WO2021136408A1 - 一种基于leaper技术治疗mps ih的方法和组合物 - Google Patents

一种基于leaper技术治疗mps ih的方法和组合物 Download PDF

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WO2021136408A1
WO2021136408A1 PCT/CN2020/141506 CN2020141506W WO2021136408A1 WO 2021136408 A1 WO2021136408 A1 WO 2021136408A1 CN 2020141506 W CN2020141506 W CN 2020141506W WO 2021136408 A1 WO2021136408 A1 WO 2021136408A1
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arrna
seq
target
rna
idua
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PCT/CN2020/141506
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English (en)
French (fr)
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袁鹏飞
赵艳霞
刘能银
易泽轩
汤刚斌
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博雅辑因（北京）生物科技有限公司
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Priority to CR20220365A priority Critical patent/CR20220365A/es
Application filed by 博雅辑因（北京）生物科技有限公司 filed Critical 博雅辑因（北京）生物科技有限公司
Priority to CN202080086708.7A priority patent/CN114846139A/zh
Priority to CA3163272A priority patent/CA3163272A1/en
Priority to US17/790,487 priority patent/US20230060518A1/en
Priority to PE2022001378A priority patent/PE20230037A1/es
Priority to IL294201A priority patent/IL294201A/en
Priority to JP2022540985A priority patent/JP2023509179A/ja
Priority to MX2022008190A priority patent/MX2022008190A/es
Priority to KR1020227025378A priority patent/KR20220119129A/ko
Priority to EP20908810.3A priority patent/EP4086345A4/en
Priority to AU2020418228A priority patent/AU2020418228A1/en
Publication of WO2021136408A1 publication Critical patent/WO2021136408A1/zh
Priority to CONC2022/0010432A priority patent/CO2022010432A2/es

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Definitions

  • This application belongs to the field of gene editing therapy. Specifically, it relates to a method for targeted editing of RNA to treat MPS IH based on LEAPER (Leveraging Endogenous ADAR for Programmable Editing on RNA) technology, which includes the use of LEAPER technology to perform RNA transfer from A to I. Site-directed editing of the base in vivo to treat diseases caused by G>A mutations, such as MPS IH.
  • LEAPER Leveraging Endogenous ADAR for Programmable Editing on RNA
  • Hurler syndrome also known as Mucopolysaccharidoses IH (MPS IH), or Mucopolysaccharidoses IH
  • MPS IH Mucopolysaccharidoses IH
  • IH/S ⁇ -L-iduronidase
  • AR Chromosomal recessive genetic disease
  • the root cause of Heller’s syndrome is the mutation of the IDUA gene located on human chromosome 4 in 4p16.3, which encodes the IDUA protein. So far, there are more than 200 pathogenic mutations, of which one is the most common type.
  • ⁇ -L-iduronidase is responsible for the degradation of Glycosaminoglycans (GAGs) in cell lysosomes.
  • GAGs Glycosaminoglycans
  • Patients with Hurler syndrome vary greatly among individuals. They can be normal at birth. The earliest sign at 3-6 months is rough facial contours, followed by symptoms such as frontal bone protrusion, skeletal deformities, growth arrest, and language disorders. Usually live less than 10 years old.
  • ERT enzyme replacement therapy
  • HSCT hematopoietic stem cell transplantation
  • HSCT hematopoietic stem cell transplantation as therapy for Hurler Syndrome.Tolar,J(2008).Bone marrow transplantation.41.531-5.10.1038/sj.bmt.1705934).
  • the principle of the gene editing technology under study for the treatment of Heller’s syndrome is to use zinc finger nuclease (ZFN) and adeno-associated virus (AAV) to encode the cDNA sequence of the normal IDUA protein.
  • ZFN zinc finger nuclease
  • AAV adeno-associated virus
  • CRISPR genome editing technology
  • Cas9 CRISPR
  • CRISPR-based DNA editing technology must express Cas9 or other nucleases with similar functions through exogenous expression, causing the following problems.
  • nucleases that usually require exogenous expression usually have a relatively large molecular weight, which makes the efficiency of their delivery into the body through a viral vector drastically reduced.
  • nuclease due to the exogenous expression of nuclease, there is a potential for off-target nuclease, which will make its application have a potential carcinogenic risk.
  • the exogenously expressed nuclease is found in bacteria, rather than naturally occurring in humans or mammals, which makes it possible to cause an immune response in the patient's body, which may cause damage to the patient himself on the one hand, on the other hand It may also neutralize the exogenously expressed nuclease, thereby losing its proper activity, or hindering further intervention and treatment.
  • RNA editing technology called REPAIR (RNA Editing for Programmable A to I Replacement) (RNA editing with CRISPR-Cas13, Cox et al., 2017), which uses exogenous expression Cas13-ADAR fusion protein and single guide RNA (sgRNA) can also achieve A to I editing of target RNA, but this method, like CRISPR technology, still requires the expression of foreign protein. Unable to solve the problems caused by the expression of foreign proteins.
  • RESTORE nucleic acid editing technology Recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing, Merkle et al., 2019.
  • This technology can get rid of the dependence on foreign proteins.
  • RESTORE technology needs to have high editing efficiency under the premise of IFN- ⁇ , and IFN- ⁇ is a key factor that determines the development and severity of autoimmunity (Interferon- ⁇ and systemic autoimmunity, Pollard et al., 2013, This makes the application of the technology in the medical field greatly reduced.
  • a guide RNA is also used in the RESTORE technology, and the guide RNA used is a chemically synthesized oligonucleotide, and the synthesized oligonucleotide A large number of chemical modifications need to be artificially introduced to ensure its stability.
  • the dRNA contains a complementary RNA sequence that hybridizes with the target RNA, and the dRNA can recruit adenosine deaminase (ADAR) that acts on the RNA to deaminate the target adenosine (A) in the target RNA.
  • ADAR adenosine deaminase
  • This application provides a brand new G to A mutation in the IDUA pathogenic gene of Hurler syndrome, especially the mutation with the highest proportion (NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter))
  • the technical solution enables it to accurately edit the mutation site on the target RNA.
  • this application provides at least the following technical solutions:
  • a method for targeted editing of target RNA in target cells based on LEAPER technology wherein the target RNA is an RNA containing a G to A mutation in the IDUA gene transcript, and the method includes:
  • RNA adenosine deaminase recruiting RNA
  • arRNA adenosine deaminase recruiting RNA
  • the arRNA includes a complementary RNA sequence that hybridizes with the target RNA
  • ADAR adenosine deaminase
  • the arRNA introduces base C paired with target A. In some embodiments, the arRNA introduces base A that is paired with target A. In some embodiments, the arRNA introduces base U paired with target A. 3. The method according to any one of items 1-2, wherein the arRNA is about 151-61 nt, 131-66 nt, 121-66 nt, 111-66 nt, 91-66 nt or 81-66 nt in length. This application discloses and covers any natural number within the stated number range.
  • RNA is an RNA containing a mutation site of NM_000203.4 (IDUA)-c.1205G-A (p.Trp402Ter).
  • arRNA comprises the following sequence: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17. SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 34.
  • arRNA comprises a sequence selected from the group consisting of SEQ ID NO: 44 or SEQ ID NO: 52.
  • the first 3 and last 3 nucleotides of the sequence were modified by 2 ⁇ -OMe,
  • the first 3 and last 3 internucleotide linkages are all phosphorothioate linkages
  • the 3'nearest neighbor of the target base is 2'-OMe modified A
  • the 5'nearest neighbor of the targeted base is 2'-OMe modified C
  • the target base and its 3’ nearest neighbor base and 5’ nearest neighbor base are respectively connected by phosphorothioate bonds
  • the first 5 and last 5 nucleotides are modified by 2 ⁇ -OMe, and
  • the first 5 and last 5 nucleotide linkages are phosphorothioate linkages.
  • the chemical modification is selected from one or more of the following:
  • CM1 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time, all U in the sequence are 2 ⁇ -OMe. ⁇ -OMe modification;
  • CM2 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 3'of the base is targeted The nearest neighbor base is 2 ⁇ -OMe modified A;
  • CM3 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 5'of the base is targeted The nearest neighbor base is 2 ⁇ -OMe modified C;
  • CM4 The first 3 and last 3 nucleotides of the sequence are modified by 2'-OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the target base and its 3' The nearest neighbor base and the 5'nearest neighbor base are respectively connected by phosphorothioate bonds; and
  • CM6 The first 5 and last 5 nucleotides of the sequence are modified by 2'-OMe respectively, and the connections between the first 5 and last 5 nucleotides are phosphorothioate linkages. 13. The method according to any one of items 1-9, wherein the construct encoding the arRNA is a linear nucleic acid strand, a viral vector or a plasmid.
  • the viral vector is an adeno-associated virus (AAV) vector or a lentiviral expression vector.
  • AAV adeno-associated virus
  • the target cells include hepatocytes or fibroblasts.
  • An arRNA or its coding sequence for targeted editing of target RNA in target cells by LEAPER technology comprising any of the following sequences or consisting of any of the following sequences: SEQ ID NO: 14, SEQ ID NO: 15. SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 44 or SEQ ID NO: 52.
  • composition, preparation, kit or biological product comprising the arRNA described in Item 18 or its coding sequence, or the plasmid, viral vector, liposome or lipid nanoparticle described in Item 19.
  • a method for treating MPS IH in an individual comprising using the method described in any one of items 1-17 to correct the G to A mutations in target cells of the individual that are associated with MPS IH disease.
  • the application also relates to the use of a sequence selected from the following in the preparation of drugs for the treatment of MPS IH disease: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 13 , SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 44, or SEQ ID NO: 52.
  • RNA in target cells such as hepatocytes or fibroblasts
  • accurately repair disease-causing mutation sites such as NM_000203.4 (IDUA)-c.1205G-A(p.Trp402Ter) mutation restores the normal expression of the protein encoded by RNA in the body, achieving the purpose of treating MSP and IH.
  • Figure 1 shows the detection of IDUA genotype on GM06214 cells.
  • FIG. 1 shows the test of cell electrotransfection conditions.
  • Figure 3A-B shows the design of arRNA for IDUA pre-mRNA and mRNA to test the cell function after editing; and the design of arRNA for IDUA pre-mRNA and mRNA to test the cell editing efficiency.
  • Figure 4A shows the design of the IDUA-reporter cell line; and Figure 4B shows the editing efficiency of detecting different lengths of arRNA (symmetrically truncated) on the 293T-IDUA-Reporter.
  • Figures 5A-B show the detection of enzyme activity and editing efficiency at different time points after transfection of different lengths (symmetrically truncated) arRNA on GM06214 cells.
  • Figure 6 shows the detection of IDUA enzyme activity and editing efficiency after arRNA (symmetric truncation, 3 ⁇ truncation and 5 ⁇ truncation) was transfected with lipofectmine RNAiMAX in GM06214 cells.
  • Figure 7A shows that the arRNA targeting the human IDUA mutation site is preferably between 55-c-25 and 55-c-10, and the bases are reduced one by one at the 3'end, and the best is selected by enzyme activity detection using GM06214 cells.
  • length. 7B shows that the arRNA targeted to the IDUA mutation site of the mouse is between 55-c-55 and 55-c-10, gradually decreasing every 5 bases at the 3'end, using MPSI mouse MEF cells (MSPI MEF (MSPI mouse embryo fibroblast)) Select the best arRNA length sequence through enzyme activity detection.
  • MSPI MEF MSPI mouse embryo fibroblast
  • Figure 8A shows that under the preferred two 3 ⁇ end lengths, the 5 ⁇ end length is gradually shortened to screen out the optimal length range.
  • Figure 8B shows that after the 3 ⁇ end length is fixed to 14 nt, the 5 ⁇ end is truncated base by base.
  • the arRNA formed after a short period of time affects the enzyme activity, and the optimal arRNA length is screened by combining Figures 8A and 8B.
  • Figure 9 shows a comparison of the effects of different chemical modifications on arRNA editing efficiency (represented by enzyme activity) under the preferred two arRNA lengths.
  • Figures 10A-D show the editing efficiency and the ability of human and mouse arRNA to edit IDUA mRNA and the ability to produce a functional IDUA protein after editing under different arRNA concentrations under a combination of preferred lengths and preferred chemical modification methods.
  • Figures 11A-D show the IDUA enzyme activity measured in human or murine cells at different times after a single transfection under the combination of the preferred length and the preferred chemical modification method.
  • Figures 12A-B show the editing efficiency of targeted sites achieved by delivering arRNA in different ways in primary liver cells of humans and mice.
  • Figure 13 shows the editing efficiency of arRNA targeting IDUA delivered by LNP on primary cultured human and mouse liver cells.
  • Figure 14 shows the editing efficiency of IDUA in mouse liver cells after the screened arRNA (SEQ ID NO: 52) targeting mouse IDUA mutations is packaged into LNP and administered to mice via the tail vein at different concentrations for 24hrs .
  • RNA editing refers to a natural process that exists in eukaryotic cells. RNA editing is the editing from base A (adenine) to I (hypoxanthine) that occurs at the RNA level after DNA transcription and before protein translation. , Hypoxanthine is recognized as G during translation, and the editing of A to I in RNA diversifies the transcriptome. Through site-specific and precise changes to RNA molecules, the total amount of RNA is increased several times. This editing is catalyzed by ADAR (Adenosine Deaminase Acting on RNA) protease, and is called site-directed RNA editing. Editing can occur in coding regions including intron and exon sequences, and can also occur in non-coding regions. Editing in coding regions can redefine protein coding sequences.
  • ADAR AdAR
  • LEAPER technology refers to a technology for RNA editing by using engineered RNA to recruit endogenous ADAR, and refers to the RNA editing technology as reported in WO2020074001A1.
  • adenosine deaminase refers to a type of adenosine deaminase that is widely expressed in various tissues of eukaryotes (including humans and other mammals), which can catalyze adenosine in RNA molecules. Conversion of A to Inosine I. In the process of protein synthesis in eukaryotes, I is usually interpreted as G for translation.
  • the "complementarity" of a nucleic acid refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10). These are respectively about 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary” means that all consecutive residues of the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence.
  • substantially complementary means that within a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, at least about 70%, 75%, 80
  • base or a single nucleotide according to the Watson-Crick base pairing principle, when A is paired with T or U, and C is paired with G or I, it is called complement or match, and vice versa; and other bases Base pairing is called non-complementarity or mismatch.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of nucleotide residues.
  • the hydrogen bonding can occur through Watson Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • a sequence that can hybridize to a given sequence is called the "complementary sequence" of the given sequence.
  • electrotransfection refers to electroporation transfection technology, which temporarily forms small holes or openings in the cell membrane after an electric field is applied to cells for a few microseconds to a few milliseconds to deliver large molecules such as DNA to the cells.
  • liposome transfection refers to a transfection technique that uses liposomes as delivery vehicles in vivo and in vitro. Liposomes include neutral liposomes and cationic liposomes.
