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WO2024131786A1 - Système permettant d'insérer un grand fragment d'adn dans un génome - Google Patents

Système permettant d'insérer un grand fragment d'adn dans un génome Download PDF

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WO2024131786A1
WO2024131786A1 PCT/CN2023/139871 CN2023139871W WO2024131786A1 WO 2024131786 A1 WO2024131786 A1 WO 2024131786A1 CN 2023139871 W CN2023139871 W CN 2023139871W WO 2024131786 A1 WO2024131786 A1 WO 2024131786A1
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sequence
utr
seq
retrotransposase
rna
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Chinese (zh)
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李伟
周琪
陈阳灿
骆胜球
胡艳萍
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Institute of Zoology of CAS
Institute for Stem Cell and Regeneration of CAS
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Institute of Zoology of CAS
Institute for Stem Cell and Regeneration of CAS
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Priority to CN202380087116.0A priority Critical patent/CN120418275A/zh
Publication of WO2024131786A1 publication Critical patent/WO2024131786A1/fr
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    • C12N2800/90—Vectors containing a transposable element

Definitions

  • the present application belongs to the field of biotechnology. More specifically, the present application relates to a retrotransposon capable of inserting a large DNA fragment into a human genome at a specific site, its use, and a system using the enzyme.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Some other methods of site-directed insertion of large DNA fragments have their own shortcomings. For example, the efficiency of large fragment insertion achieved by homologous recombination is low, and the introduced double-stranded DNA breaks pose a safety risk; recombinase systems such as Cre/loxP often require the pre-insertion of loxP sites before the second step of integration can be performed; in addition, most current technologies rely on DNA donors, making it difficult to solve problems such as in vivo delivery.
  • the reverse transcriptase complements the gap in large fragment site-specific integration technology and provides a more versatile and convenient tool for biological research.
  • the new reverse transcriptase system is mainly divided into two components. First, the reverse transcriptase protein polypeptide itself. Second, the donor RNA carrying new genetic information. The reverse transcriptase recognizes and binds to the RNA donor sequence to form a protein-RNA complex, which is then transferred to the target gene through the reverse transcriptase. Activity can convert RNA into DNA and integrate it into specific genomic sites.
  • the DNA integration method based on reverse transcription enzymes avoids the generation of double-stranded DNA breaks and the risks they bring, while avoiding the preparation and dependence on DNA donors in practical applications. Only one step of reverse transcription reaction is required to complete the integration of large fragments of DNA, which is convenient for wide application in various scenarios.
  • a reverse transposase comprising a target DNA binding domain containing a zinc finger binding motif, a reverse transcriptase domain, and an endonuclease domain, capable of reverse transcribing RNA into DNA.
  • the reverse transcriptase according to item 1 comprising 1 to 3 zinc finger domains (ZF), 1 Myb-like domain, 1 reverse transcriptase domain (RT) and 1 restriction endonuclease-like nuclease domain (RLE); further, mutations, deletions and insertions may optionally occur in the N-terminus of the protein and in the amino acid sequences that are not conservative or structural between the above four domains.
  • ZF zinc finger domains
  • RT reverse transcriptase domain
  • RLE restriction endonuclease-like nuclease domain
  • the retrotransposase according to item 1 or 2 whose amino acid sequence is as shown in any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71.
  • a system for modifying DNA comprising:
  • a donor RNA or a nucleic acid encoding the donor RNA wherein the donor RNA comprises: a sequence that binds to the retrotransposase and a heterologous sequence,
  • the heterologous sequence is at least 1-50000 bases, for example, 1 nt or more, 10 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, 100 nt or more, 150 nt or more, 200 nt or more, 250 nt or more, 300 nt or more, 350 nt or more, 400 nt or more, 450 nt or more, 500 nt or more, 550 nt or more, 600 nt or more, 650 nt or more, 700 nt or more, 750 nt or more, 800 nt or more, 850 nt or more, 900 nt or more, 950 nt or more, 1000 nt or more, 1100 nt or more, 1200 nt or more.
  • nt or more 1300nt or more, 1400nt or more, 1500nt or more, 1600nt or more, 1700nt or more, 1800nt or more, 1900nt or more, 2000nt or more, 2100nt or more, 2200nt or more, 2300nt or more, 2400nt or more, 2500nt or more, 2600nt or more, 2700nt or more, 2800nt or more, 2900nt or more, 3000nt or more, 3500nt or more, 4000nt or more, 4500nt or more, 5000nt or more, 5500nt or more, 6000nt or more, 6500nt or more, 7000nt or more, 7500nt or more, 8000nt or more, 8500nt or more, 9000nt or more, 9500nt or more, 10000nt or more, 15000nt or more, 20000nt or more, 25000nt or more, 30000nt or more, 35000
  • heterologous sequence comprises one or more of the following: a sequence encoding a polypeptide or a non-coding RNA sequence, a sequence comprising a promoter or an enhancer, a sequence encoding one or more introns, and a transcription termination sequence;
  • the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; further preferably, the polypeptide is a therapeutic protein, a membrane protein, an intracellular protein, an extracellular protein, a structural protein, a signal transduction protein, a regulatory protein, a transport protein, an organelle protein, a sensory protein, a motor protein, a defense protein, a storage protein, a reporter protein, an antibody, an enzyme, a coagulation factor, and further preferably, the number of amino acids in the polypeptide is 20 to 10000, for example, the number of amino acids is 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,
  • the intracellular protein is selected from cytoplasmic protein, nuclear protein, organelle protein, mitochondrial protein or lysosomal protein,
  • sequence encoding the polypeptide contains one or more introns.
  • the donor RNA further comprises a homology domain, preferably the homology domain comprises a first homology domain and a second homology domain,
  • the first homology domain is 5 or more bases located at the 5' end of the donor RNA and have 100% identity with the target DNA chain
  • the second homology domain is 5 or more bases located at the 3' end of the donor RNA and have 100% identity with the target DNA chain
  • the target DNA is a genomic safe harbor GSH site or the target DNA is a genomic Natural Harbor TM site.
  • nucleic acid encoding the reverse transposase according to any one of items 1 to 3 and the donor RNA or the nucleic acid encoding the donor RNA are separate nucleic acids, preferably the donor RNA does not encode the reverse transposase, and further preferably the donor RNA comprises one or more chemical modifications; or
  • nucleic acid encoding the reverse transposase described in any one of items 1 to 3 and the donor RNA or the nucleic acid encoding the donor RNA are covalently linked.
  • the nucleic acid encoding the reverse transposase described in any one of items 1 to 3 and the donor RNA or the nucleic acid encoding the donor RNA form a fusion nucleic acid.
  • the fusion nucleic acid comprises RNA or DNA.
