列进一步包括至少一个miRBS序列，所述miRBS序列与靶细胞中特异性表达的miRNA的至少一部分互补且不完全互补，所述靶细胞为所述RNA重组核酸分子待导入的细胞。
  3. 根据权利要求2所述的RNA重组核酸分子，其中，所述miRBS序列进行序列设计时，需要保证当所述miRBS序列与靶细胞中特异性表达的miRNA互补配对时，自5’至3’方向，在互补配对区的第10位和第11位的两个核苷酸的碱基设计为错配。
  4. 根据权利要求1-3中任一项所述的RNA重组核酸分子，其中，每一个miRBS序列结合相同或者不同的miRNA类型。
  5. 根据权利要求1-4中任一项所述的RNA重组核酸分子，其中，当所述第一RNA序列包括一个miRBS序列时，所述miRBS序列位于所述RS序列的上游；
    所述miRBS序列与所述RS序列通过第二间隔序列连接，且所述第二间隔序列与所述RS序列部分互补，
    任选地，当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构的至少一部分互补配对时，所述miRBS序列的至少一部分、所述第二间隔序列、所述RS序列、所述第一间隔序列的至少一部分形成第一茎环结构；
    任选地，所述RNA翻译开关元件包括至少一个所述第一茎环结构，其中，各个所述第一茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。
  6. 根据权利要求1-4中任一项所述的RNA重组核酸分子，其中，当所述第一RNA序列包括两个miRBS序列时，两个所述miRBS序列位于所述RS序列的上游；
    两个所述miRBS序列之间通过第三间隔序列连接，且所述第三间隔序列与所述RS序列部分互补配对，
    任选地，所述第三间隔序列与所述RS序列部分互补配对包括具有2-3个非互补配对区域；
    任选地，当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构中的至少一部分互补配对时，位于上游的所述miRBS序列的至少一部分、所述第三间隔序列、位于下游的所述miRBS序列、所述RS序列、所述第一间隔序列的至少一部分形成第二茎环结构；
    任选地，所述RNA翻译开关元件包括至少一个所述第二茎环结构，其中，各个所述第二茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。
  7. 根据权利要求1-4中任一项所述的RNA重组核酸分子，其中，当所述第一RNA序列包括三个miRBS序列时，三个所述miRBS序列其中的两个位于所述RS序列的上游，三个所述miRBS序列其中的另外一个位于所述RS序列和所述DS序列之间；
    任选地，当所述DS序列与所述IRES元件中含有的对翻译活性起关键作用的结构中的至少一部分互补配对时，所述RNA翻译开关元件中的DS序列上游的序列之间形成“Y”型双茎环结构，三个所述miRBS序列中的每一个的至少一部分在所述“Y”型双茎环结构中的双链茎部和/或单链环部；
    任选地，所述RS序列位于所述“Y”型双茎环结构中的两个双链茎部；
    任选地，所述“Y”型双茎环结构中的两个双链茎部分别具有1-2个非互补配对区域；
    任选地，所述RNA翻译开关元件包括至少一个所述“Y”型双茎环结构，其中，各个所述“Y”型双茎环结构中含有的miRBS序列各自独立地结合相同或者不同的miRNA类型。
  8. 根据权利要求1-7中任一项所述的RNA重组核酸分子，其中，所述RNA重组核酸分子进一步包括与非靶细胞中特异性表达的miRNA的至少一部分互补的针对非靶细胞的miRBS序列，所述RNA重组核酸分子具有至少一个针对非靶细胞的miRBS序列；
    任选地，每一个所述针对非靶细胞的miRBS序列位于(1)的上游，和/或(1)和(2)之间，和/或(2)和(3)之间，和/或(3)与终止密码子之间；
    任选地，所述针对非靶细胞的miRBS序列为多个串联的miRBS序列，且各自独立地结合相同或不同的非靶细胞中特异性表达的miRNA类型；
    任选地，所述针对非靶细胞的miRBS序列为3-5个串联的miRBS序列，
    任选地，所述针对非靶细胞的miRBS序列与非靶细胞中特异性表达的miRNA完全互补。