  • the neutral liposomes use lipid membranes to encapsulate macromolecules, such as nucleic acids, and deliver the macromolecules into the cell membrane by means of lipid membranes; cationic lipids
  • the body is positively charged, and the transferred macromolecules are not pre-embedded in it, but because the macromolecules themselves are negatively charged, they automatically bind to the positively charged liposomes, forming a macromolecule-cation liposome complex
  • the substance, which adsorbs to the negatively charged cell membrane surface, is delivered into the cell through endocytosis.
  • lipid-nanoparticle (LNP) delivery refers to the transmembrane delivery of macromolecules, such as nucleic acids, proteins, etc., into cells through lipid nanoparticles.
  • lipid nanoparticles refer to particles synthesized by mixing two phases, including an ethanol phase containing ionizable lipids, auxiliary phospholipids, cholesterol, and PEG lipids, and an acidic aqueous phase containing macromolecules such as nucleic acids and proteins.
  • LNP encapsulated with RNA can enter the cytoplasm through endocytosis.
  • the NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter) mutation refers to the mutation from G to A at position 1205 in the transcript of IDUA gene number 000203.4, which causes the transcript
  • the coding sequence of tryptophan (Trp) at position 402 of the translated peptide chain is converted into a stop codon (Ter), so that all amino acids after position 402 are deleted in the final translated amino acid, thereby losing the enzymatic activity of IDUA.
  • Patients with this mutation will affect the degradation of glucosaminoglycan in the cell lysosome due to the lack of active ⁇ -L-iduronidase, and eventually cause teratogenic or even death to the patient.
  • the scheme in this application can restore the activity of IDUA enzyme by reversing this mutation at the transcription level.
  • target RNA refers to a target RNA to be edited, which contains adenosine (A) to be edited.
  • the target RNA may be mature mRNA or mRNA precursor. In this application, mRNA precursors are more preferred.
  • the cell containing the "target RNA” is referred to as the target cell.
  • the adenosine to be edited is called “target base”, “target adenosine” or “target A”. In this application, “target base”, “target adenosine” or “target A” can be used interchangeably.
  • the base adjacent to the target adenosine at the 5'end of the target RNA is called the "5' adjacent base”; the base adjacent to the target adenosine at the 3'end of the target RNA is called the “3' adjacent base” Base”; the base triplet composed of the target base and its 3'and 5'adjacent bases is referred to herein as the "target base triplet”.
  • target base the base opposite to the target base on arRNA
  • the base adjacent to the target base at the 5'end of arRNA is called "5".
  • the base triplet of is referred to herein as the "targeted base triplet".
  • the term "construct” refers to a nucleic acid vector containing a certain nucleic acid sequence
  • the nucleic acid vector can be a linear nucleic acid molecule, a plasmid, a viral vector, or the like.
  • the nucleic acid molecule may be single-stranded or double-stranded.
  • the nucleic acid sequence may be a DNA sequence or an RNA sequence. In some embodiments, the nucleic acid sequence directly performs its function without being transcribed, translated, or expressed. In some embodiments, the nucleic acid sequence is a DNA sequence, which functions as an RNA molecule after being transcribed to form RNA.
  • the nucleic acid sequence is RNA, which functions as a polypeptide or protein after translation.
  • the nucleic acid sequence is DNA, which functions as a protein after forming a protein through the steps of transcription and translation.
  • the construct can be packaged into a target cell in the form of virus, lipid nanoparticles or exosomes, or it can enter the target cell by means of electrotransformation, microinjection, chemical transformation and the like.
  • the term “delivery” refers to the introduction of biological macromolecules such as nucleic acids and proteins into the cell membrane from outside the cell membrane through certain channels.
  • the “delivery” is, for example, electrotransfection, liposome transfection, lipid-nanoparticle delivery, virus delivery, exosomal delivery, and the like.
  • modification refers to changing the composition or structure of a nucleic acid or protein by chemical or biological methods, such as genetic engineering methods, so that one or more of the characteristics or functions of the nucleic acid or protein are changed.
  • the present application provides a method for targeted editing of IDUA target RNA containing G to A mutations in target cells based on LEAPER technology, which includes: recruiting RNA (arRNA) or encoding adenosine deaminase containing adenosine deaminase for editing target RNA
  • the arRNA construct is delivered to the target cell, wherein the arRNA includes a complementary RNA sequence that hybridizes with the target RNA, and wherein the arRNA can recruit adenosine deaminase (ADAR) that acts on RNA to make
  • the target adenosine (A) in the target RNA is deaminated.
  • the target RNA is a mRNA precursor.
  • the target RNA is mature mRNA. In some embodiments, the target RNA is an IDUA target RNA transcribed with a mutation site containing NM_000203.4 (IDUA)-c.1205G-A (p.Trp402Ter).
  • the arRNA includes bases C, A, U, or G that pair with target A.
  • the preferred sequence of the bases paired with target A is C, A, U, G. That is, when the length of the arRNA is the same, the distance between the target base and the 5'end is the same, the distance between the target base and the 3'end is the same, and the arRNA sequence except the target base is identical, the preferred base is The order is C>A>U>G.
  • the arRNA can be expressed as X nt-cY nt, where X indicates that the distance between the target base and the 5'end of the arRNA is X nt, and Y indicates the target base The distance between the base and the 3'end of the arRNA is Y nt, where X and Y can represent any natural numbers.
  • the target cell is a eukaryotic cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cells are hepatocytes or fibroblasts. In some embodiments, the target cell is a human or mouse cell.
  • the arRNA is about 151-61 nt, 131-66 nt, 121-66 nt, 111-66 nt, 91-66 nt, or 81-66 nt in length.
  • the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15n or 24nt-11nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt.
  • the length of the target base from the 3'end refers to the number of all bases from the 3'nearest neighbor base of the target base to the 3'most terminal base; the target base distance is 5'
  • the length of the end refers to the number of all bases from the 5'nearest base to the 5'most terminal base of the targeted base.
  • the target cell is a human cell
  • the target RNA is an IDUA target RNA transcribed with a mutation site of NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)
  • the full length of the arRNA is greater than or equal to 66 nt, such as about 121-66 nt, 111-66 nt, 101-66 nt, 91-66 nt, or 81-66 nt in length, that is, the full length of the arRNA is selected from any natural number in the above-mentioned length range, for example : 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86
  • the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15nt or 24nt-11nt, which is the target base distance of the arRNA
  • the distance from the 3'end is selected from any natural number in the range of the above-mentioned target base to the 3'end, for example: 12nt, 13nt, 14nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt, which is the target base distance of the arRNA
  • the distance from the 5'end is selected from any natural number in the range of the above-mentioned target base to the 5'end, for example: 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt.
  • the arRNA includes a sequence selected from: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34.
  • the target cell is a mouse cell (eg, W392X mouse cell), and wherein the target RNA is a target RNA transcribed with an IDUA mutation corresponding to the human W402X mutation. In some embodiments, the target cell is a W392X mouse cell.
  • the arRNA is about 121-53 nt, 111-61 nt, 101-61 nt, 91-61 nt, 81-61 nt, 111-66 nt, or 105-66 nt in length, that is, the full length of the arRNA is selected from the above Any natural number in the length range, for example: 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 95nt, 100nt, 110nt, 115nt, 120nt.
  • the length of the target base in the arRNA from the 3'end is 55nt-10nt or 50nt-10nt, that is, the distance between the target base of the arRNA and the 3'end is selected from the above-mentioned target base distance Any natural number in the 3'end of the length range, for example: 11nt, 12nt, 13nt, 14nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31nt , 32nt, 33nt, 34nt, 35nt, 35nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, 50nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt, which is the target base distance of the arRNA
  • the distance from the 5'end is selected from any natural number in the range of the above-mentioned target base to the 5'end, for example: 33nt, 36nt, 47nt, 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt, 60nt, 65nt , 75nt.
  • the arRNA comprises a sequence selected from: SEQ ID NO: 44 or SEQ ID NO: 52.
  • the arRNA is chemically modified.
  • the chemical modification is 2-O'-methylation and/or phosphorothioate modification.
  • the chemical modification is selected from one or more of the following:
  • the first 3 and last 3 nucleotides of the sequence were modified by 2 ⁇ -OMe,
  • the first 3 and last 3 internucleotide linkages are all phosphorothioate linkages
  • the 3'nearest neighbor of the target base is 2'-OMe modified A
  • the 5'nearest neighbor of the targeted base is 2'-OMe modified C
  • the target base and its 3’ nearest neighbor base and 5’ nearest neighbor base are respectively connected by phosphorothioate bonds
  • the first 5 and last 5 nucleotides are modified by 2 ⁇ -OMe, and
  • the first 5 and last 5 nucleotide linkages are phosphorothioate linkages.
  • a construct encoding the arRNA is a construct containing the arRNA coding sequence.
  • the arRNA is transcribed from the construct encoding the arRNA after the construct encoding the arRNA is delivered into the target cell.
  • the construct encoding the arRNA is a linear nucleic acid strand, a viral vector or a plasmid.
  • the viral vector is an adeno-associated virus (AAV) or a lentivirus.
  • the construct encoding the arRNA when the construct encoding the arRNA is delivered into the target cell, it inserts the sequence encoding the arRNA into the target cell genome through homologous recombination or non-homologous recombination, so as to continue Transcription produces the arRNA.
  • the sequence encoding the arRNA when the construct encoding the arRNA is delivered into the target cell, the sequence encoding the arRNA is present in the target cell as part of free nucleic acid, so that the sequence encoding the arRNA The arRNA can be transcribed within a certain period of time.
  • the delivery is electrotransfection, liposome transfection, or lipid-nanoparticle (LNP) delivery or infection.
  • the delivery when the target cell is a liver cell, the delivery is LNP delivery.
  • the delivery when the target cell is a fibroblast, the delivery is liposome transfection.
  • the delivery concentration of the arRNA is ⁇ 2.5-5 nM, preferably ⁇ 10-20 nM, such as ⁇ 15 nM. In the present application, the delivery concentration refers to the amount of arRNA contained in one volume unit of arRNA and the delivery system where the target cell is located when the arRNA construct is delivered to the target cell.
  • the delivery system includes arRNA or a construct thereof, target cells, and a liquid matrix surrounding the arRNA and the target cells.
  • the liquid matrix may be a cell culture medium, PBS, or other solution that can maintain a stable survival state of cells for a certain period of time and is isotonic with the cytoplasm.
  • the delivery system further includes an agent that can facilitate delivery.
  • This application also provides an arRNA, which can be used for targeted editing of target RNA in target cells based on LEAPER technology, such as transcribed with NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter) mutation site
  • the target RNA so that the target A in the target RNA is deaminated is called hypoxanthine I.
  • I will be recognized as G, so that the G>A mutation is restored to G, so that the target RNA can be translated into the correct protein after arRNA editing.
  • the target RNA is a mRNA precursor.
  • the target RNA is mature mRNA.
  • the editing efficiency of the bases (target bases) that the arRNA introduces to pair with the target A is C, A, U, G from large to small.
  • other bases can be complementary paired with the target RNA.
  • one or more bases in the arRNA form a mismatch with the target RNA.
  • the target cell is a eukaryotic cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cells are hepatocytes or fibroblasts. In some embodiments, the target cell is a human or mouse cell (e.g., W392X mouse cell).
  • the arRNA is about 151-61 nt, 131-66 nt, 121-66 nt, 111-66 nt, 91-66 nt, or 81-66 nt in length.
  • the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15n or 24nt-11nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt.
  • the length of the target base from the 3'end refers to the number of all bases from the 3'nearest neighbor base of the target base to the 3'most terminal base; the target base distance is 5'
  • the length of the end refers to the number of all bases from the 5'nearest base to the 5'most terminal base of the targeted base.
  • the target cell is a human cell
  • the target RNA is an IDUA target RNA transcribed with a mutation site of NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)
  • the full length of the arRNA is greater than or equal to 66 nt, such as about 121-66 nt, 111-66 nt, 101-66 nt, 91-66 nt, or 81-66 nt in length, that is, the full length of the arRNA is selected from any natural number in the above-mentioned length range, for example : 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86
  • the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15nt or 24nt-11nt, which is the target base distance of the arRNA
  • the distance from the 3'end is selected from any natural number in the range of the above-mentioned target base to the 3'end, for example: 12nt, 13nt, 14nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt, which is the target base distance of the arRNA
  • the distance from the 5'end is selected from any natural number in the range of the above-mentioned target base to the 5'end, for example: 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt.
  • the arRNA includes a sequence selected from: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34.
  • the target cell is a mouse cell (such as a W392X mouse cell), and wherein the target RNA is a target RNA transcribed with an IDUA mutation corresponding to the human W402X mutation, then the arRNA is about 121-53nt, 111-61nt, 101-61nt, 91-61nt, 81-61nt, 111-66nt, or 105-66nt, that is, the full length of the arRNA is selected from any natural number in the above-mentioned length range, for example: 67nt, 68nt , 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90n
  • the length of the target base in the arRNA from the 3'end is 55nt-10nt or 50nt-10nt, that is, the distance between the target base of the arRNA and the 3'end is selected from the above-mentioned target base distance Any natural number in the 3'end of the length range, for example: 11nt, 12nt, 13nt, 14nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31nt , 32nt, 33nt, 34nt, 35nt, 35nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, 50nt.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt or 55nt-45nt, which is the target base distance of the arRNA
  • the distance from the 5'end is selected from any natural number in the range of the above-mentioned target base to the 5'end, for example: 33nt, 36nt, 47nt, 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt, 60nt, 65nt , 75nt.
  • the arRNA comprises a sequence selected from: SEQ ID NO: 44 or SEQ ID NO: 52.
  • the arRNA is transcribed and expressed from a construct encoding the arRNA. In some embodiments, the arRNA is transcribed and expressed in vitro from a construct encoding the arRNA, and is obtained by purification. In some embodiments, the arRNA is directly expressed in vivo by the construct encoding the arRNA and exerts an editing function. In some embodiments, the construct is selected from viral vectors, plasmids, and linear nucleic acids. In some embodiments, the virus is AAV or lentivirus.
  • the arRNA is chemically synthesized. In some embodiments, the arRNA is chemically modified. In some embodiments, the chemical modification is 2-O'-methylation and/or phosphorothioate modification. In some embodiments, the chemical modification is selected from one or more of the following:
  • the first 3 and last 3 nucleotides of the sequence were modified by 2 ⁇ -OMe,
  • the first 3 and last 3 internucleotide linkages are all phosphorothioate linkages
  • the 3'nearest neighbor of the target base is 2'-OMe modified A
  • the 5'nearest neighbor of the targeted base is 2'-OMe modified C
  • the target base and its 3’ nearest neighbor base and 5’ nearest neighbor base are respectively connected by phosphorothioate bonds
  • the first 5 and last 5 nucleotides are modified by 2 ⁇ -OMe, and
  • the first 5 and last 5 nucleotide linkages are phosphorothioate linkages.
  • the chemical modification is selected from one or more of the following:
  • CM1 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time, all U in the sequence are 2 ⁇ -OMe. ⁇ -OMe modification;
  • CM2 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 3'of the base is targeted The nearest neighbor base is 2 ⁇ -OMe modified A;
  • CM3 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 5'of the base is targeted The nearest neighbor base is 2 ⁇ -OMe modified C;
  • CM4 The first 3 and last 3 nucleotides of the sequence are modified by 2'-OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the target base and its 3' The nearest neighbor base and the 5'nearest neighbor base are respectively connected by phosphorothioate bonds; and
  • CM6 The first 5 and last 5 nucleotides of the sequence are modified by 2'-OMe respectively, and the connections between the first 5 and last 5 nucleotides are phosphorothioate linkages.
  • the application also provides a construct encoding the aforementioned arRNA.
  • the construct is selected from viral vectors, plasmids, and linear nucleic acids.
  • the viral vector is an AAV vector or a lentiviral expression vector.
  • the application further provides viruses including the constructs, up to nanoparticles, liposomes, exosomes or cells.
  • compositions, preparations and biological products containing any of the foregoing arRNA or any of the foregoing constructs which can be used to edit target cells transcribed with NM_000203.4(IDUA)-c.1205G-A( p.Trp402Ter) target RNA at the mutation site to restore the normal function of the IDUA gene.
  • the arRNA or construct is encapsulated in liposomes.
  • the arRNA or the construct is prepared to form lipid nanoparticles.
  • the arRNA or the construct is introduced into the subject by a viral delivery method (for example, adeno-associated virus or lentivirus).
  • the arRNA-containing preparation is a therapeutic agent, which can be infused into a patient for the treatment of diseases.
  • the infusion is local injection, local infusion or intravenous infusion, local infusion or local injection.
  • the agent is in a dosage form suitable for local injection into the liver, such as hepatic arterial perfusion.
  • the medicament is in a dosage form suitable for intramuscular injection. In some embodiments, the medicament is in a dosage form suitable for intravenous injection.
  • the application also provides a kit for editing target RNA in target cells, which comprises the aforementioned arRNA, the aforementioned construct encoding the arRNA, or the aforementioned preparation.
  • the kit can be used for targeted editing of target RNA transcribed with NM_000203.4 (IDUA)-c.1205G-A (p.Trp402Ter) mutation site in target cells.
  • the kit comprises the aforementioned arRNA or the aforementioned construct encoding the arRNA, and a dye-assisting reagent, and the arRNA or construct and the dye-assisting reagent are packaged in different Container.
  • the dye-assisting reagent is a lipid solution.
  • Lipo lipofectmine RNAiMAX catalog number: 13778150
  • reagents with the same functional ingredients are packaged in different Container.
  • the kit further includes instructions for use to inform the user of the various components contained in the kit and their contents, and/or the method of using the kit.
  • the present application further provides a method for treating mucopolysaccharidosis type IH in an individual, which includes correcting the G to A mutation of IDUA gene in individual cells using the method as described above, such as NM_000203.4 (IDUA) -c.1205G-A (p.Trp402Ter) mutation.
  • the disease includes Heller's syndrome.
  • the frequency of use of arRNA is ⁇ 21 days/time, ⁇ 17 days/time, ⁇ 14 days/time, or ⁇ 10 days/time.
  • the individual is a mouse
  • the frequency of use of arRNA is ⁇ 8 days/time.
  • the therapy uses a construct encoding the arRNA, and the construct can integrate the sequence encoding the arRNA into a target cell, then the frequency of use of the arRNA is single.
  • This method does not rely on the expression of foreign proteins, so it will not be difficult to load through viral vectors and deliver in humans due to excessive protein molecular weight; it will not cause off-target effects due to overexpression of foreign proteins; no Causes the body's immune response and damage caused by the expression of foreign proteins; it will not neutralize the foreign editing enzymes or effector proteins due to the pre-existing antibodies in the body, which will cause gene editing to fail.
  • RNA editing is reversible and controllable.
  • diseases can be treated, and protein and RNA functions can be studied. Because the potential side effects of RNA editing are reversible, it is safer.
  • this method can not only chemically synthesize arRNA and complete it by electrotransfection or liposome transfection, but also can be delivered to patients through adeno-associated virus (AAV), lentivirus and other vectors to perform functions , which makes the choice of delivery methods more flexible and more efficient in editing.
  • AAV adeno-associated virus
  • the editing arRNA was synthesized by Synthego or Suzhou Beixin Biotechnology Co., Ltd., Sanger sequencing was completed by Beijing Ruibo Biotechnology Co., Ltd., and the second-generation sequencing was completed by Nuohe Zhiyuan Bioinformatics Co., Ltd. or the sequencing platform of Rice Research Institute of Chinese Academy of Sciences. .
  • GM06214 cells fibroblasts derived from Hurler syndrome patients
  • fibroblast culture medium ScienCell, FM medium, catalog number: 23011
  • Fibroblast growth supplement ScienCell, GFS, catalog number: 2301
  • NCBI-Primer blast URL: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
  • SEQ ID NO 1 CGCTTCCAGGTCAACAACAC (forward primer hIDUA-F1)
  • SEQ ID NO 2 CTCGCGTAGATCAGCACCG (reverse primer hIDUA-R1).
  • the PCR reaction was performed, and the PCR product was subjected to Sanger sequencing. It is determined that the mutant type of the cell is the pathogenic type where G at position 15704 in the IDUA genome changes to A, as shown in Figure 1.
  • Example 2 Screening of electrotransfection conditions for GM06214 cells
  • Cells of each condition are divided into 2 wells (6-well plates) and seeded in the culture plate. Place the cells Cultivate in a 37°C, 5% CO2 incubator. 24 hours after electrotransfection, digest one well of cells in each of the 2-well cells under electrotransfection conditions, and use flow cytometry to measure the proportion of GFP-positive cells. 48 hours after electrotransfection After hours, digest the cells in the other well of the two-well cells in each electrotransfection condition, and measure the proportion of GFP-positive cells by flow cytometry. The best electrotransfection conditions for the cells are the electrotransfection conditions of the CA-137 group ,as shown in picture 2.
  • Example 3 Study on IDUA enzyme activity and editing efficiency of GM06214 cells electrotransfected with arRNA
  • SEQ ID NO 3 GACGCCCACCGUGUGGUUGCUGUCCAGGACGGUCCCGGCCUGCGACACUUCGGCCCAGAGCUGCUCCUCAUCCAGCAGCGCCAGCAGCCCCAUGGCCGUGAGCACCGGCUU (Pre-55nt-c-55nt);
  • SEQ ID NO 4 GACGCCCACCGUGUGGUUGCUGUCCAGGACGGUCCCGGCCUGCGACACUUCGGCCCAGAGCUGCUCCUCAUCUGCGGGGCGGGGGGGGGCCGUCGCCGCGUGGGGUCGUUG (m-55nt-c-55nt);
  • SEQ ID NO 5 UACCGCUACAGCCACGCUGAUUUCAGCUAUACCUGCCCGGUAUAAAGGGACGUUCACACCGCGAUGUUCUCUGCUGGGGAAUUGCGCGAUAUUCAGGAUUA
  • Buffer RLT Plus 0.35ml of Buffer RLT Plus to 5 ⁇ 10 5 cells by pipetting and mix well (if cryopreserved cells directly extract RNA, it is recommended to wash once with PBS).
  • RNA concentration of the extracted RNA was measured with Nanodrop (Thermo, article number: Nanodrop2000), and 1ug of RNA was used for reverse transcription (Thermo, article number of reverse transcriptase: 28025013).
  • the GM06214 cells were digested and centrifuged, and resuspended in 28 ul of 1 ⁇ PBS containing 0.1% Triton X-100 on ice for 30 minutes. Then add 25ul of cell lysate to 25ul containing 190 ⁇ m 4-methylumbelliferyl- ⁇ -L-iduronidase (Cayman, 2A-19543-500) , Dissolved in 0.4M sodium formate buffer, containing 0.2% Triton X-100, PH3.5). Incubate at 37°C for 90 minutes in the dark.
  • GM01323 cells are fibroblasts derived from patients with Scheie syndrome.
  • Scheie syndrome is a milder form of mucopolysaccharidosis, and the symptoms are much milder than Hurler syndrome.
  • Patients with Scheie syndrome tend to have a better prognosis, with a normal life span and can live to adulthood.
  • the IDUA enzyme activity in fibroblasts of patients with Scheie syndrome is 0.3% of that in healthy human wild-type fibroblasts.
  • the results show that when arRNA targets mRNA precursor (pre-mRNA), it can show higher enzyme activity and editing efficiency, while arRNA targeting mature mRNA (mature-mRNA) shows significantly lower Enzyme activity and editing efficiency. Therefore, the arRNAs involved in the following examples are all targeting mRNA precursors (pre-mRNA).
  • Example 4 IDUA target site editing efficiency after electrotransfection of arRNA on IDUA-reporter cell line Detection
  • a segment carrying the IDUA mutation site and the upstream and downstream sequences of about 100 bp were inserted to construct the plasmid.
  • the above constructed plasmid was packaged into a virus, and 293T cells were infected. After it was integrated into the genome, IDUA-reporter monoclonal cells were screened out. This monoclonal cell only expresses mCherry protein because of the influence of the TAG stop codon at the IDUA mutation site in the inserted sequence, and when the cell is edited by arRNA, TAG->TGG occurs, and the GFP protein behind it can be expressed normally.
  • GFP Protein expression can be regarded as the editing efficiency of arRNA editing cells.
  • Example 5 After arRNA of different lengths are electrotransfected into GM06214 cells, the detection of different time points Intracellular IDUA enzyme activity and intracellular RNA editing efficiency
  • Example 2 Using the electrotransfection conditions of Example 2 to electrotransfect arRNA of different lengths on GM06214 cells (see Table 4), according to the method in Example 3, the second, fourth, sixth, eighth, and tenth sections were respectively electrotransfected after electrotransfection. , The enzyme activity in the cell was tested on the 12th and 14th day, and the editing efficiency of the intracellular RNA was tested on the 2nd and 4th day. From the results, as shown in Figure 5, 91nt:45-c-45 has the highest enzyme activity, and the IDUA enzyme activity is still maintained at a high level on the 6th day after electrotransfection. In terms of editing efficiency, 91nt and 111nt show roughly the same editing efficiency.
  • Example 6 Editing efficiency of editing site A corresponding to different positions of arRNA
  • both ends of the mutation site are simultaneously truncated and the 5'end or the 3'end are respectively truncated to study the editing efficiency of the target base corresponding to different positions of the arRNA.
  • lipofectmine RNAiMAX was used to introduce arRNA into cells.
  • RNA editing efficiency is better than other sequences, as shown in Figure 6.
  • Example 7 The effect of the length from the 3'end of the target base on editing efficiency
  • Example 6 higher IDUA enzyme activity and editing efficiency were detected on the 81nt:55-c-25 and 71nt:55-c-15 sequences.
  • the 3'end starts from 25nt (81nt:55-c-25) to 10nt (66nt:55nt-c-10nt) from the target base. Truncate one by one, as shown in Table 6.
  • the optimal length of the target base from the 3'end of the IDUA enzyme activity assay was selected to be 24 nt to 11 nt, as shown in Figure 7A.
  • the arRNA targeting the mouse IDUA mutation site (the mutation site corresponding to the human IDUA-W402X mutation) for the optimal length of the target base distance from the 3'end, and arRNA's 3 ⁇ The length of the target base starts from 55nt and is truncated every 5 bases, as shown in Table 7.
  • IDUA enzyme activity it was selected that the length of the target base from the 3'end in the mouse was 55 nt-10 nt, and the optimal length was 55 nt-10 nt, as shown in Figure 7B.
  • 111nt: 55nt-c-50nt (SEQ ID NO: 44) and 66nt: 55nt-c-10nt (SEQ ID NO: 52) show superior editing efficiency.
  • RNA modifications can increase the stability of RNA and reduce the possibility of off-target.
  • the more common chemical modifications to RNA are 2 ⁇ -OMe and thiosulfide.
  • CM1 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time, all U in the sequence are 2 ⁇ -OMe. ⁇ -OMe modification.
  • CM2 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 3'of the base is targeted The nearest neighbor base is 2'-OMe modified A.
  • CM3 The first 3 and last 3 nucleotides of the sequence are modified by 2 ⁇ -OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the 5'of the base is targeted The nearest neighbor base is 2'-OMe modified C.
  • CM4 The first 3 and last 3 nucleotides of the sequence are modified by 2'-OMe respectively, the connections between the first 3 and the last 3 nucleotides are all phosphorothioate linkages; at the same time the target base and its 3' The nearest neighbor base and the 5'nearest neighbor base are respectively connected by phosphorothioate bonds.
  • CM5 All nucleotides are modified by 2 ⁇ -OMe except for the target base, the 5 bases adjacent to the 5'end and the 5 bases adjacent to the 3'end, all nucleotides are modified by 2 ⁇ -OMe; at the same time the first 3 of the sequence The connections between the first and last 3 nucleotides are all phosphorothioate linkages.
  • CM6 The first 5 and last 5 nucleotides of the sequence are modified by 2'-OMe respectively, and the connections between the first 5 and last 5 nucleotides are phosphorothioate linkages.
  • ro represents that the nucleotide is not modified and the ester bond between the nucleotide is not modified
  • r* represents that the nucleotide is not modified and the nucleotide is connected by phosphorothioate bond
  • mo It represents that the nucleotides are modified by 2'-OMe and the ester bonds between the nucleotides are not modified
  • m* represents that the nucleotides are modified by 2'-OMe and the nucleotides are connected by phosphorothioate bonds.
  • This example relates to 3 preferred arRNAs targeting the mutation site of human IDUA and 1 preferred arRNA targeting the mutation site of mouse IDUA.
  • the chemical modification method in CM1 mode is used in GM06214 cells and MSPI MEF (MSPI mouse embryos).
  • Fibroblasts (MSPI mouse embryo fibroblast, MEF), isolated from IDUA homozygous mutant fetal mice (idua W392X mouse, B6.129S-Idua tm1.1Kmke /J) (Wang D, Shukla C, Liu X, et al .Characterization of an MPS IH knock-in mouse that carries a nonsense mutation analogous to the human IDUA-W402X mutation[published correction appears in Mol Genet Metab.2010Apr;99(4):439].Mol Genet Metab.2010;99( 1): 62-71. doi: 10.1016/j.ymgme.2009.08.002)) Concentration gradient experiment on cells.
  • Example 7 From the experimental results of Example 7, we selected 3 arRNAs that target human IDUA, the lengths are: 55nt-c-16nt, 55nt-c-14nt, 55nt-c-11nt, 1 target mouse IDUA ArRNA: 55nt-c-10nt. In addition, we also selected the random arRNA sequence RM-67CM1 as a control. From Example 9, we selected the chemical modification method of CM1 (all u: 2'-OMe) to synthesize the above-mentioned IDUA-targeted arRNA, as shown in Table 9. We performed arRNA concentration gradient transfection on human GM06214 cells compared with MSPI mouse MEF.
  • the concentration of arRNA is: 160nM, 80nM, 40nM, 20nM, 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM in 9 concentrations.
  • the cells were spread in a 6-well plate and transfected 24hrs after plating. Cells were digested 48hrs after transfection. Half of the cells were tested for IDUA enzyme activity and half of the cells were extracted for RNA editing efficiency testing.
  • ro represents no modification on the nucleotides and no modification of the ester bond between the nucleotides
  • r* represents no modification on the nucleotides and the connection between the nucleotides with phosphorothioate bond
  • mo represents the nucleus The nucleotides are connected with 2 ⁇ -OMe modified nucleotides without modification
  • m* represents that the nucleotides are connected with 2 ⁇ -OMe modified nucleotides with phosphorothioate bonds.
  • Example 11 IDUA can sustain protease activity after arRNA editing
  • Fibroblast cells have significantly improved IDUA enzyme activity, and it can last for more than 3 weeks.
  • Example 10 we performed a comparison of the arRNA targeting IDUA in humans and mice at different concentrations 48hrs after transfection.
  • FIG 11A after arRNA was transfected into GM06214 cells, we continuously detected IDUA enzyme activity for 14 days. The peak of enzyme activity after transfection was from day 4 to day 9, and the enzyme activity on day 14 was still higher than that on day 2. , And then we tested at a longer time point for 2 days, namely the 17th day and the 21st day after transfection. From Figure 10A, it can be seen that the enzyme activity on day 21 is still higher than that on day 1 after transfection.
  • the enzyme activity is about 6 to 10 times that of GM01323. (Editing efficiency needs to be supplemented, Figure 11B).
  • Figure 11C the enzymatic activity at 24hrs after arRNA transfection is about twice that of GM1323 cells until day 8. From Figure 11D It can be seen from the editing efficiency test of IDUA that IDUA's editing efficiency peaked at 24hrs, and then continued to decline.
  • This experiment involves using LEAPER technology to edit wild-type PPIB gene loci on primary cultured human and mouse liver cells, and deliver arRNA in different ways to screen out the optimal delivery method.
  • PPIB refers to the wild site in the UTR region of human NM_000942 (PPIB Genomic chr15(-): 64163082) or the wild site in the UTR region of mouse NM_011149 (PPIB Genomic chr9(+): 66066490). It can be mature mRNA or mRNA precursor. There is a TAG in the UTR segment of PPIB. In this embodiment, A in the TAG is used as a target for editing to test the editing efficiency of the arRNA in this application in liver cells.
  • Human liver cells were resuscitated 24hrs for arRNA delivery, and the delivery concentrations of LNP and Lipo were both 20nM.
  • Mouse liver cells were isolated and cultured for 24hrs and then delivered arRNA. The delivery concentrations of LNP and Lipo were both 20nM.
  • Human and mouse liver cells collected RNA at 24hrs and 48hrs after arRNA delivery, and the editing efficiency was tested by next-generation sequencing. It can be seen from Fig. 12A that in human liver cells, the editing efficiency of 48hrs after the two methods of arRNA delivery is higher than 24hrs, and the editing efficiency of arRNA delivered by LNP is better than that of Lipo delivery at 24hrs. Both 48hrs. The editing efficiency of the delivery method is similar.
  • This experiment involves studying the editing efficiency of IDUA using LNP to deliver IDUA arRNA on primary cultured human and mouse liver cells.
  • mice homozygous for this mutation are viable and fertile, their average life span is 69 weeks. Homozygotes show a progressive increase in the excretion of glycosaminoglycans (GAGs) in the urine and a progressive accumulation of GAGs in the tissues. Steady state Idua mRNA levels are reduced by 30-50%.
  • GAGs glycosaminoglycans
  • mice liver cells were taken for IDUA editing efficiency test, as shown in Figure 14.
  • the editing efficiency of about 2% can be detected in the 10 mg/kg group 24 hours after administration.
  • the result of this example proves that the arRNA directed to the IDUA of the MSPI model mouse can achieve precise editing of the mutated IDUA gene in liver cells in vivo, correct the IDUA mutation, and achieve the purpose of treating MPSI.

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Abstract

一种基于LEAPER技术靶向编辑RNA的方法，其包括利用LEAPER技术安全有效地进行RNA上由腺苷到次黄嘌呤碱基的体内编辑，精准地修复致病的突变位点，实现治疗所有由于G>A突变导致的疾病，例如MPS IH的目的。

Description

一种基于LEAPER技术治疗MPS IH的方法和组合物 技术领域
本申请属于基因编辑治疗领域，具体地，涉及一种基于LEAPER(Leveraging Endogenous ADAR for Programmable Editing on RNA)技术靶向编辑RNA治疗MPS IH的方法，其包括利用LEAPER技术进行RNA上由A到I碱基的体内位点定向编辑，来治疗由于G>A突变导致的疾病，例如MPS IH。
背景技术
赫勒氏综合征(Hurler syndrome)，又称粘多糖贮积症IH型(Mucopolysaccharidoses IH，MPS IH)，或称粘多糖病IH，是MPSI型三种亚型IH、IH/S、IS中最严重的一种，它是由于该病患者体内α-L-艾杜糖醛酸酶(α-L-iduronidase，IDUA)缺乏而引起的一种致残、致死性的遗传性代谢病，为常染色体隐性遗传病(autosomal recessive，AR)。导致赫勒氏综合征的根本原因，是位于人4号染色体上4p16.3编码IDUA蛋白的IDUA基因突变导致的，到目前为止它的致病突变有200多种，其中最为常见的一种类型是位于α-L-艾杜糖醛酸苷酶cDNA上1205位G到A的突变，该突变导致原本的色氨酸变为终止密码子，进而使最终翻译出来的蛋白上缺乏该位点之后的全部氨基酸(NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter))而丧失IDUA全部酶活性。该突变类型可占到总发病人群的63％(Worldwide distribution of common IDUA pathogenic variants，Poletto,Edina(2018).Clinical Genetics.94.10.1111/cge.13224.)。α-L-艾杜糖醛酸酶是负责细胞溶酶体(lysosomes)内葡糖胺聚糖(Glycosaminoglycans，GAGs)降解的。Hurler综合征的病人个体间差异较大，出生时可为正常，3-6个月时最早出现的征兆为面部轮廓粗糙，随后出现额骨突出、骨骼畸形、生长停滞、语言障碍等症状，病人通常活不过10岁。
赫勒氏综合征没有治愈方法，已知目前批准了两种治疗方法：酶替代疗法(ERT)和造血干细胞移植(HSCT)。ERT在内脏表型方面取得了良好的效果，包括减少肝脏大小，呼吸功能改善，患者活动性全面改善。不利的是，它不能到达中枢神经系统，因此不能阻止认知障碍。另一方面，成功的HSCT可预防大多数临床症状，包括神经系统症状，但必须在出现临床症状前进行治疗(最好在8个月大之前)，但是由于这种治疗方法的死亡率高所以只适用于病情严重的患者(Combination of enzyme replacement and hematopoietic stem cell transplantation as therapy for Hurler Syndrome.Tolar,J(2008).Bone marrow transplantation.41.531-5.10.1038/sj.bmt.1705934)。
目前，在研究的基因编辑技术治疗赫勒氏综合征的原理是利用锌指核糖核酸酶(zinc finger nuclease，ZFN)和腺相关病毒(Adeno-associated virus，AAV)将编码正常IDUA蛋白的cDNA序列定点插入到肝细胞的基因组中，但这种方法仍旧无法解决赫勒氏综合征中大脑骨骼等系统的症状，另外DNA编辑所产生的脱靶(off-target)是需要高度关注的问题。
理论上，近年来正在飞速发展的基因组编辑技术CRISPR(Clustered regularly interspaced short palindromic repeats)也可以用来治疗赫勒氏综合征。许多科研工作者和生物技术公司也在致力于将该技术推上临床。例如，2019年9月，首次报道了利用CRISPR技术编辑干细胞并将其回输至患者，以治疗其艾滋病和白血病的临床实验结果，为CRISPR技术在基因治疗方向的转化做出了巨大的贡献。尽管CRISPR技术存在极大的潜在应用前景，但该技术也存在一系列缺陷，导致该技术从科研阶段向临床治疗应用中的转化步履维艰。问题之一便是CRISPR技术用到的核心作用酶：Cas9。基于CRISPR的DNA编辑技术，必须通过外源表达Cas9或拥有相似功能的其它核酸酶，从而造成了以下几个问题。首先，通常需要外源表达的核酸酶通常具有较大分子量，这使得通过病毒载体将其递送至体内的效率急剧下降。其次，由于核酸酶的外源表达，使得其存在潜在的核酸酶脱靶可能，这将使得其应用中具有潜在的致癌风险。最后，外源表达的核酸酶是从细菌中发现的，而非人类或哺乳动物天然存在的，这使得其可能引起患者体内的免疫反应，这一方面可能会对患者自身造成损伤，另一方面也可能会使外源表达的核酸酶被中和，从而失去应有的活性，或者阻碍进一步的干预治疗。
2017年，张锋课题组曾报道一种名为REPAIR(RNA Editing for Programmable A to I Replacement)的RNA编辑技术(RNA editing with CRISPR-Cas13，Cox et al.,2017)，该技术通过外源表达Cas13-ADAR融合蛋白及单独向导RNA(single guide RNA,sgRNA)同样可以实现靶向目标RNA的A到I的编辑，但是该方法同CRISPR技术一样，仍需要外源蛋白的表达。无法解决外源蛋白表达造成的问题。
2019年1月，Thorsten Stafforst课题组曾报道一种名为RESTORE核酸编辑技术(recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing,Merkle et al.,2019)。该技术能够摆脱对外源蛋白的依赖。但首先RESTORE技术需要在IFN-γ存在的前提下才能有较高的编辑效率，而IFN-γ是决定自体免疫发展和严重程度的关键因子(Interferon-γand systemic autoimmunity,Pollard et al.,2013，这使得该技术在医学领域的应用大打折扣。另一方面，RESTORE技术中同样也用到一段向导RNA，而其使用的向导RNA是化学合成的寡核苷酸，并且其合成的寡核苷酸需要人为引入大量的化学修饰以保证其稳定性。
2019年，PCT/CN2019/110782和PCT/CN2020/084922申请提供了一种工程改造的RNA，其与靶转录物部分互补以募集天然ADAR1或ADAR2以在靶标RNA中的特定位点将腺苷变为肌苷，该方法称为“LEAPER(Leveraging Endogenous ADAR for Programmable Editing on RNA)”(利用内源性ADAR进行RNA的可编程编辑)，而募集ADAR的RNA可称为“dRNA”或“arRNA”。该dRNA包含与靶标RNA杂交的互补RNA序列，并且所述dRNA能够募集作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷(A)脱氨基。
发明内容
本申请针对Hurler综合征IDUA致病基因中的G到A的突变，尤其是占比最高的突变型(NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter))提供了一种全新的技术方案使其能够精准地在靶标RNA上编辑突变位点。
具体地，本申请提供了至少以下技术方案：
1.一种基于LEAPER技术靶向编辑靶标细胞中靶标RNA的方法，其中所述靶标RNA为IDUA基因转录本中含有G到A突变的RNA，该方法包括：
将包含用于编辑靶标RNA的腺苷脱氨酶募集RNA(arRNA)或编码所述arRNA的构建体递送至所述靶标细胞，其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且其中所述arRNA能够募集作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷(A)脱氨基。
2.如项1中所述的方法，其中所述arRNA引入与靶标A配对的碱基C、A、U或G。
在一些实施方案中，所述arRNA引入与靶标A配对的碱基C。在一些实施方案中，所述arRNA引入与靶标A配对的碱基A。在一些实施方案中，所述arRNA引入与靶标A配对的碱基U。3.如项1-2中任一项所述的方法，其中所述arRNA长约151-61nt、131-66nt、121-66nt、111-66nt、91-66nt或81-66nt。本申请公开和涵盖所述数字范围内的任何自然数。
4.如项3中所述的方法，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15n或24nt-11nt。本申请公开和涵盖所述数字范围内的任何自然数。
5.如项3或4中所述的方法，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt。本申请公开和涵盖所述数字范围内的任何自然数。
6.如项1-5中所述的方法，其中所述靶标细胞是人的细胞。
7.项1-6中任一项的方法，其中所述靶标RNA为包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的RNA。
8.项1-7中任一项的方法，其中所述arRNA包含以下的序列：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31或SEQ ID NO：34。
9.如项1-5中任一项所述的方法，其中所述arRNA包含选自以下的序列：SEQ ID NO：44或SEQ ID NO：52。
10.如项1-9中任一项所述的方法，其中所述arRNA是经化学修饰的。
11.如项10中所述的方法，其中所述化学修饰包含2-O’-甲基化(2`-OMe)或硫代磷酸酯修饰。
12.如项11中所述的方法，其中所述化学修饰选自如下的一项或多项：
序列前3个和后3个核苷酸分别被2`-OMe修饰，
前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
序列中全部U均被2`-OMe修饰，
靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
前5个和后5个核苷酸分别被2`-OMe修饰，和
前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
在一些具体实施方案中，所述化学修饰选自如下的一项或多项：
CM1：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时序列中全部U均被2`-OMe修饰；
CM2：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的3’最近邻碱基为2`-OMe修饰的A；
CM3：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的5’最近邻碱基为2`-OMe修饰的C；
CM4：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接；以及
CM6：序列前5个和后5个核苷酸分别被2`-OMe修饰，并且前5个和后5个核苷酸间连接为硫代磷酸酯键连接。13.如项1-9中任一项所述的方法，其中所述编码所述arRNA的构建体为线性核酸链、病毒载体或质粒。
14.如项13中所述的方法，其中所述病毒载体为腺相关病毒(AAV)载体或慢病毒表达载体。
15.如项1-14中任一项所述的方法，其中所述递送方式为电转染、脂质体转染、脂质-纳米颗粒(lipid nanoparticle,LNP)递送或感染。
16.如项15中所述的方法，通过LNP将包含用于编辑靶标RNA的腺苷脱氨酶募集RNA(arRNA)或编码所述arRNA的构建体递送至所述靶标细胞。
17.如项1-16中任一项所述的方法，其中所述arRNA的递送浓度≥2.5，≥5nM，≥10nM，≥15nM，或≥20nM。
在上述实施方案中，所述靶标细胞包括肝细胞或成纤维细胞。
18.一种用于通过LEAPER技术靶向编辑靶标细胞中靶标RNA的arRNA或其编码序列，所述arRNA包含如下任一序列或由如下任一序列组成：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：34、SEQ ID NO：44或SEQ ID NO：52。
19.包含项18所述arRNA或其编码序列的质粒、病毒载体、脂质体或脂质纳米颗粒。
20.包含项18所述arRNA或其编码序列、或项19所述质粒、病毒载体、脂质体或脂质纳米颗粒的组合物、制剂、试剂盒或生物制品。
21.一种治疗个体中MPS IH的方法，包括用项1-17中任一项所述的方法校正所述个体的靶标细胞中与MPS IH疾病相关的G到A的突变。
22.项20的方法，其中所述突变为NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变。
23.如项20或21所述的方法，其中所述arRNA的使用频次为≥21天/次、≥17天/次、≥14天/次或≥10天/次。
在一些实施方案中，本申请还涉及选自如下的序列在制备治疗MPS IH疾病的药物中的用途：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：34、SEQ ID NO：44或SEQ ID NO：52。
利用本申请的技术方案，可以安全有效地进行靶细胞(例如肝细胞或成纤维细胞)内RNA上由A到I碱基的体内编辑，精准地修复致病的突变位 点，例如NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变，恢复由RNA编码的蛋白在体内的正常表达，实现治疗MSP IH的目的。
附图说明
图1显示了在GM06214细胞上检测IDUA基因型。
图2显示了细胞电转染条件测试。
图3A-B显示了针对IDUA pre-mRNA和mRNA设计arRNA，对编辑后细胞功能进行检测；和针对IDUA pre-mRNA和mRNA设计arRNA，对细胞编辑效率进行检测。
图4A显示了IDUA-reporter细胞系的设计；和图4B显示了在293T-IDUA-Reporter上检测不同长度arRNA(对称截短)的编辑效率。
图5A-B显示了在GM06214细胞上转染不同长度(对称截短)arRNA后，不同时间点的酶活和编辑效率检测。
图6显示了在GM06214细胞用lipofectmine RNAiMAX转染arRNA(对称截短、3`截短和5`截短)后，IDUA酶活性和编辑效率检测。
图7A显示了靶向人IDUA突变位点的arRNA在优选的55-c-25到55-c-10之间，在3`端逐个减少碱基，使用GM06214细胞通过酶活性检测选出最佳长度。7B显示了靶向小鼠的IDUA突变位点的arRNA在55-c-55到55-c-10之间，在3`端每5个碱基逐渐减少，使用MPSI小鼠MEF细胞(MSPI MEF(MSPI mouse embryo fibroblast))通过酶活性检测选出最佳arRNA长度序列。
图8A显示了在优选的2个3`端长度下，逐渐截短5`端长度以筛选出最优长度范围，图8B显示了3`端长度固定为14nt后，5`端逐个碱基截短后形成的arRNA对酶活性的影响，综合图8A和8B筛选出最优的的arRNA长度。
图9显示了在优选的2个arRNA长度下，比较不同种化学修饰对arRNA编辑效率(以酶活性体现)的影响。
图10A-D显示了在优选的长度和优选的化学修饰方式组合下，在不同的arRNA浓度下，人和小鼠arRNA编辑IDUA mRNA所产生的编辑效率和编辑后产生有功能IDUA蛋白的能力。
图11A-D显示了在优选的长度和优选化学修饰方式组合下，一次转染 后，在不同时间在人或鼠的细胞中测得的IDUA酶活性。
图12A-B显示在人和小鼠的原代肝脏细胞中，用不同的方式进行arRNA的递送，所实现的靶向位点的编辑效率。
图13显示在原代培养的人和小鼠肝脏细胞上利用LNP递送的靶向IDUA的arRNA的编辑效率。
图14显示经筛选的靶向小鼠IDUA突变的arRNA(SEQ ID NO：52)在包装成LNP后，以不同浓度通过尾静脉向小鼠给药24hrs后，小鼠肝脏细胞中的IDUA编辑效率。
具体实施方式
定义
RNA编辑是指存在于真核细胞中的一种天然过程，RNA编辑是在DNA转录之后，蛋白质翻译之前在RNA水平上发生的由碱基A(腺嘌呤)到I(次黄嘌呤)的编辑，次黄嘌呤在翻译的过程中被识别为G，RNA中A到I的编辑使转录组多样化。通过位点特异和精确改变RNA分子的方式将RNA的全部数量增加几倍。这种编辑是被ADAR(Adenosine Deaminase Acting on RNA)蛋白酶催化产生的，被称为位点定向RNA编辑。编辑可以发生的编码区包括内含子和外显子序列，也可以发生在非编码区序列，编码区的编辑可以重新定义蛋白质编码序列。
如本文所用，“LEAPER技术”即通过使用工程化RNA募集内源性ADAR进行RNA编辑的技术，是指如WO2020074001A1中所报道的RNA编辑技术。所述工程化RNA即腺苷脱氨酶募集RNA(arRNA)，如本文所用，是指能通过招募ADAR或某些包含ADAR结构域的复合物在RNA中使靶标腺苷脱脱氨基的RNA。
如本文所用，术语“腺苷脱氨酶(ADAR)”是指是一类在真核生物(包括人等哺乳动物)各组织中广泛表达的腺苷脱氨酶，能够催化RNA分子中腺苷A到肌苷I的转换。而I在真核生物蛋白质合成过程中，通常被当做G进行翻译。
如本文所用，核酸的“互补”是指一条核酸通过传统的Watson-Crick碱基配对与另一条核酸形成氢键的能力。百分比互补性表示核酸分子中可与另一 核酸分子形成氢键(即，Watson-Crick碱基配对)的残基的百分比(例如，10个中的约5、6、7、8、9、10个分别为约50％，60％，70％，80％，90％和100％互补)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数量的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250或更多个核苷酸的区域内，至少约70％，75％，80％，85％，90％，95％，97％，98％，或99％中的任何一个的互补程度，或指在严格条件下杂交的两条核酸。对于单个碱基或单个核苷酸，按照Watson-Crick碱基配对原则，A与T或U、C与G或I配对时，被称为互补或匹配，反之亦然；而除此以外的碱基配对都称为不互补或不匹配。
“杂交”是指其中一种或多种多核苷酸反应形成复合物的反应，所述复合物通过核苷酸残基的碱基之间的氢键稳定。所述氢键可以通过Watson Crick碱基配对，Hoogstein结合或以任何其他序列特异性的方式发生。能够与给定序列杂交的序列称为给定序列的“互补序列”。
如本文所用，术语“电转染”即电穿孔转染技术，其通过电场作用于细胞几微秒到几毫秒之后，在细胞膜上暂时形成小孔或开口，把大分子如DNA等递送至细胞的技术。
如本文所用，术语“脂质体转染(Lipo)”是指以脂质体作为体内和体外输送载体的转染技术。脂质体包括中性脂质体和阳离子脂质体，其中中性脂质体是利用脂质膜包裹大分子，例如核酸，借助脂质膜将所述大分子递送至细胞膜内；阳离子脂质体带正电荷，其转载的大分子并没有预先包埋其中，而是由于所述大分子本身带负电荷而自动结合到带正电的脂质体上，形成大分子-阳离子脂质体复合物，从而吸附到带负电的细胞膜表面，经过内吞被递送入细胞。
如本文所用，术语“脂质-纳米颗粒(LNP)递送”是指将大分子，例如核酸、蛋白质等物质通过脂质纳米颗粒向细胞内进行跨膜递送。其中，脂质纳米颗粒是指由两相混合合成的颗粒包括含有可电离脂质、辅助磷脂、胆固醇和PEG脂的乙醇相和含有核酸、蛋白质等大分子的酸性水相。例如，包裹有RNA的LNP可通过内吞进入细胞质内。
在本文中，NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变是指IDUA基因编号000203.4的转录本中，1205位的G至A的突变，所述突变 导致所述转录本翻译的肽链的402位色氨酸(Trp)编码序列转变为终止密码子(Ter)，使得最终翻译的氨基酸缺失了402位之后的全部氨基酸，从而丧失IDUA的酶活性。发生这一突变的患者会因为缺乏具有活性的α-L-艾杜糖醛酸酶而影响细胞溶酶体内葡糖胺聚糖的降解，最终对患者致畸甚至致死。而本申请中的方案可通过在转录水平逆转这一突变，恢复IDUA酶的活性。
如本文所用，术语“靶标RNA”是指待编辑的目标RNA，其包含待编辑的腺苷(A)。所述靶标RNA可以是成熟的mRNA，也可以是mRNA前体。在本申请中更优选mRNA前体。在本文中，包含所述“靶标RNA”的细胞称为靶标细胞。所述待编辑的腺苷称为“靶标碱基”，“靶标腺苷”或“靶标A”。在本申请中，“靶标碱基”，“靶标腺苷”或“靶标A”可以互换使用。在靶标RNA的5’端与靶标腺苷相邻的碱基称为“5’相邻碱基”；在靶标RNA的3’端与靶标腺苷相邻的碱基称为“3’相邻碱基”；靶标碱基及其3’和5’相邻碱基组成的碱基三联体在本文中被称为“靶标碱基三联体”。当arRNA与靶标RNA杂交时，在arRNA上与所述靶标碱基相对的碱基称为“靶向碱基”，在arRNA的5’端与靶向碱基相邻的碱基称为“5’最近邻碱基”；在arRNA的3’端与靶向碱基相邻的碱基称为“3’最近邻碱基”；靶向碱基及其3’和5’最近邻碱基组成的碱基三联体在本文中被称为“靶向碱基三联体”。
如本文所用，术语“构建体”是指包含某一核酸序列的核酸载体，所述核酸载体可以是线性核酸分子、质粒或病毒载体等。所述核酸分子可以是单链也可以是双链分子。所述核酸序列可以是DNA序列也可以是RNA序列。在一些实施方案中，所述核酸序列不经过转录、翻译或表达而直接发挥其功能。在一些实施方案中，所述核酸序列为DNA序列，其经过转录形成RNA后以RNA分子形式发挥功能。在一些实施方案中，所述核酸序列为RNA，其经过翻译后以多肽或蛋白质的形式发挥作用。在一些实施方案中，所述核酸序列为DNA，其经过转录和翻译步骤形成蛋白质后以蛋白质的形式发挥功能。所述构建体可以通过包装为病毒、脂质纳米颗粒或外泌体等形式进入靶标细胞，亦可以通过电转化、显微注射、化学转化等方式进入靶标细胞。
如本文所用，术语“递送”是指将核酸、蛋白等生物大分子通过某些途径从细胞膜外引入细胞膜内。所述“递送”例如电转染、脂质体转染、脂质-纳米 颗粒递送、病毒递送、外泌体递送等方式。
本申请所用术语“修饰”是指通过化学或生物学方法，例如基因工程方法改变核酸或蛋白的组成或结构，从而使所述核酸或蛋白的一项或多项特性或功能发生改变。
除非本文另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同含义。
RNA编辑方法
本申请提供了一种基于LEAPER技术靶向编辑靶标细胞中含有G到A突变的IDUA靶标RNA的方法，其包括：将包含用于编辑靶标RNA的腺苷脱氨酶募集RNA(arRNA)或编码所述arRNA的构建体递送至所述靶标细胞，其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且其中所述arRNA能够募集作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷(A)脱氨基。在一些实施方案中，所述靶标RNA是mRNA前体。在一些实施方案中，所述靶标RNA是成熟mRNA。在一些实施方案中，所述靶标RNA为转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的IDUA靶标RNA。
在一些实施方案中，所述arRNA包含与靶标A配对的碱基C、A、U或G。所述与靶标A配对的碱基优选顺序为C、A、U、G。即在arRNA长度一致、靶向碱基距离5’端的距离一致、靶向碱基距离3’端的距离一致、并且除靶向碱基以外的arRNA序列完全一致的情况下，靶向碱基的优选顺序为C＞A＞U＞G。当所述靶向碱基为C时，所述arRNA可以表示为X nt-c-Y nt，其中X表示所述靶向碱基距离arRNA的5’端的距离为X nt，Y表示所述靶向碱基距离arRNA的3’端的距离为Y nt，其中X和Y可以表示任何自然数。
在一些实施方案中，所述靶标细胞是真核细胞。在一些实施方案中，所述靶标细胞是哺乳动物细胞。在一些实施方案中，所述靶标细胞是肝细胞或成纤维细胞。在一些实施方案中，所述靶标细胞是人或小鼠细胞。
在一些实施方案中，所述arRNA长约151-61nt、131-66nt、121-66nt、111-66nt、91-66nt或81-66nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15n或24nt-11nt。在一 些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt。其中，所述靶向碱基距离3’端的长度是指将靶向碱基的3’最近邻碱基至3’最末端碱基的所有碱基个数；所述靶向碱基距离5’端的长度是指靶向碱基的5’最近邻碱基至5’最末端碱基的所有碱基个数。
在一些实施方案中，所述靶标细胞是人的细胞，且其中所述靶标RNA为转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的IDUA靶标RNA，则所述arRNA的全长≥66nt，例如长约121-66nt、111-66nt、101-66nt、91-66nt或81-66nt，即所述arRNA的全长选自上述长度范围的任意自然数，例如：67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、77nt、78nt、79nt、80nt、81nt、82nt、83nt、84nt、85nt、86nt、87nt、88nt、89nt、90nt、91nt、95nt、100nt、110nt、115nt、120nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt或24nt-11nt，即所述arRNA的靶向碱基距离3’端的距离选自上述靶向碱基距离3’端的长度范围的任意自然数，例如：12nt、13nt、14nt、16nt、17nt、18nt、19nt、20nt、21nt、22nt、23nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt，即所述arRNA的靶向碱基距离5’端的距离选自上述靶向碱基距离5’端的长度范围的任意自然数，例如：46nt、47nt、48nt、49nt、50nt、51nt、52nt、53nt、54nt。在一些实施方案中，所述arRNA包含选自以下的序列：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：34。
在一些实施方案中，所述靶标细胞是小鼠细胞(例如W392X小鼠细胞)，且其中所述靶标RNA为转录有包含与人W402X突变相应的IDUA突变的靶标RNA。在一些实施方案中，所述靶标细胞为W392X小鼠细胞。在一些实施方案中，所述arRNA长约121-53nt、111-61nt、101-61nt、91-61nt、81-61nt、111-66nt、或105-66nt，即所述arRNA的全长选自上述长度范围的任意自然数，例如：67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、77nt、78nt、79nt、80nt、81nt、82nt、83nt、84nt、85nt、86nt、87nt、88nt、 89nt、90nt、91nt、95nt、100nt、110nt、115nt、120nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为55nt-10nt或50nt-10nt，即所述arRNA的靶向碱基距离3’端的距离选自上述靶向碱基距离3’端的长度范围的任意自然数，例如：11nt、12nt、13nt、14nt、16nt、17nt、18nt、19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt、27nt、28nt、29nt、30nt、31nt、32nt、33nt、34nt、35nt、35nt、37nt、38nt、39nt、40nt、41nt、42nt、43nt、44nt、45nt、46nt、47nt、48nt、49nt、50nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt，即所述arRNA的靶向碱基距离5’端的距离选自上述靶向碱基距离5’端的长度范围的任意自然数，例如：33nt、36nt、47nt、46nt、47nt、48nt、49nt、50nt、51nt、52nt、53nt、54nt、60nt、65nt、75nt。在一些实施方案中，所述arRNA包含选自以下的序列：SEQ ID NO：44或SEQ ID NO：52。
在一些实施方案中，所述arRNA是经化学修饰的。在一些实施方案中，所述化学修饰为2-O’-甲基化和/或硫代磷酸酯修饰。在一些实施方案中，所述化学修饰选自如下的一项或多项：
序列前3个和后3个核苷酸分别被2`-OMe修饰，
前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
序列中全部U均被2`-OMe修饰，
靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
前5个和后5个核苷酸分别被2`-OMe修饰，和
前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
如本文所述，编码所述arRNA的构建体即包含所述arRNA编码序列的构建体。在一些实施方案中，所述arRNA在所述编码所述arRNA的构建体被递送入靶标细胞后，由编码所述arRNA的构建体转录而成。在一些实施方案中，所述编码所述arRNA的构建体为线性核酸链、病毒载体或质粒。在一些实施方案中，所述病毒载体为腺相关病毒(AAV)或慢病毒。在一些实施方案中，当所述编码所述arRNA的构建体被递送入靶标细胞时，其通过 同源重组或非同源重组等方式将编码所述编码arRNA的序列插入靶标细胞基因组，从而持续转录生成所述arRNA。在一些实施方案中，所述编码所述arRNA的构建体被递送入靶标细胞时，其使编码所述arRNA的序列作为游离核酸的一部分存在于靶标细胞中，从而所述编码所述arRNA的序列可以在一定的时间内转录生成所述arRNA。
在一些实施方案中，所述递送为电转染、脂质体转染或脂质-纳米颗粒(lipid nanoparticle,LNP)递送或感染。在一些实施方案中，当所述靶标细胞为肝脏细胞时，所述递送为LNP递送。在一些实施方案中，当所述靶标细胞为成纤维细胞时，所述递送为脂质体转染。在一些实施方案中，所述arRNA的递送浓度≥2.5-5nM，优选地≥10-20nM，例如≥15nM。在本申请中，所述递送浓度是指在将所述arRNA构建体递送至所述靶标细胞时，一体积单位arRNA与所述靶标细胞所在的递送体系中所含有的arRNA的量。所述递送体系包含arRNA或其构建体、靶标细胞及所述arRNA及靶标细胞周围的液体基质。在一些实施方案中，所述液体基质可以是细胞培养基、PBS或其他可在一定时间内维持细胞稳定生存状态的，与胞浆等渗的溶液。在一些实施方案中，所述递送体系还包含可促进递送的试剂。
arRNA
本申请还提供了一种arRNA，其可用于基于LEAPER技术靶向编辑靶标细胞中的靶标RNA，例如转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的靶标RNA，以使靶标RNA中的靶A脱氨基称为次黄嘌呤I。而在靶标细胞后续的翻译过程中，I会被识别为G，从而将G>A的突变恢复为G，使得经arRNA编辑后，靶标RNA可翻译为正确的蛋白。在一些实施方案中，所述靶标RNA是mRNA前体。在一些实施方案中，所述靶标RNA是成熟mRNA。
在一些实施方案中，所述arRNA引入与靶标A配对的碱基(靶向碱基)编辑效率从大到小为C、A、U、G。在一些实施方案中，所述arRNA中除靶向碱基外，其他碱基均可与靶标RNA互补配对。在一些实施方案中，所述arRNA中除靶向碱基外，还有一个或多个碱基与靶标RNA形成错配。
在一些实施方案中，所述靶标细胞是真核细胞。在一些实施方案中，所 述靶标细胞是哺乳动物细胞。在一些实施方案中，所述靶标细胞是肝细胞或成纤维细胞。在一些实施方案中，所述靶标细胞是人或小鼠细胞(例如W392X小鼠细胞)。
在一些实施方案中，所述arRNA长约151-61nt、131-66nt、121-66nt、111-66nt、91-66nt或81-66nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15n或24nt-11nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt。其中，所述靶向碱基距离3’端的长度是指将靶向碱基的3’最近邻碱基至3’最末端碱基的所有碱基个数；所述靶向碱基距离5’端的长度是指靶向碱基的5’最近邻碱基至5’最末端碱基的所有碱基个数。
在一些实施方案中，所述靶标细胞是人的细胞，且其中所述靶标RNA为转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的IDUA靶标RNA，则所述arRNA的全长≥66nt，例如长约121-66nt、111-66nt、101-66nt、91-66nt或81-66nt，即所述arRNA的全长选自上述长度范围的任意自然数，例如：67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、77nt、78nt、79nt、80nt、81nt、82nt、83nt、84nt、85nt、86nt、87nt、88nt、89nt、90nt、91nt、95nt、100nt、110nt、115nt、120nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt或24nt-11nt，即所述arRNA的靶向碱基距离3’端的距离选自上述靶向碱基距离3’端的长度范围的任意自然数，例如：12nt、13nt、14nt、16nt、17nt、18nt、19nt、20nt、21nt、22nt、23nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt，即所述arRNA的靶向碱基距离5’端的距离选自上述靶向碱基距离5’端的长度范围的任意自然数，例如：46nt、47nt、48nt、49nt、50nt、51nt、52nt、53nt、54nt。在一些实施方案中，所述arRNA包含选自以下的序列：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：34。
在一些实施方案中，所述靶标细胞是小鼠细胞(例如W392X小鼠细胞)， 且其中所述靶标RNA为转录有包含与人W402X突变相应的IDUA突变的靶标RNA，则所述arRNA长约121-53nt、111-61nt、101-61nt、91-61nt、81-61nt、111-66nt、或105-66nt，即所述arRNA的全长选自上述长度范围的任意自然数，例如：67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、77nt、78nt、79nt、80nt、81nt、82nt、83nt、84nt、85nt、86nt、87nt、88nt、89nt、90nt、91nt、95nt、100nt、110nt、115nt、120nt。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为55nt-10nt或50nt-10nt，即所述arRNA的靶向碱基距离3’端的距离选自上述靶向碱基距离3’端的长度范围的任意自然数，例如：11nt、12nt、13nt、14nt、16nt、17nt、18nt、19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt、27nt、28nt、29nt、30nt、31nt、32nt、33nt、34nt、35nt、35nt、37nt、38nt、39nt、40nt、41nt、42nt、43nt、44nt、45nt、46nt、47nt、48nt、49nt、50nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt，即所述arRNA的靶向碱基距离5’端的距离选自上述靶向碱基距离5’端的长度范围的任意自然数，例如：33nt、36nt、47nt、46nt、47nt、48nt、49nt、50nt、51nt、52nt、53nt、54nt、60nt、65nt、75nt。在一些实施方案中，所述arRNA包含选自以下的序列：SEQ ID NO：44或SEQ ID NO：52。
在一些实施方案中，所述arRNA由编码所述arRNA的构建体转录表达而成。在一些实施方案中，所述arRNA由编码所述arRNA的构建体在体外转录表达，并经纯化获得。在一些实施方案中，所述arRNA由编码所述arRNA的构建体在体内直接表达并发挥编辑作用。在一些实施方案中，所述构建体选自病毒载体、质粒和线性核酸。在一些实施方案中，所述病毒为AAV或慢病毒。
在一些实施方案中，所述arRNA由化学合成。在一些实施方案中，所述arRNA是经化学修饰的。在一些实施方案中，所述化学修饰为2-O’-甲基化和/或硫代磷酸酯修饰。在一些实施方案中，所述化学修饰选自如下的一项或多项：
序列前3个和后3个核苷酸分别被2`-OMe修饰，
前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
序列中全部U均被2`-OMe修饰，
靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
前5个和后5个核苷酸分别被2`-OMe修饰，和
前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
在一些实施方案中，所述化学修饰选自如下的一项或多项：
CM1：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时序列中全部U均被2`-OMe修饰；
CM2：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的3’最近邻碱基为2`-OMe修饰的A；
CM3：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的5’最近邻碱基为2`-OMe修饰的C；
CM4：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接；以及
CM6：序列前5个和后5个核苷酸分别被2`-OMe修饰，并且前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
构建体
本申请还提供了一种编码前述arRNA的构建体。在一些实施方案中，所述构建体选自病毒载体、质粒和线性核酸。在一些实施方案中，所述病毒载体为AAV载体或慢病毒表达载体。
本申请还进一步提供了包含所述构建体的病毒、直至纳米颗粒、脂质体、外泌体或细胞。
制剂和生物制品
本申请还提供了包含前述任一种arRNA或前述任一种构建体的组合物、制剂和生物制品，其可用于编辑靶标细胞中转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的靶标RNA，以恢复IDUA基因的正常功能。在一些实施方案中，所述arRNA或构建体包裹在脂质体中。在一些实施方案中，所述arRNA或所述构建体经制备形成脂质纳米颗粒。在一些实施方案中，所述arRNA或所述构建体通过病毒递送方式(例如腺相关病毒或慢病毒)导入受试者体内。
在一些实施方案中，所述包含arRNA的制剂为一种治疗药剂，可输入患者体内用于疾病的治疗。在一些实施方案中，所述输入为局部注射、局部灌注或静脉输注、局部灌注或局部注射。在一些实施方案中，所述药剂为适于肝脏局部注射的剂型，例如肝脏动脉灌注。在一些实施方案中，所述药剂为适于肌肉注射的剂型。在一些实施方案中，所述药剂为适于静脉注射的剂型。
试剂盒
本申请同时还提供了一种用于编辑靶标细胞中的靶标RNA的试剂盒，其包含如前所述的arRNA、如前所述的编码所述arRNA的构建体或如前所述的制剂。所述试剂盒可用于靶向编辑靶标细胞中转录有包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的靶标RNA。
在一些实施方案中，所述试剂盒包含如前所述的arRNA或如前所述的编码所述arRNA的构建体，以及助染试剂，所述arRNA或构建体以及助染试剂分装于不同的容器。在一些实施方案中，所述助染试剂为脂质溶液。例如：英潍捷基的Lipo(lipofectmine RNAiMAX货号：13778150)或与其具有相同功能成分的试剂。
在一些实施方案中，所述试剂盒还包含使用说明，以告知使用者所述试剂盒中包含的各种成分及其含量，和/或所述试剂盒的使用方法。
疗法
本申请还进一步提供了一种治疗个体中粘多糖贮积症IH型的方法，包括使用如前所述的方法校正个体细胞中与IDUA基因的G到A的突变，例 如NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变。在一些实施方案中，所述疾病包括赫勒氏综合征。在一些实施方案中，当所述个体为人时，其中arRNA的使用频次为≥21天/次、≥17天/次、≥14天/次或≥10天/次。在一些实施方案中，当所述个体为小鼠时，其中arRNA的使用频次为≥8天/次。在一些实施方案中，所述疗法使用编码所述arRNA的构建体，并且所述构建体可将编码所述arRNA的序列整合入靶标细胞，则所述arRNA的使用频次为单次。
本申请提供的利用LEAPER技术对RNA进行靶向编辑的方法，具有以下优点：
1.该方法不依赖于外源蛋白的表达，因此，不会由于蛋白分子量过大使得通过病毒载体进行装载及人体内递送十分困难；不会由于外源蛋白过表达引起的脱靶效应；不会导致由外源蛋白表达引起的机体免疫反应及损伤；也不会由于机体内的预存抗体使外源编辑酶或效应蛋白被中和从而导致基因编辑失败。
2.本申请提供的这种酶导向特定位点RNA编辑方法与DNA编辑不同，RNA编辑是可逆和可调控的。通过重新编码氨基酸密码子可以对疾病进行治疗，对蛋白质和RNA功能进行研究。由于RNA编辑潜在的副作用是可逆的，因此更安全。
3.与现有技术相比，该方法不仅可以通过化学合成arRNA后，经电转染或脂质体转染完成，也可以通过腺相关病毒(AAV)、慢病毒等载体递送至患者发挥功能，这使得其递送手段的选择上更加灵活多变，编辑效率更高。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
下面结合具体实施例，对本发明的技术方案作进一步的详述，但本发明并不限于以下实施例。如未特别指出，以下所涉及的试剂均可通过商业途径购得。为简便起见，部分操作未详述操作的参数、步骤及所使用的仪器，应当理解，这些都是本领域技术人员所熟知并可重复再现的。本文中所使用的细胞(GM06214)够买于美国Coriell公司，是一株来源于Hurler综合征病 人的成纤维细胞。编辑用arRNA是由美国Synthego公司或苏州贝信生物技术有限公司合成，Sanger测序由北京睿博生物技术有限公司完成，二代测序由诺禾致源生物信息科技有限公司或中科院水稻所测序平台完成。
实施例
实施例1：检测GM06214突变基因型
将GM06214细胞(赫尔勒氏综合症(Hurler syndrome)患者来源的成纤维母细胞)放入含有15％血清的成纤维细胞培养液(ScienCell，FM培养基，货号：2301)中，添加1％成纤维细胞生长添加物(ScienCell，GFS，货号：2301)，在37℃、5％CO 2培养箱里培养2-3天。待细胞融合度达到90％时，用0.25％的胰酶消化后，含有15％血清的成纤维细胞培养液终止消化。用
Figure PCTCN2020141506-appb-000001
(TIANGEN Biotech(Beijing)Co.,Ltd.)细胞DNA提取试剂盒(货号：DP304-03)根据操作说明进行DNA提取。
利用NCBI-Primer blast(网址：https：//www.ncbi.nlm.nih.gov/tools/primer-blast/)进行IDUA突变位点上下游序列的引物设计。SEQ ID NO 1：CGCTTCCAGGTCAACAACAC(正向引物hIDUA-F1)；SEQ ID NO 2：CTCGCGTAGATCAGCACCG(反向引物hIDUA-R1)。进行PCR反应，PCR产物进行Sanger测序。确定细胞的突变型是IDUA基因组上第15704位点G变为A的致病型，如图1所示。
实施例2：GM06214细胞电转染条件筛选
待GM06214细胞生长至约90％汇合度时消化细胞，终止消化后计数。电转染时，用预混合电转染液(Lonza，货号：V4XP-3024)400ul重悬600万个细胞，再加20ug的GFP质粒(Lonza，货号：V4XP-3024，混匀后，取每20ul为一个电转染体系，用Lonza核电转染仪测试7个电转染条件(参见图2)加一个阴性对照共8个，每个条件做2个重复。电转染后，将细胞迅速转入2ml含有15％血清的成纤维细胞培养液(ScienCell，FM培养基，货号：2301)中，每个条件的细胞分2个孔(6孔板)接种在培养板中，将细胞放入37℃、5％CO2培养箱培养。电转染后24小时，消化每个电转染条件2孔细胞中的一孔细胞，利用流式细胞仪测GFP阳性细胞的比例。电转染后 48小时，消化每个电转染条件2孔细胞中的另一孔细胞，利用流式细胞仪测GFP阳性细胞的比例。得到细胞的最佳电转染条件为CA-137组的电转染条件，如图2所示。
实施例3：GM06214细胞电转染arRNA后IDUA酶活性和编辑效率研究
针对IDUA基因转录后pre-mRNA(mRNA前体)和mature-mRNA(成熟mRNA)突变位点上下游序列，设计合成以下arRNA序列：SEQ ID NO 3：GACGCCCACCGUGUGGUUGCUGUCCAGGACGGUCCCGGCCUGCGACACUUCGGCCCAGAGCUGCUCCUCAUCCAGCAGCGCCAGCAGCCCCAUGGCCGUGAGCACCGGCUU(Pre-55nt-c-55nt)；SEQ ID NO 4：GACGCCCACCGUGUGGUUGCUGUCCAGGACGGUCCCGGCCUGCGACACUUCGGCCCAGAGCUGCUCCUCAUCUGCGGGGCGGGGGGGGGCCGUCGCCGCGUGGGGUCGUUG(m-55nt-c-55nt)；SEQ ID NO 5：UACCGCUACAGCCACGCUGAUUUCAGCUAUACCUGCCCGGUAUAAAGGGACGUUCACACCGCGAUGUUCUCUGCUGGGGAAUUGCGCGAUAUUCAGGAUUAAAAGAAGUGC(Random-111nt)，其中，合成的arRNA中突变位点对应的碱基由T变成C，形成一个A-C错配。合成arRNA的长度优选在111nt。利用实施例2得到的最佳电转染条件对细胞进行电转染，电转染后48小时，收集细胞，进行酶活测定和编辑效率检测。
编辑效率检测：
用无RNase的水(全式金，货号：GI201-01，)将设计合成的arRNA溶解至需要的浓度，-80℃保存。待GM06214细胞生长至约90％汇合度时消化细胞，终止消化后计数。取100万个细胞，添加200pmol的arRNA，至100ul体积，CA-137条件下电转染。电转染后48小时，对细胞计数，并测活率。将细胞移入无RNA酶的离心管中离心后弃上清，用QIAGEN RNA提取试剂盒提RNA(QIAGEN，货号：74134)。根据说明书，按5×10 5个细胞加入0.35ml的Buffer RLT Plus吹打混匀(若是冻存细胞直接提RNA，建议用PBS洗一次)。将裂解后的细胞液加入gDNA Eliminator旋转离心柱(spin column)，在≥8000g离心30s，扔掉柱子留下液体。加入一倍体积的70％的乙醇，吹打 混匀，立即进行下一步。将液体加入RNeasyMinElute旋转离心柱中，在≥8000g离心15s，弃废液。向RNeasyMinElute旋转离心柱中添加700μl的Buffer RW1，在≥8000g离心15s，弃废液。向RNeasyMinElute旋转离心柱中添加500μl的Buffer RPE，在≥8000g离心15s，弃废液。向RNeasyMinElute旋转离心柱中添加500μl 80％的乙醇，在≥8000g离心2分钟，弃废液。将RNeasyMinElute旋转离心柱放到一个新的2ml的收集柱中，开盖最高速度离心5分钟以便柱子干燥。将RNeasyMinElute旋转离心柱放到一个新的1.5ml的收集柱中，在柱膜中心位置滴加14μl无RNase水，最大速度离心1分钟洗脱RNA。
将提取的RNA用Nanodrop(Thermo，货号：Nanodrop2000)测浓度，用1ug RNA做反转录(Thermo，逆转录酶货号：28025013)。如表1配置反转录体系，65℃温育5分钟后，立即冰浴冷却。37℃继续温育50分钟。70℃，15分钟灭活反转录酶。在表3所示条件下，进行PCR。PCR结束后，取2ul PCR产物进行琼脂糖凝胶电泳，根据电泳结果初步判定PCR产物的浓度以及条带大小是否正确等。纯化后，PCR产物建库，送二代测序。
表1.反转录体系配置-1
  体积(ul)
总RNA(1ug) X
Oligo dT 1
10nM dNTP 1
RNase-Free Water 10-X
总体积 12
65℃，5分钟，立即转移到冰上。
表2.反转录体系配置-2
Figure PCTCN2020141506-appb-000002
Figure PCTCN2020141506-appb-000003
表3.PCR条件
Figure PCTCN2020141506-appb-000004
酶活性检测：
将GM06214细胞消化后离心，用28ul含0.1％Triton X-100的1×PBS重新悬浮在冰上裂解30分钟。然后将25ul的细胞裂解液添加到25ul含190μm 4-甲基伞形酮-α-L-异丁烯酸酶(4-methylumbelliferyl-α-L-iduronidase)的底物中(Cayman,2A-19543-500，溶解于0.4M甲酸钠缓冲液中，含有0.2％Triton X-100，PH3.5)。在暗处，37℃孵育90分钟。加入200ul 0.5M NaOH/Glycine溶液(北京化工，NAOH，货号：AR500G；索莱宝，Glycine货号：G8200，)，PH 10.3，灭活催化反应。在4℃离心2分钟。上清液被转移到96孔板上，荧光值测量用365nm激发波长和450nm激发射波长，使用Infinite M200仪器(TECAN)。
在本申请中，所有酶活性检测结果数据均以GM01323细胞中酶活性的倍数来表示。其中，GM01323细胞是来源于Scheie综合征患者的成纤维细胞。Scheie综合征是黏多糖贮积症中较为轻微的一种，症状比Hurler综合征轻得多。Scheie综合征患者往往预后较好，寿命一般为正常，可活至成年。Scheie综合征患者的成纤维细胞中IDUA酶活性是健康人野生型成纤维细胞的0.3％。
如图3所示，结果显示，当arRNA靶向mRNA前体(pre-mRNA)时能够表现出较高的酶活性和编辑效率，而靶向成熟mRNA(mature-mRNA)的arRNA表现出明显低的酶活性和编辑效率。因此在后面的实施例中所涉及到的arRNA均是靶向mRNA前体(pre-mRNA)的。
实施例4：在IDUA-报告子细胞系上电转染arRNA后IDUA靶位点编辑效率 的检测
如图4A所示，在慢病毒质粒上表达mCherry和GFP蛋白的序列之间插入了一段携带IDUA突变位点及上下游有各100bp左右的序列，构建质粒。将上述构建后的质粒包装成病毒，感染293T细胞，待其整合进基因组后，筛选出IDUA-报告子(reporter)单克隆细胞。这个单克隆细胞因为受到插入序列中IDUA突变位点TAG终止密码子的影响，只表达mCherry蛋白，而当细胞被arRNA编辑后，发生TAG->TGG，其后面的GFP蛋白才能够正常表达，GFP蛋白的表达可以看作是arRNA编辑细胞的编辑效率。我们优选设计了4条从51nt-111nt不同长度的arRNA，如下表4所示，细胞以实施例2中的电转染条件电转染不同长度的arRNA后，分别在第1-第7天检测细胞中GFP的比率，对编辑效率进行初步检测。从图4B中可以看编辑效率最高的序列是91nt：45-c-45，编辑的最高峰出现在第2天(48h)。由此可见，在arRNA的长度方面，并不是序列越长编辑效率越高。
表4：
Figure PCTCN2020141506-appb-000005
Figure PCTCN2020141506-appb-000006
实施例5：不同长度的arRNA电转染GM06214细胞后，检测不同时间点的 细胞内IDUA酶活性和细胞内RNA编辑效率
利用实施例2的电转染条件在GM06214细胞上电转染不同长度的arRNA(参见表4)，以实施例3中的方法，分别在电转染后第2，4，6，8，10，12，14天检测了细胞内的酶活性，在第2和4天检测了细胞内RNA被编辑的效率。从结果上看，如图5所示，91nt：45-c-45酶活性最高，在电转染后第6天IDUA酶活性仍然维持在高水平。从编辑效率上看，91nt和111nt呈现了大致相同的编辑效率。
实施例6：编辑位点A对应于arRNA的不同位置的编辑效率
以111nt长度的arRNA为例，从突变位点两端同时截断和从5’端或3’端二端分别截断，研究靶向碱基对应于arRNA不同位置的编辑效率。
本实验研究以lipofectmine RNAiMAX方式将arRNA导入细胞。首先我们将arRNA序列从两端同时截短，之后扩展到固定一端从另一端截短，得到14条arRNA和4个等长的随机序列，如下表5所示。通过转染后48小时IDUA酶活性和RNA编辑效率的检测发现，81nt：55nt-c-25nt(SEQ ID NO：14)，71nt：55nt-c-15nt(SEQ ID NO：15)，91nt：45nt-c-45nt(SEQ ID NO：9)，91nt：55nt-c-35nt(SEQ ID NO：13)，101nt：45nt-c-55nt(SEQ ID NO：17)在所检测的序列中IDUA酶活性和RNA编辑效率优于其它序列，如图6所示。
表5：
Figure PCTCN2020141506-appb-000007
Figure PCTCN2020141506-appb-000008
Figure PCTCN2020141506-appb-000009
实施例7：距离靶向碱基3’端的长度对编辑效率的影响
在实施例6中，在81nt：55-c-25、71nt：55-c-15序列上检测到了较高的IDUA酶活性和编辑效率。为找到最短且最优的3’端的长度，对3’端从距离靶向碱基的25nt开始(81nt：55-c-25)到10nt(66nt：55nt-c-10nt)之间的序列进行逐个截短，如表6所示。最终通过IDUA酶活测定筛选出靶向碱基距离3’端的最优长度为24nt-11nt，如图7A所示。此外，通过比较可见80nt：55nt-c-24nt(SEQ ID NO：22)、79nt：55nt-c-23nt(SEQ ID NO：23)、72nt：55nt-c-16nt(SEQ ID NO：30)、70nt：55nt-c-14nt(SEQ ID NO：31)、67nt：55nt-c-11nt(SEQ ID NO：34)可导致与SEQ ID NO：14及SEQ ID NO：15类似或更高的IDUA酶活性。
除此之外我们还在靶向鼠的IDUA突变位点(与人IDUA-W402X突变相对应的突变位点)的arRNA进行了靶向碱基距离3`端最优长度的筛选，arRNA的3`距离靶向碱基的长度从55nt开始，每5个碱基截短一次，如表7所示。通过IDUA酶活性的测定筛选出小鼠中靶向碱基距离3`端的长度为55nt-10nt，最优长度为55nt-10nt，如图7B所示。其中，111nt：55nt-c-50nt(SEQ ID NO：44)和66nt：55nt-c-10nt(SEQ ID NO：52)表现出较为优异的编辑效率。
表6：
Figure PCTCN2020141506-appb-000010
Figure PCTCN2020141506-appb-000011
Figure PCTCN2020141506-appb-000012
表7：
Figure PCTCN2020141506-appb-000013
Figure PCTCN2020141506-appb-000014
实施例8：5’端长度对编辑效率的影响
我们选取了2个不同长度的arRNA：76nt：55-c-20、71nt：55-c-15，在3’端长度固定不变的基础上，5’端逐渐截短，如表8所示。通过IDUA酶活性实验测定当5’端长度在55nt—45nt之间时，利用arRNA编辑后的细胞的IDUA酶活性较高，同时arRNA总长度在65nt—61nt之间IDUA酶活性有一个显著下降，如图8A所示。当3`端的长度固定在14nt时，5`端长度从51nt(即总长度66nt)开始逐个碱基截短，当5`端长度从51nt截短到50nt(即总长度为65nt)时，IDUA酶活性出现了显著下降，因此5`端的arRNA的截短要求总arRNA长度不能低于66nt,如图8B所示。
表8：
Figure PCTCN2020141506-appb-000015
Figure PCTCN2020141506-appb-000016
实施例9：RNA化学修饰对编辑效率的影响
RNA合成时对RNA进行不同类型的化学修饰，可以增加RNA的稳定性和减少脱靶的可能性。比较常见的对RNA进行的化学修饰有2`-OMe和硫代，我们实验选取了2个不同长度的arRNA：71nt、76nt，进行了2种化学修饰方式的不同组合，如表8所示。具体修饰方式所表示的意义如下所示：
CM1：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时序列中全部U均被2`-OMe修饰。
CM2：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的3’最近邻碱基为2`-OMe修饰的A。
CM3：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基的5’最近邻碱基为2`-OMe修饰的C。
CM4：序列前3个和后3个核苷酸分别被2`-OMe修饰，前3个和后3个核苷酸间连接均为硫代磷酸酯键连接；同时靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接。
CM5：除靶向碱基及与5’端与其邻近的5个碱基以及3’端与其最邻近的5个碱基之外，所有核苷酸均被2`-OMe修饰；同时序列前3个和后3个核苷酸间连接均为硫代磷酸酯键连接。
CM6：序列前5个和后5个核苷酸分别被2`-OMe修饰，并且前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
在GM06214细胞中转染不同的arRNA对细胞内IDUA进行编辑，收集转染后48小时的细胞进行IDUA酶活性测定，从实验结果上看，除CM5(第5种修饰：全部2`-OMe，除靶向碱基附近11nt)其余几种修饰组合均具体较好的酶活性，如图9所示。
表9
Figure PCTCN2020141506-appb-000017
Figure PCTCN2020141506-appb-000018
Figure PCTCN2020141506-appb-000019
注：ro代表核苷酸上不做修饰核和核苷酸之间连接酯键不修饰；r*代表核苷酸上不做修饰核和核苷酸之间用硫代磷酸酯键连接；mo代表核苷酸做2`-OMe修饰核苷酸之间连接酯键不修饰；m*代表核苷酸做2`-OMe修饰核苷酸之间用硫代磷酸酯键连接。
实施例10：化学修饰对编辑效率的影响
本实施例涉及靶向人IDUA的突变位点的3个优选arRNA和靶向小鼠IDUA突变位点1个优选arRNA，应用CM1方式的化学修饰方法，在GM06214细胞和MSPI MEF(MSPI小鼠胚胎成纤维细胞(MSPI mouse embryo fibroblast,MEF)，分离自IDUA纯合突变的胎鼠(idua W392X小鼠，B6.129S-Idua tm1.1Kmke/J)(Wang D,Shukla C,Liu X,et al.Characterization of an MPS I-H knock-in mouse that carries a nonsense mutation analogous to the human IDUA-W402X mutation[published correction appears in Mol Genet Metab.2010Apr；99(4):439].Mol Genet Metab.2010；99(1):62-71.doi:10.1016/j.ymgme.2009.08.002))细胞上进行浓度梯度实验。
从实施例7的实验结果中，我们选取了3个靶向人IDUA的arRNA，长度分别是：55nt-c-16nt,55nt-c-14nt,55nt-c-11nt，1个靶向小鼠IDUA的arRNA：55nt-c-10nt。此外，我们还选取了随机arRNA序列RM-67CM1作为对照。从实施例9中我们选取了CM1(全部u：2`-OMe)的化学修饰方式合成了上述靶向IDUA的arRNA，如表9所示。我们在人GM06214细胞和 MSPI小鼠MEF相比上进行了arRNA浓度梯度的转染。arRNA浓度为：160nM，80nM，40nM，20nM，10nM，5nM，2.5nM，1.25nM，0.625nM共9个浓度。细胞铺在6孔板中，铺板后24hrs进行转染，转染后48hrs消化细胞，取一半细胞进行IDUA酶活性检测，一半细胞提RNA进行编辑效率检测。酶活性检测的结果显示arRNA的转染浓度在大于等于2.5-5nM时，便能达到一个较高的酶活，而在大于等于10-20nM时其酶活达到平台期，如图10A和10C所示，同时相同浓度的arRNA转染在人细胞(GM06214)和小鼠细胞(MSPI MEF)中所产生的IDUA酶活性和编辑效率不同，如图10B和10D所示。
表10：
Figure PCTCN2020141506-appb-000020
Figure PCTCN2020141506-appb-000021
注：ro代表核苷酸上不做修饰和核苷酸之间连接酯键不修饰；r*代表核苷酸上不做修饰和核苷酸之间用硫代磷酸酯键连接；mo代表核苷酸做2`-OMe修饰核苷酸之间连接不修饰；m*代表核苷酸做2`-OMe修饰核苷酸之间用硫代磷酸酯键连接。
实施例11：通过arRNA编辑后IDUA可持续保持蛋白酶活性
本实验通过使用靶向人IDUA的突变位点的3个优选arRNA和靶向小鼠IDUA突变位点1个优选arRNA，并对其进行CM1方式的化学修饰，在GM06214细胞和MSPI MEF(mouse embryo fibroblast)细胞上获得显著提高的IDUA酶活性，并且可持续约3周以上。