  • a 5' untranslated sequence (5'UTR) to which the retrotransposase binds,
  • the promoter is located between the 5' untranslated sequence (5'UTR) to which the reverse transposase binds and the heterologous sequence, or preferably, the promoter is located between the 3' untranslated sequence (3'UTR) to which the reverse transposase binds and the heterologous sequence.
  • 5'UTR 5' untranslated sequence
  • 3'UTR 3' untranslated sequence
  • heterologous sequence comprises an open reading frame or its reverse complement sequence oriented in a 5' to 3' direction on the donor RNA; or the heterologous sequence comprises an open reading frame or its reverse complement sequence oriented in a 3' to 5' direction on the donor RNA.
  • the donor RNA further comprises a nuclear localization signal or the nucleic acid encoding the retrotransposase according to any one of items 1 to 3 comprises a nuclear localization signal and/or a nucleolar localization signal and/or a nuclear export signal.
  • the nucleic acid encoding the retrotransposase and the nucleic acid encoding the donor RNA are present in a ratio of 10:1 to 1:10, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the donor RNA comprises a stem-loop sequence or helix 5' of the pseudoknot sequence, preferably comprises one or more (e.g. 2, 3 or more) stem-loop sequences or helices 3' of the pseudoknot sequence, such as 3' of the pseudoknot sequence and 5' of the heterologous sequence, and further preferably the donor RNA of the pseudoknot has catalytic activity, such as RNA cleavage activity, such as cis-RNA cleavage activity, or
  • the donor RNA comprises, e.g., at least one stem-loop sequence or helix, e.g., 1, 2, 3, 4, 5 or more stem-loop sequences, hairpin or helix sequences, e.g., 3' to the heterologous sequence.
  • the 5' untranslated sequence (5'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No.7 to 12 or SEQ ID No.44 to 55;
  • the 3’ non-translated sequence (5’UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence described in any one of SEQ ID No.13 to 18 or SEQ ID No.56 to 67.
  • the first homology domain is the first homology domain
  • 3' untranslated sequence (3'UTR) to which the retrotransposase binds
  • the second homology domain is the second homology domain
  • the first homology domain is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more bases located at the 5' end of the donor RNA and having 100% identity with the target DNA chain
  • the second homology domain is 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more bases located at the 3' end of the donor RNA and having 100% identity with the target DNA chain. or more than 60 or more than 70 or more than 80 or more than 90 or more than 100 bases;
  • the 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of any one of SEQ ID No.7 to 12;
  • the 3’ untranslated sequence (5’UTR) in the donor RNA that binds to the retrotransposase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence described in any one of SEQ ID No. 13 to 18.
  • the 5' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 5' untranslated sequence (5'UTR); or
  • the 3' untranslated sequence (5'UTR) to which the retrotransposase binds is a non-natural 3' untranslated sequence (5'UTR);
  • non-native 5' untranslated sequences having additions, deletions and/or substitutions of nucleotides relative to the native 5'UTR sequences;
  • non-native 3' untranslated sequences having additions, deletions and/or substitutions of nucleotides relative to the native 3'UTR sequences;
  • non-native 5' untranslated sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide sequence of SEQ ID No.19-21;
  • the non-natural 3’ untranslated sequence (3’UTR) is further preferred, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence described in any one of SEQ ID No.22-23.
  • the heterologous sequence is inserted into the target site at a copy number of 1 insertion in about 1%-80% of the cells (e.g., about 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, or 70%-80% of the cells) in a population of cells contacted with the system, for example, as measured using colony isolation and ddPCR.
  • a non-natural 5' untranslated sequence having additions, deletions and/or substitutions of nucleotides relative to a natural 5'UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of SEQ ID No. 19-21.
  • a non-natural 3’ untranslated sequence (3’UTR) having additions, deletions and/or substitutions of nucleotides relative to a natural 3’UTR sequence, preferably having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of SEQ ID No. 22-23.
  • An engineered transposable element comprising, from 5' to 3':
  • 5' untranslated sequence (5'UTR), heterologous sequence and 3' untranslated sequence (3'UTR),
  • the 5' untranslated sequence comprises a nucleotide sequence selected from SEQ ID No. 19-21 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity;
  • the non-natural 3’ untranslated sequence (3’UTR) is further preferred, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence described in any one of SEQ ID No.22-23.
  • a host cell comprising the system according to any one of items 4 to 18 or the element according to item 21 or 22, wherein the host cell is preferably a mammalian cell or a plant cell, and more preferably a human cell.
  • a method for modifying a target DNA strand in a cell, tissue or subject comprising applying the system of any one of items 4 to 18 to the cell, tissue or subject, wherein the The system reverse transcribes the donor RNA sequence into the target DNA strand, thereby modifying the target DNA strand in a cell, tissue or subject.
  • the cells and tissues are mammalian cells and tissues, preferably human cells and tissues, and the subject is a mammal, preferably a human.
  • a method for modifying the genome of a mammalian cell or inserting DNA into the genome of a mammal comprising applying the system of any one of items 4 to 18 to the cell, preferably the mammal is a human.
  • the method comprises contacting a cell, a tissue or a subject with a retrotransposase as described in any one of items 1 to 3 or a nucleic acid encoding the retrotransposase as described in any one of items 1 to 3 and a donor RNA or a nucleic acid encoding the donor RNA,
  • the contacting comprises contacting the cell, tissue or subject with a plasmid, virus, virus-like particle, virosome, liposome, vesicle, exosome or lipid nanoparticle;
  • said contacting comprises the use of non-viral delivery, such as electroporation.
  • the contacting comprises intravenously administering to the subject, preferably at least twice, the retrotransposase shown in any one of Items 1 to 3 or a nucleic acid encoding the retrotransposase shown in any one of Items 1 to 3 and a donor RNA or a nucleic acid encoding the donor RNA.
  • the retrotransposase described in any one of Items 1 to 3 or the nucleic acid encoding the retrotransposase described in any one of Items 1 to 3 and the donor RNA or the nucleic acid encoding the donor RNA are administered separately; or
  • the retrotransposase described in any one of Items 1 to 3 or a nucleic acid encoding the retrotransposase described in any one of Items 1 to 3 is administered together with a donor RNA or a nucleic acid encoding the donor RNA.
  • a vector comprising the nucleic acid described in item 34.
  • a host cell comprising the vector of item 35.
  • a pharmaceutical composition comprising the system described in any one of items 4 to 18, or the nucleic acid described in item 34, or the vector described in item 35, or the host cell described in item 23 or 36.
  • the system is placed in a pharmaceutically acceptable carrier, and further preferably, the carrier is a vesicle (including liposomes, natural or synthetic lipid bilayers, exosomes), lipid nanoparticles, viruses or plasmid vectors.
  • the system and method constructed by the present application can realize gene writing at the DNA level by using only RNA donors, which is a technical innovation.
  • the system and method of the present application can meet but are not limited to:
  • Treatment needs for example, by providing expression of a therapeutic transgene in an individual with a loss-of-function mutation, by replacing a gain-of-function mutation with a normal transgene, by providing a regulatory sequence to eliminate expression of a gain-of-function mutation, and/or by controlling the expression of operably linked genes, transgenes, and systems thereof.
  • the RNA sequence template encodes a promoter region specific to the host cell's therapeutic needs, such as a tissue-specific promoter or enhancer.
  • the promoter can be operably linked to a coding sequence.
  • plants can be given new economic traits (such as stress resistance, insect resistance, etc.).
  • FIG. 1 shows the structures of the vector expressing the retrotransposase protein and the vector expressing the donor RNA.
  • Figure 2 Activity results of various retrotransposase systems in mammals using the system constructed in Example 2. The results showed that compared with the negative control, the six novel retrotransposase systems (#3, #21, #23, #24, #31, #33) had significant activity.
  • FIG. 3 GFP after #21 retrotransposase achieves GFP gene integration in mammalian cells The expression results were obtained and the proportion of GFP-positive cells was quantified by flow cytometry.
  • FIG. 4 shows the GFP expression results after the #21 retrotransposase achieves GFP gene integration in mammalian cells, and the GFP expression is observed by fluorescence microscopy.
  • FIG5 shows that #21 retrotransposase achieves GFP gene integration in mammalian cells, and explores the effect of donor RNA containing non-natural 5’UTR and/or non-natural 3’UTR on the efficiency of gene integration of #21 retrotransposase in mammalian cells.
  • FIG. 6 Display of the positions of PCR primers in the examples.
  • Figure 7 PCR amplification results of the junction between the 3’ end of the integrated DNA sequence and the genome (inside the 28s rDNA gene) in mammalian cells using different retrotransposase systems.
  • FIG8 PCR amplification results of sequences spanning both ends of introns of integration sequences in mammalian cells using different retrotransposase systems.
  • FIG. 9 shows the expression results of GFP after the GFP gene was integrated into mammalian cells by four types of #21 retrotransposases, and the expression level of GFP was observed by fluorescence microscopy.
  • nucleic acid refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and their analogs.
  • Oligonucleotide and “oligonucleotide” are used interchangeably and refer to short polynucleotides having no more than about 50 nucleotides.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid via traditional Watson-Crick base pairing.
  • the complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10 out of 10, which are about 50%, 60%, 70%, 80%, 90%, 100% complementary, respectively).
  • “Completely complementary” means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence.
  • substantially complementary means that the degree of complementarity is at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • Percentage (%) sequence identity for nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a particular nucleic acid sequence after alignment of the sequences (if necessary) by allowing gaps to achieve the maximum percentage of sequence identity.
  • Percentage (%) sequence identity for peptide, polypeptide or protein sequences is the percentage of amino acid residues in a candidate sequence that are identically replaced with the amino acid residues in a particular peptide or amino acid sequence after alignment of the sequences (if necessary) by allowing gaps to achieve the maximum percentage of sequence homology.
  • alignment can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software such as mafft, muscle, Clustal, needle, BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software, for example, preferably using methods such as mafft, muscle, Clustal, needle.
  • suitable parameters for measuring alignment including any algorithm required for achieving maximum alignment over the full length of the compared sequence.
  • polypeptide and “peptide” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids.
  • a protein may have one or more polypeptides.
  • the term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component).
  • variant is interpreted as a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, respectively, but retains the necessary properties.
  • a typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the variant nucleic acid sequence may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below.
  • a typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence.
  • the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions.
  • the amino acid sequences of the variant and the reference polypeptide may differ by any combination of one or more substitutions, additions, deletions.
  • the substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code.
  • Variants of polynucleotides or polypeptides may be naturally occurring (such as allelic variants), or may be unknown naturally occurring variants.
  • Non-naturally occurring variants of nucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.
  • wild type has a meaning generally understood by those skilled in the art, and refers to an organism, strain, gene or characteristic in a typical form that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from resources in nature and has not been intentionally modified.
  • nucleic acid molecule or polypeptide As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated or naturally occurring.
  • Cell as used herein should be understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in progeny due to mutation or environmental influences, such progeny may not in fact be the same as the parent cell, but are still included within the scope of the term herein.
  • transduction and “transfection” include methods known in the art for introducing DNA into cells using infectious agents (such as viruses) or other means to express a protein or molecule of interest.
  • infectious agents such as viruses
  • virus-like agents there are chemical-based transfection methods, such as transfection methods using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposon; particle-based methods, such as using a gene gun, magnetofection or magnet-assisted transfection, particle bombardment; and hybrid methods (such as nuclear transfection).
  • transfected refers to the process of transferring or introducing exogenous nucleic acid into a host cell.
  • a “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid.
  • in vivo refers to inside the organism from which the cell was obtained.
  • Ex vivo or “in vitro” refers to outside the organism from which the cell was obtained.
  • treatment is a method for obtaining beneficial or desired results (including clinical results).
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) remission of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving the quality of life, and/or prolonging Survival.
  • Treatment also includes reducing the pathological consequences of a disorder, condition, or disease. The methods of the present invention contemplate any one or more of these aspects of
  • the term "effective amount” refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease (such as improving, alleviating, alleviating and/or delaying one or more symptoms thereof).
  • an "effective amount” can be administered in one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired treatment endpoint.
  • Subject “Subject,” “individual,” or “patient” are used interchangeably herein for purposes of treatment and refer to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, farm, or pet animals such as dogs, horses, cats, cows, etc.
  • the individual is a human individual.
  • reference to "not" a value or parameter generally means and describes an "except” value or parameter.
  • the method is not used to treat cancer type X, meaning that the method is used to treat cancers other than type X.
  • the term “and/or” in phrases such as "A and/or B” is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” in phrases such as "A, B, and/or C” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the mutations described herein may include one or more of: insertion, deletion, substitution, and may be mutations of a single amino acid or multiple amino acids.
  • a “vector” is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses.
  • suitable vectors are The vector comprises a replication origin, a promoter sequence, a convenient restriction endonuclease site and one or more selective markers that function in at least one organism.
  • the term “vector” should also be interpreted as including non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells, such as, for example, polylysine compounds, liposomes, etc.
  • the vector is a viral vector.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.
  • the vector is a bacteriophage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals.
  • the rAAV construct can be administered to a subject enterally. In some embodiments, the rAAV construct can be administered to a subject parenterally. In some embodiments, the rAAV particles can be administered subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intraventricularly, intramuscularly, intrathecally (IT), intracisternal, intraperitoneally, via inhalation, topically, or by direct injection into one or more cells, tissues, or organs. In some embodiments, the rAAV particles can be administered to a subject by injection into the hepatic artery or portal vein.
  • Vectors can be transferred into host cells by physical, chemical or biological methods.
  • vectors into host cells include: calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.
  • Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • the vector is introduced into the cell by electroporation.
  • the cell is a bacterium, a yeast cell, a fungal cell, an algae cell, a plant cell or an animal cell (e.g., a mammalian cell, such as a human cell).
  • the cell is a cell of natural origin, such as a cell isolated by a tissue biopsy.
  • the cell is a cell isolated from a cell line cultured in vitro.
  • the cell is from a primary cell line.
  • the cell is from an immortalized cell line.
  • the cell is a genetically engineered cell.
  • the nuclear localization signal herein is a domain of a protein, usually a short amino acid sequence, which can interact with a nuclear import carrier to enable the protein to be transported into the cell nucleus.
  • the nuclear localization signal may also be a RNA sequence.
  • the nuclear localization signal is located on the donor RNA.
  • the retrotransposase polypeptide is encoded on the first RNA, and the donor RNA is a second separate RNA, and the nuclear localization signal is located on the donor RNA instead of on the RNA encoding the retrotransposase polypeptide.
  • the RNA encoding the retrotransposase is mainly targeted to the cytoplasm to promote its translation, while the donor RNA is mainly targeted to the nucleus to promote its retrotransposition into the genome.
  • the nuclear localization signal is at the 3' end, 5' end or inside of the donor RNA. In some embodiments, the nuclear localization signal is at the 3' end of the heterologous sequence (e.g., directly at the 3' end of the heterologous sequence) or at the 5' end of the heterologous sequence (e.g., directly at the 5' end of the heterologous sequence).
  • the nuclear localization signal is placed outside the 5' UTR of the donor RNA or outside the 3' UTR. In some embodiments, the nuclear localization signal is placed between the 5'UTR and the 3'UTR, wherein optionally, the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is in an antisense orientation or downstream of a transcription termination signal or a polyadenylation signal). In some embodiments, the nuclear localization sequence is located within an intron. In some embodiments, a plurality of identical or different nuclear localization signals are in RNA, such as in a donor RNA.
  • the length of the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp.
  • RNA nuclear localization sequences can be used.
  • domain refers to the structure of a biomolecule that contributes to a specific function of a biomolecule.
  • a domain can comprise a continuous region (e.g., a continuous sequence) or different non-continuous regions (e.g., a non-continuous sequence) of a biomolecule.
  • protein domains include, but are not limited to, endonuclease domains, target DNA binding domains, reverse transcription domains; examples of domains of nucleic acids are regulatory domains, such as transcription factor binding domains.
  • the present application relates to a target DNA binding domain containing a zinc finger binding motif, a reverse transcriptase domain, and an endonuclease domain.
  • the reverse transcriptase domain refers to a domain with reverse transcription function, and those skilled in the art can use conventional tools as basic local comparison search tools (e.g., BLAST) to identify the reverse transcription domain based on homology with other known reverse transcription domains.
  • the reverse transcriptase domain is modified, for example, by site-specific mutation.
  • the reverse transcriptase domain is engineered to bind to a heterologous sequence.
  • the endonuclease domain refers to a domain with endonuclease function
  • the endonuclease element is a heterologous endonuclease element, such as Fok1 nuclease, type II restriction endonuclease (RLE type nuclease) or another RLE type endonuclease (also referred to as REL).
  • the heterologous endonuclease activity has nickase activity and does not form double-strand breaks.
  • BLAST basic local comparison search tools
  • websites or software to predict domains e.g., using InterPro website, hhpred website, CDD website, psi-blast software, blastp software or hh-suite software to predict domains
  • endonuclease domains based on homology with other known endonuclease domains.
  • the target DNA binding domain is a target DNA binding domain containing a zinc finger binding motif, wherein the zinc finger binding motif is an amino acid sequence responsible for binding to a target DNA of a specific sequence.
  • exogenous when used with respect to a biomolecule (e.g., a nucleic acid sequence or a polypeptide), refers to the artificial introduction of the biomolecule into a host genome, cell, or organism.
  • a nucleic acid added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods is exogenous to the existing nucleic acid sequence, cell, tissue, or subject.
  • heterologous means that when used to describe a first element with reference to a second element, the term heterologous means that the first element and the second element do not exist in the arrangement as described in nature.
  • a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or a portion of a polypeptide or nucleic acid molecule sequence that is not natural for the cell expressing it, (b) a polypeptide or nucleic acid molecule or a portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its natural state, or (c) a polypeptide or nucleic acid molecule with expression that is altered compared to the natural expression level under similar conditions.
  • a heterologous regulatory sequence e.g., a promoter, an enhancer
  • a heterologous domain of a polypeptide or nucleic acid sequence e.g., a DNA binding domain of a polypeptide or a nucleic acid encoding a DNA binding domain of a polypeptide
  • a heterologous domain of a polypeptide or nucleic acid sequence can be arranged relative to other domains, or can be a different sequence or relative to other domains or portions of a polypeptide or its encoding nucleic acid from different sources.
  • the heterologous nucleic acid molecule may be present in the native host cell genome, but may have an altered expression level or a different sequence, or both.
  • the heterologous nucleic acid molecule may not be endogenous to the host cell or the host genome, but may have been introduced into the host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may be It may be integrated into the host genome or may exist transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vectors, plasmids, or other self-replicating vectors) as extrachromosomal genetic material.
  • the term "gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • the promoter or enhancer is operably linked to the coding sequence.
  • Operably linked DNA sequences can be continuous or non-continuous. In the case where it is necessary to connect two protein coding regions, the operably linked sequences can be in the same reading frame.
  • the term "host genome or host cell” refers to a cell and/or its genome into which proteins and/or genetic material have been introduced. It should be understood that these terms are intended to refer not only to specific subject cells and/or genomes, but also to the offspring of such cells and/or the genomes of the offspring of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from parental cells, but are still included in the scope of the term "host cell” used herein.
  • the host genome or host cell can be a separated cell or cell line grown in culture, or a genomic material separated from such a cell or cell line, or can be a host cell or host genome that constitutes a living tissue or organism.
  • the host cell can be an animal cell or a plant cell, for example, as described herein.
  • the host cell can be a cattle cell, a horse cell, a pig cell, a goat cell, a sheep cell, a chicken cell or a turkey cell.
  • the host cell can be a corn cell, a soybean cell, a wheat cell or a rice cell.
  • genomic safe harbor sites are sites in the host genome that can accommodate the integration of new genetic material, for example, so that the inserted genetic elements do not cause significant changes in the host genome that pose a risk to the host cell or organism.
  • GSH sites typically meet 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the following conditions: (i) >300 kb from cancer-related genes; (ii) >300 kb from miRNA/other functional small RNAs; (iii) >50 kb from the 5' gene end; (iv) >50 kb from the replication origin; (v) >50 kb from any extremely conserved element; (vi) low transcriptional activity (i.e., no mRNA +/- 25 kb); (vii) not in a copy number variable region; (viii) in open chromatin; and/or (ix) is unique, with 1 copy in the human genome.
  • GSH sites in the human genome that meet some or all of these criteria include: (i) adeno-associated virus site 1 (AAVS1), which is the natural site of integration of the AAV virus on chromosome 19; (ii) the chemokine (CC motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as the HIV-1 co-receptor; (iii) the mouse Rosa26 locus Human ortholog; (iv) rDNA locus. Additional GSH sites are known and described, for example, in Pellenz et al., electronic publication on August 20, 2018 (https://doi.org/10.1101/396390).
  • the genomic safe harbor site is a Natural Harbor TM site.
  • the Natural Harbor TM site is a ribosomal DNA (rDNA).
  • the Natural Harbor TM site is a 5S rDNA, 18S rDNA, 5.8S rDNA, or 28S rDNA.
  • the Natural Harbor TM site is a Mutsu site in 5S rDNA.
  • the Natural Harbor TM site is an R2 site in 28S rDNA.
  • a "pseudoknot sequence” refers to a nucleic acid (eg, RNA) having a sequence with appropriate self-complementarity to form a pseudoknot structure.
  • a “stem-loop sequence” refers to a nucleic acid sequence (e.g., an RNA sequence) having sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and having a loop having at least three (e.g., four) base pairs.
  • the stem may contain mismatches or bulges.
  • the cell is an animal cell of an organism selected from the group consisting of cow, sheep, goat, horse, pig, deer, chicken, duck, goose, rabbit, and fish.
  • the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a human embryonic kidney 293T (HEK293T or 293T) cell or a HeLa cell. In some embodiments, the cell is a human embryonic kidney (HEK293T) cell. In some embodiments, the cell is a mouse Hepa1-6 cell. In some embodiments, the mammalian cell is selected from the group consisting of an immune cell, a hepatocyte, a tumor cell, a stem cell, a blood cell, a neural cell, a zygote, a muscle cell (such as a cardiomyocyte) and a skin cell.
  • an immune cell a hepatocyte, a tumor cell, a stem cell, a blood cell, a neural cell, a zygote, a muscle cell (such as a cardiomyocyte) and a skin cell.
  • the cell is an immune cell selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, ⁇ T cells, tumor-infiltrating T cells, and dendritic cells (DC) activated T cells.
  • the method produces modified immune cells, such as CAR-T cells or TCR-T cells.
  • the cell is an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a gamete progenitor cell, a gamete, a zygote, or a cell in an embryo.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • the reverse transcriptase of the present application comprises a target DNA binding domain containing a zinc finger binding motif, a reverse transcriptase domain, and a nuclease endonuclease domain, and can reverse transcribe RNA into DNA.
  • the amino acid sequence of the retrotransposase of the present application is as shown in any one of SEQ ID No.1-6 or SEQ ID No.32-43 or SEQ ID No.68-71, or has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of any one of SEQ ID No.1-6 or SEQ ID No.32-43 or SEQ ID No.68-71.
  • the amino acid sequence of the reverse transposase of the present application is a conservative mutant of the amino acid sequence shown in any one of SEQ ID No. 1 to 6 or SEQ ID No. 32 to 43 or SEQ ID No. 68 to 71.
  • the reverse transcriptase of the present application comprises 1 to 3 zinc finger domains (ZF), 1 Myb-like domain, 1 reverse transcriptase domain (RT) and 1 restriction endonuclease-like nuclease domain (RLE).
  • ZF zinc finger domains
  • RT reverse transcriptase domain
  • RLE restriction endonuclease-like nuclease domain
  • the N-terminus of the protein and the amino acid sequences of different lengths between the domains do not have obvious structural or conservative properties. Therefore, the conservative mutants of the amino acid sequences shown in any one of SEQ ID No. 1 to 6 or SEQ ID No. 32 to 43 or SEQ ID No. 68 to 71 of the present application include the truncated sequences of the amino acid sequences shown in any one of SEQ ID No. 1 to 6 or SEQ ID No. 32 to 43 or SEQ ID No.
  • 68 to 71 obtained by removing the N-terminus of the protein and the amino acid sequences that do not have conservatism or structure between the four domains, namely, 1 to 3 zinc finger domains (ZF), 1 Myb-like domain, 1 reverse transcriptase domain (RT) and 1 restriction endonuclease-like nuclease domain (RLE), but they still have reverse transposase activity. Or, a conservative mutant still having retrotransposase activity obtained by mutation, deletion, insertion, etc.
  • ZF zinc finger domains
  • RT reverse transcriptase domain
  • RLE restriction endonuclease-like nuclease domain
  • ZF zinc finger domains
  • RT reverse transcriptase domain
  • RLE restriction endonuclease-like nuclease domain
  • the structural property of a region in a protein refers to the region having an obvious rigid structure ( ⁇ -helix or ⁇ -fold) in the resolved three-dimensional structure of the protein (which can be obtained from databases such as PDB) or in the three-dimensional structure predicted by protein three-dimensional structure prediction software (e.g. Alphafold).
  • it may be a truncated form of protein #21, such as a truncated form after truncating the amino acids 401-467, which still has the activity of the retrotransposase required by the present application.
  • truncated form of protein #21 such as a truncated form after truncating amino acids 401-467 and further adding 32 linked amino acids after amino acid 400, which still has the activity of the retrotransposase required by the present application.
  • it may be a truncated form of protein #21, for example, a truncated form after truncating amino acids 1-100 thereof, which still has the activity of the retrotransposase required by the present application.
  • it may be a truncated form of protein #21, for example, a truncated form after truncating the amino acids 1-200 thereof, which still has the activity of the retrotransposase required by the present application.
  • it may be a truncated form of protein #21, such as a truncated form after truncating amino acids 1-200 and amino acids 401-467, which still has the activity of the retrotransposase required by the present application.
  • the present application relates to a system for modifying DNA, the system comprising: the reverse transposase of the present application or a nucleic acid encoding the reverse transposase of the present application; and a donor RNA or a nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the reverse transposase and a heterologous sequence, preferably the heterologous sequence is at least 1-50000 bases, for example, more than 1nt, more than 10nt, more than 50nt, more than 60nt, more than 70nt, more than 80nt, more than 90nt, more than 10 ...
  • 0nt or more 150nt or more, 200nt or more, 250nt or more, 300nt or more, 350nt or more, 400nt or more, 450nt or more, 500nt or more, 550nt or more, 600nt or more, 650nt or more, 700nt or more, 750nt or more, 800nt or more, 850nt or more, 900nt or more, 950nt or more, 1000nt or more, 1100nt or more, 1200nt or more, 1300nt or more 1400nt or more, 1500nt or more, 1600nt or more, 1700nt or more, 1800nt or more, 1900nt or more, 2000nt or more, 2100nt or more, 2200nt or more, 2300nt or more, 2400nt or more, 2500nt or more, 2600nt or more, 2700nt or more, 2800nt or more, 2900nt or more, 3000nt or more
  • the retrotransposase and the donor RNA are encoded separately by two plasmids, the retrotransposase gene is expressed by the CAG promoter, and the donor RNA is expressed by the CAG promoter.
  • the expression frame of the donor RNA is also embedded with a CMV promoter. In the expression frame initiated by the CMV promoter, CMV expresses GFP, but the GFP here is separated by an intron sequence inserted in the opposite direction.
  • the final mode of action of the system of the present application is: after the donor RNA is expressed by the CAG promoter, the intron sequence contained in the donor RNA is sheared off from the expressed RNA, and the GFP sequence in the expression frame initiated by the CMV promoter at this time returns to normal at the RNA level.
  • the GFP sequence at this time is an antisense RNA chain and does not have the ability to translate GFP protein.
  • the reverse expression frame initiated by the CMV promoter can express the normal mRNA of GFP without introns, and then express the GFP protein with green fluorescence.
  • the heterologous sequence of the present application is selected from one or more of the following: a sequence encoding a polypeptide, a sequence containing an organizational promoter or enhancer, and a sequence encoding one or more introns.
  • the polypeptide is a therapeutic polypeptide or a mammalian polypeptide; further preferably, the polypeptide is a therapeutic protein, a membrane protein, an intracellular protein, an extracellular protein, a structural protein, a signal transduction protein, a regulatory protein, a transport protein, an organelle protein, a sensory protein, a motor protein, a defense protein, a storage protein, a reporter protein, an antibody, an enzyme, or a coagulation factor.
  • the number of amino acids in the polypeptide is 20 to 10,000, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 , 330, 340, 350, 360, 370, 380, 390, 400,
  • the intracellular protein is selected from cytoplasmic proteins, nuclear proteins, organelle proteins, mitochondrial proteins or lysosomal proteins.
  • one or more introns are included in the sequence encoding the polypeptide.
  • the systems described herein can be used in vitro or in vivo.
  • the system or system components are delivered to cells (e.g., mammalian cells, such as human cells), for example, in vitro or in vivo.
  • the cells are eukaryotic cells, such as cells of multicellular organisms, such as animals, such as mammals (e.g., humans, pigs, cattle), birds (e.g., poultry, such as chickens, turkeys, or ducks), or fish.
  • the cells are non-human animal cells (e.g., experimental animals, livestock, or companion animals).
  • the cells are stem cells (e.g., hematopoietic stem cells), fibroblasts, or T cells.
  • the cells are non-dividing cells, such as non-dividing fibroblasts or non-dividing T cells. In some embodiments, the cells are plant cells.
  • the components of the Gene Writer system can be delivered in the form of polypeptides, nucleic acids (e.g., DNA, RNA), and combinations thereof.
  • delivery can use any combination of the following to deliver the retrotransposase (e.g., as DNA encoding the retrotransposase protein, as RNA encoding the retrotransposase protein, or as the protein itself) and the donor RNA (e.g., as DNA encoding RNA, or as RNA):
  • the retrotransposase e.g., as DNA encoding the retrotransposase protein, as RNA encoding the retrotransposase protein, or as the protein itself
  • the donor RNA e.g., as DNA encoding RNA, or as RNA
  • a virus is used to deliver DNA encoding a retrotransposase protein.
  • RNA and in some embodiments, a virus is used to deliver the donor RNA (or DNA encoding the donor RNA).
  • the system and/or components of the system are delivered in the form of nucleic acids.
  • the retrotransposase polypeptide can be delivered in the form of DNA or RNA encoding the polypeptide, and the donor RNA can be delivered in the form of RNA or its complementary DNA to be transcribed into RNA.
  • the system or components of the system are delivered on 1, 2, 3, 4 or more different nucleic acid molecules.
  • the system or components of the system are delivered as a combination of DNA and RNA.
  • the system or components of the system are delivered as a combination of DNA and protein.
  • the system or components of the system are delivered as a combination of RNA and protein.
  • the retrotransposase polypeptide is delivered as a protein.
  • a system or a component of a system is delivered to a cell, such as a mammalian cell or a human cell, using a vector.
  • the vector can be, for example, a plasmid or a virus.
  • delivery is in vivo, in vitro, ex vivo or in situ.
  • the virus is an adeno-associated virus (AAV), a lentivirus, an adenovirus.
  • a system or a component of a system is delivered to a cell together with a virus-like particle or a virion. In some embodiments, delivery uses more than one virus, virus-like particle or virion.
  • compositions and systems described herein can be formulated in liposomes or other similar vesicles.
  • Liposomes are spherical vesicle structures, which are composed of a monolayer or multilayer lipid bilayer around an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their loads across biomembranes and blood-brain barriers (BBB).
  • BBB blood-brain barriers
  • Vesicles can be made of several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers.
  • Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids).
  • U.S. Patent No. 6,693,086 which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids.
  • Extruded lipids can be prepared by extrusion through a filter with a reduced size.
  • Lipid nanoparticles are another example of carriers that provide a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein.
  • Nanostructured lipid carriers are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improving the stability and Drug loading, and prevent drug leakage.
  • Polymer nanoparticles are an important component of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release.
  • Lipopolymer nanoparticles (PLN) can also be used, which is a new type of carrier that combines liposomes and polymers. These nanoparticles have the complementary advantages of PNP and liposomes.
  • PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. In this way, these two components improve the drug encapsulation efficiency, promote surface modification, and prevent the leakage of water-soluble drugs.
  • the 5' untranslated sequence (5'UTR) in the donor RNA that binds to the retrotransposase is a natural sequence or a non-natural sequence.
  • the non-natural 5' untranslated sequence (5'UTR) has a sequence obtained by adding, deleting and/or replacing nucleotides relative to the natural 5'UTR sequence.
  • the 3' untranslated sequence (3'UTR) in the donor RNA that binds to the retrotransposase is a natural sequence or a non-natural sequence.
  • the non-natural 3' untranslated sequence (5'UTR) has a sequence obtained by adding, deleting and/or replacing nucleotides relative to the natural 3'UTR sequence.
  • the 5' untranslated sequence (5'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of any one of SEQ ID No. 7 to 12 or SEQ ID No. 44 to 55.
  • the 3' untranslated sequence (5'UTR) in the donor RNA to which the retrotransposase binds has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of any one of SEQ ID No. 13 to 18 or SEQ ID No. 56 to 67.
  • the non-natural 5' untranslated sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity with the nucleotide sequence of SEQ ID No.19-21.
  • the non-natural 3' untranslated sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of any one of SEQ ID No.22-23.
  • the present application also relates to the use of the above-mentioned non-native 3' untranslated sequence (3'UTR) and non-native 5' untranslated sequence (5'UTR).
  • the protein sequence of the retrotransposase of the present application is codon-optimized (human) and synthesized, and the DNA coding fragment is loaded between the XmaI and NheI restriction sites of the pCAG-SV40poly(A) vector by Gibson cloning.
  • the expression donor RNA plasmid is amplified by overlapping PCR method to contain multiple sequences of GFP(N)-intron-GFP(C) (Seq ID No.24), 5'-UTR (as described in any one of Seq ID No.7 to 12), 3'-UTR sequence (as described in any one of Seq ID No.13 to 18), and CMV promoter (Seq ID No.25), and finally the DNA coding fragment is loaded into the pSV40-mCherry vector by Gibson cloning to construct a plasmid expressing the donor RNA.
  • HEK293T cell line cells (sourced from ATCC) were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in a 24-well plate-cell culture dish (Corning) for 16 hours until the cell density reached 70%-90%.
  • DMEM Gibco
  • Gibco penicillin-streptomycin
  • Gibco 10% fetal bovine serum
  • Lipofectamine 3000 Invitrogen
  • 250 ng of a plasmid encoding a retrotransposase protein as described in any one of Seq ID No. 1 to 6
  • 250 ng of a plasmid expressing a donor RNA were transfected into each 24-well plate-cell culture dish.
  • This embodiment designs a reporter system that can accurately reflect whether the retrotransposase system of the present application can work in mammalian cells ( Figure 1).
  • the retrotransposase protein and the donor RNA are separately encoded by two plasmids, respectively, reflecting the modularity of the novel retrotransposase system.
  • the donor RNA is expressed by the CAG promoter. It is worth noting that the expression frame of the donor RNA initiated by the CAG promoter also contains a reverse expression frame initiated by the CMV promoter. In the expression frame initiated by the CMV promoter, CMV expresses GFP, but the GFP here is separated by an intron sequence inserted in the reverse direction.
  • the final mode of action of the system of the present application is: after the donor RNA is expressed by the CAG promoter, the intron sequence contained in the donor RNA is sheared off from the expressed RNA, and the GFP sequence in the expression frame expressed by the CMV promoter returns to normal at the RNA level.
  • the GFP sequence at this time is an antisense RNA chain and does not have the ability to translate GFP protein.
  • the reverse expression frame expressed by the CMV promoter can express normal GFP mRNA without introns, and then express GFP protein with green fluorescence. Therefore, by detecting the presence and proportion of the final GFP cells by fluorescence microscopy and flow cytometry, we can judge whether the new retrotransposase can play an effect in mammalian cells and how high the activity is.
  • Example 2 Novel retrotransposases are able to function in mammalian cells
  • the donor RNA of the novel retrotransposase system generally includes 5 parts, homology arm-left (Seq ID No. 26), 5'UTR sequence, 3'UTR sequence, a sequence between the 5'UTR sequence and the 3'UTR sequence carrying new gene information, and homology arm-right (Seq ID No. 27) ( Figure 1).
  • the protein polypeptide sequence of the novel retrotransposase tested in this example was codon-optimized (human) and synthesized, and the DNA coding fragment was loaded between the XmaI and NheI restriction sites of the pCAG-SV40poly(A) vector by Gibson cloning.
  • the expression donor RNA plasmid was amplified by overlapping PCR to amplify multiple sequences containing GFP sequence, intron sequence, 5'-UTR, 3'-UTR sequence, and CMV promoter, and finally the DNA coding fragment was loaded into the pSV40-mCherry vector by Gibson cloning to construct a plasmid expressing the donor RNA.
  • HEK293T cell line cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-well plates-cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%.
  • DMEM fetal bovine serum
  • Gibco penicillin-streptomycin
  • Gibco 10% fetal bovine serum
  • 250ng of plasmid encoding retrotransposase protein and 250ng of plasmid expressing donor RNA were transfected into each 24-well plate-cell culture dish. After transfection for 24 hours, cells were digested with trypsin-EDTA (0.05%) (Gibco).
  • Example 1 we used the reporter system constructed in Example 1 to test 33 different retrotransposase systems.
  • the sequences of the retrotransposases of some systems and the corresponding 5'UTR sequences and 3'UTR sequences are shown in Table 1 below.
  • 6 new systems had GFP signals that were significantly higher than the negative control ( Figure 2).
  • the protein sequences, 5'UTR and 3'UTR sequences of these 6 new retrotransposase systems (#3, #21, #23, #24, #31, #33) are shown in the sequence table.
  • Example 3 Use of donors containing non-native 5'UTR and/or non-native 3'UTR RNA boosts efficiency of new retrotransposase
  • this example tests the effects of multiple donor RNAs containing non-natural 5'UTR and/or non-natural 3'UTR on the efficiency of the novel retrotransposase (#21).
  • the donor RNA of the novel retrotransposase system generally contains 5 parts, homology arm-left (Seq ID No.26), 5'UTR sequence (Seq ID No.8, Seq ID No.19-21), 3'UTR sequence (Seq ID No.14, Seq ID No.22-23), a sequence between the 5'UTR sequence and the 3'UTR sequence carrying new gene information, and homology arm-right (Seq ID No.27)) ( Figure 1).
  • the protein polypeptide sequence of the novel retrotransposase tested in this example was codon optimized (human) and synthesized, and the DNA coding fragment was loaded between the XmaI and NheI restriction sites of the pCAG-SV40poly(A) vector by Gibson cloning.
  • the expression donor RNA plasmid was amplified by overlapping PCR method to contain multiple sequences of GFP(N)-intron-GFP(C), non-natural 5'-UTR, non-natural 3'-UTR sequence, and CMV promoter, and finally the DNA coding fragment was loaded into the pSV40-mCherry vector by Gibson cloning to construct a plasmid expressing donor RNA.
  • the cells of the HEK293T cell line were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in a 24-well plate-cell culture dish (Corning) for 16 hours until the cell density reached 70%-90%.
  • DMEM fetal bovine serum
  • 250ng of the plasmid encoding the retrotransposase protein and 250ng of the plasmid expressing the donor RNA were transfected into each 24-well plate-cell culture dish. After 24 hours of transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco).
  • the cells with mCherry signals were then sorted using a MoFlo XDP (Beckman Coulter) instrument and re-seeded back into a 12-well plate. After continuing to culture for 6 days, the cells were digested with trypsin-EDTA (0.05%) (Gibco) and then stained using a BD FACSAria TM Fusion Cell The ratio of cells with GFP positive signals was analyzed by Sorter (BD) instrument. The ratio of cells with GFP positive signals was compared with that of negative control and combined with the results observed under a fluorescence microscope to confirm whether the new retrotransposase system can function in mammalian cells.
  • BD Sorter
  • Example 4 The novel retrotransposase can amplify the corresponding DNA writing fragment
  • FIG. 7 shows the PCR amplification results of different retrotransposases at the junction of the 3' end of the integrated DNA sequence and the genome (inside the 28s rDNA gene) in mammalian cells.