  9. 根据权利要求2-8中任一项所述的RNA重组核酸分子，其中，所述RS序列的长度为9-25nt，优选为9-13nt；
    任选地，所述DS序列的长度为10-25nt，优选为10-22nt；
    任选地，与靶细胞或非靶细胞中特异性表达的miRNA结合的miRBS序列的长度为18-25nt，优选20-24nt。
  10. 根据权利要求1-9中任一项所述的RNA重组核酸分子，其中，所述与IRES元件具有类似功能的元件为具有翻译起始功能的元件；
    任选地，所述与IRES元件具有类似功能的元件包括与IRES元件相比，具有至少一个核酸的缺失、插入或替换的元件；
    任选地，所述IRES元件源自类丙型肝炎病毒；
    任选地，所述类丙型肝炎病毒包括选自丙型肝炎病毒、禽脑脊髓炎病毒、猪瘟病毒、猪肠病毒中的至少之一；
    任选地，所述IRES元件中含有的对翻译活性起关键作用的结构为3’端假结结构；
    任选地，所述与IRES元件具有类似功能的元件为截短的IRES元件；
    任选地，所述截短的IRES元件的核酸序列为去除野生型IRES的5’端30-40个碱基的序列。
  11. 根据权利要求1-10中任一项所述的RNA重组核酸分子，其中，所述RNA重组核酸分子为线性RNA分子或者环状RNA分子。
  12. 一种分离的DNA序列，其中，包括能够转录为权利要求1-11中任一项所述的RNA重组核酸分子的DNA序列。
  13. 一种载体，其中，包括权利要求12所述的分离的DNA序列。
  14. 一种细胞，其中，包括权利要求1-11中任一项所述的RNA重组核酸分子和/或权利要求12所述的分离的DNA序列和/或权利要求13所述的载体。
  15. 一种组合物，其中，包括权利要求1-11中任一项所述的RNA重组核酸分子和/或权利要求12所述的分离的DNA序列和/或权利要求13所述的载体。
  16. 根据权利要求15所述的组合物，其中，所述组合物进一步包括药学上可接受的载体；
    任选地，所述载体为脂质纳米颗粒或脂质体，所述脂质纳米颗粒或脂质体将权利要求1-11中任一项所述的RNA重组核酸分子和/或权利要求12所述的分离的DNA序列和/或权利要求13所述的载体包裹其中。
  17. 权利要求1-11中任一项所述的RNA重组核酸分子、权利要求12所述的分离的DNA序列、权利要求13所述的载体、权利要求15或16所述的组合物在制备治疗或预防疾病的药物组合物中的应用，其中，所述疾病与所述目标多肽的表达相关，所述目标多肽的表达能够治疗或者预防所述疾病。
  18. 根据权利要求17所述的应用，其中，当所述疾病为肿瘤时，所述目标多肽选自诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽中的至少之一。
  19. 一种在细胞内表达目标多肽的方法，其中，所述方法包括：
    将权利要求1-11中任一项所述的RNA重组核酸分子和/或权利要求12所述的分离的DNA序列和/或权利要求13所述的载体和/或权利要求15或16所述的组合物导入细胞中，表达目标多肽；
    任选地，所述细胞中特异性表达的miRNA的至少一部分与导入外源分子后细胞中表达的RNA重组核酸分子中的第一RNA序列中的miRBS序列互补且不完全互补。
  20. 一种治疗肿瘤的方法，其中，所述方法包括：
    向受试者施用权利要求1-11中任一项所述的RNA重组核酸分子和/或权利要求12所述的分离的DNA序列和/或权利要求13所述的载体和/或权利要求15或16所述的组合物，所述目标多肽在肿瘤细胞中表达且在健康细胞中不表达，
    其中，所述靶细胞为肿瘤细胞，所述非靶细胞为健康细胞。
  21. 根据权利要求20所述的方法，其中，所述目标多肽选自诱导细胞凋亡的多肽、对肿瘤细胞有毒性或杀伤性的多肽中的至少之一。
PCT/CN2023/112676 2023-08-11 2023-08-11 基于miRNA调控的细胞可编程RNA翻译开关 Pending WO2025035296A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/112676 WO2025035296A1 (zh) 2023-08-11 2023-08-11 基于miRNA调控的细胞可编程RNA翻译开关