在实施例10中我们进行了人和小鼠中优选出的靶向IDUA的arRNA转染后48hrs在不同浓度下的比较，此实施例中我们选取20nM的浓度在不同时间通过IDUA酶活性和编辑效率进行比较。如图11A所示，arRNA转染GM06214细胞后我们连续检测了14天的IDUA酶活，转染后酶活的高峰为第4天到第9天，第14天酶活性仍高于第2天，随后我们又在更长的时间点上检测了2天分别是转染后第17天和21天，从10A图上可以看到第21天的酶活仍高于转染后第1天，酶活约为GM01323的6到10倍。(编辑效率待补充，图11B)。在MSPI MEF细胞中arRNA转染后我们连续检测了8天IDUA酶活性，由图11C中可以看到，arRNA转染后24hrs开始酶活性约 为GM1323细胞的2倍直至第8天，从图11D的编辑效率检测中可以看到，IDUA的编辑效率高峰为24hrs，而后持续下降。
通过人和小鼠数据的比较我们发现，arRNA在小鼠中的编辑高峰是转染后24hrs，而人中是48hrs，编辑后IDUA蛋白酶活性的持续时间在人的细胞中大于21天，在小鼠中大于8天。
实施例12：arRNA递送方式对编辑效率的影响
本实验涉及在原代培养的人和小鼠的肝脏细胞上利用LEAPER技术对野生型的PPIB基因位点进行编辑，通过不同的方式进行arRNA递送，筛选出最优的递送方式。
“PPIB”是指人NM_000942(PPIB Genomic chr15(-)：64163082)UTR区域野生位点或小鼠NM_011149(PPIB Genomic chr9(+)：66066490)UTR区域野生位点。其可以是成熟的mRNA也可以是mRNA前体。PPIB的UTR区段存在一个TAG，本实施例中将所述TAG中的A作为靶标进行编辑，以测试本申请中的arRNA在肝脏细胞中的编辑效率。
我们设计并合成了靶向PPIB UTR区域的arRNA(55nt-c-15nt)，如表11所示。合成的arRNA部分溶解分装后存于-80度用于Lipo(lipofectmine RNAiMAX)的转染，其余包装成LNP。LNP的包装参考文献中方法(参考文献：Witzigmann D,Kulkarni J A,Leung J,et al.Lipid nanoparticle technology for therapeutic gene regulation in the liver[J].Advanced Drug Delivery Reviews,2020.；Kauffman K J,Dorkin J R,Yang J H,et al.Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs[J].Nano letters,2015,15(11)：7300-7306.；Reis J,Kanagaraj S,Fonseca A,et al.In vitro studies of multiwalled carbon nanotube/ultrahigh molecular weight polyethylene nanocomposites with osteoblast-like MG63cells[J].Brazilian Journal of Medical and Biological Research,2010,43(5)：476-482.)。
人肝脏原代细胞购买于LONZA(货号：HUCPI)，细胞按照厂家说明书复苏培养(复苏培养基货号：MCHT50；铺板培养基货号：MP100)。细胞贴壁后更换为肝细胞维持培养基5C(参考文献：Xiang C,Du Y,Meng G,et  al.Long-term functional maintenance of primary human hepatocytes in vitro[J].Science,2019,364(6438)：399-402)。
小鼠肝脏原代细胞分离于C57BJ小鼠(参考文献：Charni-Natan M,Goldstein I.Protocol for Primary Mouse Hepatocyte Isolation[J].STAR protocols,2020,1(2)：100086.)分离肝脏细胞贴壁后更换为维持培养基5C。
人的肝脏细胞复苏后24hrs进行arRNA递送，LNP和Lipo的递送浓度均为20nM。小鼠肝脏细胞分离培养24hrs后进行arRNA递送，LNP和Lipo的递送浓度均为20nM。人和小鼠的肝脏细胞分别于arRNA递送后24hrs和48hrs收取RNA，通过二代测序检测编辑效率。从图12A中可以看到，在人的肝脏细胞中，2种方式的arRNA递送后48hrs编辑效率均高于24hrs，同时LNP递送的arRNA产生的编辑效率在24hrs优于Lipo的递送，48hrs两种递送方式编辑效率近似。从图12B中可以看到，在小鼠的肝脏细胞中，2种方式的arRNA递送后24hrs编辑效率均高于48hrs，同时Lipo递送arRNA产生的编辑效率在24hrs和48hrs均高于同时间LNP的递送。
因此，通过肝脏原代细胞中PPIB位点编辑效率的比较，我们可以得出小鼠肝脏细胞中编辑的高峰位于arRNA递送后24hrs，而人的肝细胞中编辑高峰位于48hrs，这与在人的GM06214细胞和小鼠的MSPI MEF细胞中的数据一致。人肝脏细胞中arRNA的递送，LNP优于或等于Lipo的递送。小鼠的肝脏细胞中arRNA的递送，Lipo远优于LNP的递送。
表11：
Figure PCTCN2020141506-appb-000022
实施例13：LNP递送的编辑效率
本实验涉及在原代培养的人和小鼠的肝脏细胞上利用LNP递送IDUA的arRNA对IDUA的编辑效率的研究。
我们设计并合成了靶向人IDUA CDS区域野生位点的arRNA,如表12所示。将人和小鼠的arRNA(20nM)分别通过LNP递送至人原代肝脏细胞和 小鼠原代肝脏细胞，递送后分别于24小时和48小时检测编辑效率，如图13所示。从图中我们可以看到在体外原代培养的肝脏细胞中IDUA的编辑效率中，人的IDUA在48小时编辑效率可以达到约30％，小鼠中最高编辑效率出现在24小时约为15％。进一步表明LNP可以在人的细胞中，尤其是人的肝脏原代细胞中，达到更高的递送效率。
表12
Figure PCTCN2020141506-appb-000023
实施例14：针对MSPI模型小鼠IDUA的arRNA的治疗作用
本实验使用的是MPSI模型小鼠(idua W392X小鼠，B6.129S-Idua tm1.1Kmke/J)(Wang D,Shukla C,Liu X,et al.Characterization of an MPS I-H knock-in mouse that carries a nonsense mutation analogous to the human IDUA-W402X mutation[published correction appears in Mol Genet Metab.2010 Apr；99(4):439].Mol Genet Metab.2010；99(1):62-71.doi:10.1016/j.ymgme.2009.08.002)，该突变与人IDUA-W402X突变相对应，该突变可提前终止蛋白质合成，在Hurler黏多多糖症(MPS I-H)综合征患者中普遍存在。α-l-iduronidase酶的缺乏导致这种溶酶体储存疾病。5周龄、10周龄和30周龄纯合子小鼠的大脑和肝脏组织中均未检测到α-l-iduronidase活性。尽管这种突变纯合子的小鼠是有活力和可育的，但它们的平均寿命是69周。纯合子表现出尿中糖胺聚糖(GAGs)排泄量的进行性增加，以及组织中GAGs的进行性积累。稳态Idua mRNA水平降低30-50％。组织学分析显示浦肯野细胞、髓质神经元的胞质中溶酶体储存内含物的累进积累，以及随年龄增长而增加的泡沫状巨噬细胞浸润。x线片显示颧骨弓和股骨增厚在15周龄时可见，35周龄时增厚。35周龄时，股骨骨密度增加，体脂百分比减少。我们将筛选出的靶向小鼠突变IDUA的arRNA：55nt-c-10ntCM1(SEQ ID NO：52),包装成LNP。将不同浓度的arRNA通过尾静脉进行注射，所述不同浓度分别为0.1mg/kg；0.5mg/kg；2mg/kg；10mg/kg。给药后24hrs取小鼠肝脏细胞进行IDUA编辑效率检测，如图14 所示。给药后24小时可以在10mg/kg组检测到约2％的编辑效率。该实施例结果证明了针对MSPI模型小鼠IDUA的arRNA在体内可实现对肝脏细胞中突变IDUA基因的精准编辑，纠正IDUA突变，达到治疗MPSI的目的。

Claims (23)

  1. 一种基于LEAPER技术靶向编辑靶标细胞中靶标RNA的方法，其中所述靶标RNA为IDUA基因转录本中含有G到A突变的RNA，该方法包括：
    将包含用于编辑靶标RNA的腺苷脱氨酶募集RNA(arRNA)或编码所述arRNA的构建体递送至所述靶标细胞，其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且其中所述arRNA能够募集作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷(A)脱氨基。
  2. 如权利要求1中所述的方法，其中所述arRNA包含与靶标A配对的碱基C、A、U或G。
  3. 如权利要求1-2中任一项所述的方法，其中所述arRNA长约151-61nt、131-66nt、121-66nt、111-66nt、91-66nt或81-66nt。
  4. 如权利要求3中所述的方法，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15n或24nt-11nt。
  5. 如权利要求3或4中所述的方法，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt或55nt-45nt。
  6. 如权利要求1-5中所述的方法，其中所述靶标细胞是人的细胞。
  7. 权利要求1-6中任一项的方法，其中所述靶标RNA为包含NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变位点的RNA。
  8. 权利要求1-7中任一项的方法，其中所述arRNA包含以下的序列：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31或SEQ ID NO：34。
  9. 如权利要求1-5中任一项所述的方法，其中所述arRNA包含选自以下的序列：SEQ ID NO：44或SEQ ID NO：52。
  10. 如权利要求1-9中任一项所述的方法，其中所述arRNA是经化学修饰的。
  11. 如权利要求10中所述的方法，其中所述化学修饰包含2-O’-甲基化(2`-OMe)或硫代磷酸酯修饰。
  12. 如权利要求11中所述的方法，其中所述化学修饰选自如下的一项或多项：
    序列前3个和后3个核苷酸分别被2`-OMe修饰，
    前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
    序列中全部U均被2`-OMe修饰，
    靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
    靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
    靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
    前5个和后5个核苷酸分别被2`-OMe修饰，和
    前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
  13. 如权利要求1-9中任一项所述的方法，其中所述编码所述arRNA的构建体为线性核酸链、病毒载体或质粒。
  14. 如权利要求13中所述的方法，其中所述病毒载体为腺相关病毒(AAV)载体或慢病毒表达载体。
  15. 如权利要求1-14中任一项所述的方法，其中所述递送方式为电转染、脂质体转染、脂质-纳米颗粒(lipid nanoparticle,LNP)递送或感染。
  16. 如权利要求15中所述的方法，通过LNP将包含用于编辑靶标RNA的腺苷脱氨酶募集RNA(arRNA)或编码所述arRNA的构建体递送至所述靶标细胞。
  17. 如权利要求1-16中任一项所述的方法，其中所述arRNA的递送浓度≥2.5，≥5nM，≥10nM，≥15nM，或≥20nM。
  18. 一种用于通过LEAPER技术靶向编辑靶标细胞中靶标RNA的arRNA或其编码序列，所述arRNA包含如下任一序列或由如下任一序列组成：SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：34、SEQ ID NO：44或SEQ ID NO：52。
  19. 包含权利要求18所述arRNA或其编码序列的质粒、病毒载体、脂质体或脂质纳米颗粒。
  20. 包含权利要求18所述arRNA或其编码序列、或权利要求19所述质粒、病毒载体、脂质体或脂质纳米颗粒的组合物或生物制品。
  21. 一种治疗个体中MPS IH的方法，包括用权利要求1-17中任一项所述的方法校正所述个体的靶标细胞中与MPS IH疾病相关的G到A的突变。
  22. 权利要求20的方法，其中所述突变为NM_000203.4(IDUA)-c.1205G-A(p.Trp402Ter)突变。
  23. 如权利要求20或21所述的方法，其中所述arRNA的使用频次为≥21天/次、≥17天/次、≥14天/次或≥10天/次。
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US12018257B2 (en) 2016-06-22 2024-06-25 Proqr Therapeutics Ii B.V. Single-stranded RNA-editing oligonucleotides
US11702658B2 (en) 2019-04-15 2023-07-18 Edigene Therapeutics (Beijing) Inc. Methods and compositions for editing RNAs
US11661596B2 (en) 2019-07-12 2023-05-30 Peking University Targeted RNA editing by leveraging endogenous ADAR using engineered RNAs
US11827880B2 (en) 2019-12-02 2023-11-28 Shape Therapeutics Inc. Therapeutic editing
WO2023152371A1 (en) 2022-02-14 2023-08-17 Proqr Therapeutics Ii B.V. Guide oligonucleotides for nucleic acid editing in the treatment of hypercholesterolemia
WO2024013361A1 (en) 2022-07-15 2024-01-18 Proqr Therapeutics Ii B.V. Oligonucleotides for adar-mediated rna editing and use thereof
WO2024013360A1 (en) 2022-07-15 2024-01-18 Proqr Therapeutics Ii B.V. Chemically modified oligonucleotides for adar-mediated rna editing
WO2024084048A1 (en) 2022-10-21 2024-04-25 Proqr Therapeutics Ii B.V. Heteroduplex rna editing oligonucleotide complexes
WO2024110565A1 (en) 2022-11-24 2024-05-30 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of hereditary hfe-hemochromatosis
WO2024115635A1 (en) 2022-12-01 2024-06-06 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of aldehyde dehydrogenase 2 deficiency
WO2024121373A1 (en) 2022-12-09 2024-06-13 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of cardiovascular disease
WO2024153801A1 (en) 2023-01-20 2024-07-25 Proqr Therapeutics Ii B.V. Delivery of oligonucleotides
WO2024175550A1 (en) 2023-02-20 2024-08-29 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of atherosclerotic cardiovascular disease
WO2024206175A1 (en) 2023-03-24 2024-10-03 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of neurological disorders
WO2024200278A1 (en) 2023-03-24 2024-10-03 Proqr Therapeutics Ii B.V. Chemically modified antisense oligonucleotides for use in rna editing
WO2024200472A1 (en) 2023-03-27 2024-10-03 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of liver disease

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