  • the donor RNA of the novel retrotransposase system generally comprises five parts, homology arm-left (Seq ID No. 26), 5'UTR sequence, 3'UTR sequence, a sequence between the 5'UTR sequence and the 3'UTR sequence carrying new gene information, and homology arm-right (Seq ID No. 27) ( Figure 1).
  • the protein polypeptide sequence of the novel retrotransposase tested in this example was codon optimized (human) and synthesized, and the DNA coding fragment was loaded between the XmaI and NheI restriction sites of the pCAG-SV40poly(A) vector by Gibson cloning.
  • the expression donor RNA plasmid was amplified by overlapping PCR to amplify multiple sequences containing GFP sequence, intron sequence, 5'-UTR, 3'-UTR sequence, and CMV promoter, and finally the DNA coding fragment was loaded into the pSV40-mCherry vector by Gibson cloning to construct a plasmid expressing the donor RNA.
  • the cells of the HEK293T cell line were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plate-cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%.
  • DMEM fetal bovine serum
  • 250 ng of the plasmid encoding the retrotransposase protein and 250 ng of the plasmid expressing the donor RNA were transfected into each 24-well plate-cell culture dish. After 24 hours of transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco).
  • the cells were then sorted using a MoFlo XDP (Beckman Coulter) instrument.
  • the cells with mCherry signal were re-seeded into 12-well plates and cultured for 6 days before being digested with trypsin-EDTA (0.05%) (Gibco).
  • the left side is the experimental group that was transfected with plasmids expressing the donor and the retrotransposase protein at the same time
  • the right side is the control group that was transfected with only the donor plasmid.
  • the black triangle indicates the corresponding PCR-amplified positive fragment.
  • some of the retrotransposase systems are able to amplify the corresponding written fragments.
  • Example 5 A novel retrotransposase can achieve integration of the GFP gene
  • FIG. 8 shows the results of PCR amplification of sequences spanning both ends of introns of integration sequences in mammalian cells using different retrotransposase systems.
  • the donor RNA of the novel retrotransposase system generally comprises five parts, homology arm-left (Seq ID No. 26), 5'UTR sequence, 3'UTR sequence, a sequence between the 5'UTR sequence and the 3'UTR sequence carrying new gene information, and homology arm-right (Seq ID No. 27) ( Figure 1).
  • the protein polypeptide sequence of the novel retrotransposase tested in this example was codon-optimized (human) and synthesized, and the DNA coding fragment was loaded into the pCAG-SV40 poly (A) vector between the XmaI and NheI restriction sites by Gibson cloning.
  • the expression donor RNA plasmid containing the GFP sequence, intron sequence, 5'-UTR, 3'-UTR sequence, CMV promoter sequence was cloned by overlapping PCR. Multiple sequences of the promoter were amplified separately, and finally the DNA coding fragments were loaded into the pSV40-mCherry vector using Gibson cloning to construct a plasmid expressing the donor RNA.
  • Cells of the HEK293T cell line were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). The cells were seeded in 24-well plates-cell culture dishes (Corning) for 16 hours until the cell density reached 70%-90%.
  • DMEM fetal bovine serum
  • Lipofectamine3000 Invitrogen
  • 250 ng of plasmid encoding retrotransposase protein and 250 ng of plasmid expressing donor RNA were transfected into each 24-well plate-cell culture dish. After 24 hours of transfection, the cells were digested with trypsin-EDTA (0.05%) (Gibco).
  • the cells with mCherry signal were then sorted using a MoFlo XDP (Beckman Coulter) instrument and re-seeded back into 12-well plates. After 6 days of continuous culture, the cells were digested with trypsin-EDTA (0.05%) (Gibco).
  • Figure 8 shows that in the two channels of each group of gel images, the left side is the experimental group that simultaneously transfected the plasmid expressing the donor and the retrotransposase protein, and the right side is the control group that only transfected the expression of the donor plasmid.
  • some retrotransposase systems proteins #3, #4, #5, #8, #10, #11, #14, #21, #25, #29, #31, #32) can amplify the corresponding written fragments.
  • #3, #4, #5, #8, #10, #11, #14, #17, #21, #24, #25, #27, #29, #31, and #32 proteins can achieve site-specific integration of DNA in mammalian cells in combination with their respective donors.
  • the fluorescence flow sorting experiment combined with GFP shows that #3, #21, #23, #24, #31, and #33 can achieve complete integration of the GFP gene.
  • Example 6 Exploring the activity of truncated retrotransposase protein in mammalian cells
  • the sequence of the retrotransposase protein was aligned using the mafft software (muscle software, Clustal software, and blast software also have similar functions), and then the needle software (blast software also has similar functions) was used to calculate the similarity between proteins.
  • the retrotransposase protein was predicted using the InterPro website (hhpred website, NCBI CDD website, psi-blast software, blastp software, and hh-suite software also have similar functions).

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Abstract

La présente demande a trait à une rétrotransposase. La rétrotransposase comprend un domaine de liaison à l'ADN cible contenant un motif de liaison à doigt de zinc, un domaine de transcriptase inverse et un domaine d'endonucléase, et peut transcrire de manière inverse un ARN dans un ADN. Une séquence d'acides aminés de la rétrotransposase selon la présente demande est telle que représentée dans l'une quelconque des SEQ ID NO : 1-6, ou SEQ ID NO : 32-43, ou SEQ ID NO : 68-71, ou a au moins 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, ou 99% d'identité avec la séquence d'acides aminés dans l'une quelconque des SEQ ID NO : 1-6, ou SEQ ID NO : 32-43, ou SEQ ID NO : 68-71. La présente demande concerne en outre un système de modification d'ADN. Le système comprend : la rétrotransposase selon la présente demande ou un acide nucléique codant pour la rétrotransposase selon la présente demande ; et un ARN donneur ou un acide nucléique codant pour l'ARN donneur, l'ARN donneur comprenant une séquence et une séquence hétérologue qui se lient à la séquence hétérologue.
PCT/CN2023/139871 2022-12-19 2023-12-19 Système permettant d'insérer un grand fragment d'adn dans un génome Ceased WO2024131786A1 (fr)

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WO2021178717A2 (fr) * 2020-03-04 2021-09-10 Flagship Pioneering Innovations Vi, Llc Procédés et compositions améliorés pour moduler un génome
WO2021178720A2 (fr) * 2020-03-04 2021-09-10 Flagship Pioneering Innovations Vi, Llc Procédés et compositions pour moduler un génome
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CN113286880A (zh) * 2018-08-28 2021-08-20 旗舰先锋创新Vi有限责任公司 调控基因组的方法和组合物
CN114981409A (zh) * 2019-09-03 2022-08-30 美洛德生物医药公司 用于基因组整合的方法和组合物
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