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/112676 WO2025035296A1 (zh) 2023-08-11 2023-08-11 基于miRNA调控的细胞可编程RNA翻译开关

Publications (1)

Publication Number Publication Date
WO2025035296A1 true WO2025035296A1 (zh) 2025-02-20

Family

ID=94632058

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/112676 Pending WO2025035296A1 (zh) 2023-08-11 2023-08-11 基于miRNA调控的细胞可编程RNA翻译开关

Country Status (1)

Country Link
WO (1) WO2025035296A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120252876A1 (en) * 2009-05-22 2012-10-04 Tenenbaum Scott A Trans-acting rna switches
US20160369271A1 (en) * 2013-09-17 2016-12-22 Hocuslocus, Llc Methods of using structurally interacting rna
JP2017209030A (ja) * 2016-05-23 2017-11-30 国立大学法人京都大学 リボソーム侵入サイト（ｉｒｅｓ）を用いた人工リボスイッチ
WO2018009923A1 (en) * 2016-07-08 2018-01-11 F1 Oncology, Inc. Methods and compositions for transducing lymphocytes and regulating the activity thereof
WO2020257655A1 (en) * 2019-06-21 2020-12-24 Kernal Biologics, Inc. Engineered oncoselective protein expression
US20230212592A1 (en) * 2020-06-12 2023-07-06 President And Fellows Of Harvard College Riboswitch modules and methods for controlling eukaryotic protein translation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120252876A1 (en) * 2009-05-22 2012-10-04 Tenenbaum Scott A Trans-acting rna switches
US20160369271A1 (en) * 2013-09-17 2016-12-22 Hocuslocus, Llc Methods of using structurally interacting rna
JP2017209030A (ja) * 2016-05-23 2017-11-30 国立大学法人京都大学 リボソーム侵入サイト（ｉｒｅｓ）を用いた人工リボスイッチ
WO2018009923A1 (en) * 2016-07-08 2018-01-11 F1 Oncology, Inc. Methods and compositions for transducing lymphocytes and regulating the activity thereof
WO2020257655A1 (en) * 2019-06-21 2020-12-24 Kernal Biologics, Inc. Engineered oncoselective protein expression
US20230212592A1 (en) * 2020-06-12 2023-07-06 President And Fellows Of Harvard College Riboswitch modules and methods for controlling eukaryotic protein translation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHIGETOSHI KAMEDA ET AL.: "Synthetic Circular RNA Switches and Circuits that Control Protein Expression in Mammalian Cells", NUCLEIC ACIDS RESEARCH, vol. 51, no. 4, 16 January 2023 (2023-01-16), pages e24, XP093084983, DOI: 10.1093/nar/gkac1252 *

Similar Documents

Publication Publication Date Title
JP7623423B2 (ja) 選択的スプライシングのアプタマー媒介性調節による遺伝子発現の調節
CN114616336B (zh) 用于将货物递送至靶细胞的组合物和方法
US20230075045A1 (en) Engineered crispr/cas13 system and uses thereof
JP2013500339A (ja) 癌を治療するための送達ツールとしての脂肪由来間質細胞（ａｓｃ）
CA2580189C (en) Sirna-mediated gene silencing of alpha synuclein
US10093923B2 (en) Modulation of HSP47 expression
JP4804467B2 (ja) 多重ｒｎａポリメラーゼｉｉｉプロモーター発現構築物
US9388425B2 (en) Tunable genetic switch for regulating gene expression
US10202601B2 (en) C/EBPα short activating RNA compositions and methods of use
JP2021527405A (ja) 合成肝臓指向性アデノ随伴ウイルスカプシドおよびその使用
TW201249991A (en) Modulation of TIMP1 and TIMP2 expression
CN112662674B (zh) 靶向编辑VEGFA基因外显子区域的gRNA及其应用
KR20220078650A (ko) 이중 바이러스 및 이중 종양용해 바이러스 및 치료 방법
JP2011507554A (ja) 遺伝子発現を増加させるための方法および組成物
JP2015221026A (ja) 人工合成ｍＲＮＡの翻訳効率化方法
JP2022523806A (ja) 閉端ｄｎａ（ｃｅｄｎａ）および免疫調節化合物
CA2551100A1 (en) Compositions and methods for combined therapy of disease
CN119630786A (zh) 用于位点特异性基因组修饰的多组件系统
CN109415733A (zh) 用于治疗乙型肝炎病毒(hbv)感染的药剂及其使用
Hoffmann et al. Selectively expressed RNA molecules: a new dimension in functionalized cell targeting
RU2575056C2 (ru) Модуляция экспрессии hsp47
CN118660966A (zh) 用于rna可编程细胞编辑的组合物和系统及其制备和使用方法
HK40102897A (zh) 使用工程化rna通过利用内源性adar进行靶向rna编辑
HK1235821B (zh) Hsp47表达的调节

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23948732

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE