WO2021136404A1 - 一种治疗Usher综合征的方法和其组合物 - Google Patents

一种治疗Usher综合征的方法和其组合物 Download PDF

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WO2021136404A1
WO2021136404A1 PCT/CN2020/141501 CN2020141501W WO2021136404A1 WO 2021136404 A1 WO2021136404 A1 WO 2021136404A1 CN 2020141501 W CN2020141501 W CN 2020141501W WO 2021136404 A1 WO2021136404 A1 WO 2021136404A1
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seq
arrna
rna
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editing
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French (fr)
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刘能银
易泽轩
袁鹏飞
赵艳霞
汤刚斌
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Edigene Inc
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Edigene Inc
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Priority to EP20909536.3A priority patent/EP4086346A4/en
Priority to MX2022008197A priority patent/MX2022008197A/es
Priority to AU2020418026A priority patent/AU2020418026A1/en
Priority to JP2022540983A priority patent/JP2023509177A/ja
Priority to CA3163280A priority patent/CA3163280A1/en
Priority to PE2023003048A priority patent/PE20250386A1/es
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Priority to PH1/2022/551594A priority patent/PH12022551594A1/en
Priority to CN202080086703.4A priority patent/CN114829598A/zh
Priority to BR112022012921A priority patent/BR112022012921A2/pt
Priority to KR1020227024955A priority patent/KR20220118512A/ko
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Definitions

  • This application belongs to the field of gene editing therapy, and specifically relates to a method for targeted editing of Usher Syndrome Type II related gene mutations based on LEAPER (Leveraging Endogenous ADAR for Programmable Editing on RNA) technology.
  • Usher syndrome also known as hereditary deafness - retinitis pigmentosa syndrome, was described in 1914 by the name of Charles Howard Usher and 12, is a mutation caused by the gene, resulting in congenital patients or gradual loss of vision and hearing ability An autosomal recessive rare disease.
  • the incidence of Usher syndrome is about 1 in 12,500 8 in Germany, 1/28,000 4 in Norway, and 1/23,000 in the United States. It is estimated that more than half of the 16,000 blind and deaf people in the United States suffer from Usher syndrome 1 .
  • Usher syndrome 1 There are tens of thousands or even hundreds of thousands of patients in the world who are in urgent need of treatment.
  • Type I is severe congenital deafness, severe visual impairment occurs around 10 years old, and type II is moderate to severe congenital deafness, with a gradual loss of vision between the ages of 10 and 50.
  • Types III and IV are relatively rare, and the condition is relatively mild.
  • the auditory manifestations are sensorineural hearing loss, and the time of visual loss is uncertain.
  • type I has an early onset, the intervention window is small, while types III and IV are relatively mild.
  • the visual loss of type II patients will start gradually from the age of 10 to 20. The initial stage of the onset is night blindness and eventually loss of vision ( Figure 1). This allows us to have a longer therapeutic intervention window and hopefully terminate the gradual loss of vision in patients. .
  • the USH2A gene encodes a protein called Usherin, which plays an important function in vision, mainly in the photosensitive cone and rod cells, which connect the inner segment and outer segment ( The important role of Outer Segment).
  • Usherin a protein that plays an important function in vision, mainly in the photosensitive cone and rod cells, which connect the inner segment and outer segment ( The important role of Outer Segment).
  • USH2A mutations cause degeneration of cone cells and rod cells 5 death.
  • a 2017 study by Neuhaus et al. showed that 13% of the USH2A gene mutation cases sequenced were NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter), and this type of mutation is all mutation types.
  • USH2A gene therapy Another common research and development direction for USH2A gene therapy is through exon skipping. Because some of the mutations of the USH2A gene are caused by a frame shift or nonsense mutation of an exon, as long as the exon can be specifically skipped during the RNA splicing process , The sequence after the exon can be translated normally.
  • a common practice is to introduce a short segment of anti-sense nucleotides (Anti-Sense Oligo, ASO), so as to specifically skip the exons targeted by the translated nucleotides during the splicing process. This method is similar to the previous method, because the mutated exons are skipped, the full-length Usherin is still not obtained in the end.
  • Anti-Sense Oligo ASO
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • CRISPR-based DNA editing technology requires the exogenous expression of Cas9 or other nucleases with similar functions, causing the following problems.
  • nucleases that require exogenous expression usually have a relatively large molecular weight, which makes the efficiency of delivering them into the body through viral vectors drastically reduced.
  • this method has potential for off-target nucleases, which will lead to potential carcinogenic risks in its applications.
  • exogenously expressed Cas9 and other similar nucleases are 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 other hand, it may also neutralize the exogenously expressed nuclease, thereby losing its proper activity and affecting the therapeutic effect.
  • RNA editing technology called REPAIR (RNA Editing for Programmable A to I Replacement), which expresses Cas13-ADAR fusion protein and single guide RNA ( Single guide RNA, sgRNA) can achieve the editing of target RNA from A to I, but this method, like CRISPR technology, still requires the expression of foreign proteins. Unable to solve the problems caused by the expression of foreign proteins.
  • RESTORE Recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing, Merkle et al., 2019.
  • RESTORE can get rid of the dependence on foreign proteins, but the RESTORE technology needs to have high editing efficiency under the premise of the existence of IFN- ⁇ , and IFN- ⁇ is a key factor that determines the development and severity of autoimmunity, which makes this technology useful
  • IFN- ⁇ is a key factor that determines the development and severity of autoimmunity, which makes this technology useful
  • the application in the medical field is greatly reduced.
  • a guide RNA is also used in the RESTORE technology, and the guide RNA it uses is a chemically synthesized oligonucleotide, and the synthesized oligonucleotide requires artificial introduction of a large number of chemical modifications to ensure its stability. .
  • these chemical modifications some of them are unnatural modifications, which make the oligonucleotide may be toxic or immunogenic; and some of the modifications will result in different conformations of the same base chain, making it possible for the same RNA sequence to have different conformations. Dozens of different conformational combinations, which increase the difficulty of its delivery into cells.
  • the technology has greater advantages in the process of transforming into the medical field; on the other hand, the technology only realizes the editing of adenosine A to creatinine I at the transcription level (creatinine I will be recognized as guanine during protein translation. G), without changing the sequence in the genome, so it is safer and more convenient.
  • LEAPER technology can not only be completed by chemical synthesis of RNA, 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.
  • AAV adeno-associated virus
  • This application relates to a method for targeted editing of target RNA containing G to A mutations in the USH2A gene transcript based on LEAPER technology, by recruiting the adenosine deaminase used for editing target RNA to recruit RNA (arRNA) or the construction of encoding the arRNA
  • arRNA adenosine deaminase
  • ADAR adenosine deaminase
  • this application creatively applies it to target RNA containing G to A mutations in the USH2A gene transcript, for example, with NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutation
  • the USHER syndrome II related target, RAN achieves the purpose of treating USHER syndrome II by reversing and repairing the mutation site.
  • the purpose of this application is to target Usher syndrome type II pathogenic genes, such as the mutant NM_206933.2(USH2A)_c.11864G>A (p.Trp3955Ter) pathogenic gene, which accounts for the highest proportion of human USH2A, and to provide a new kind of pathogenic gene.
  • the technical solution does not require the introduction of excessively large nucleic acid or foreign protein molecules, so that it can accurately edit the mutation site on the target RNA and restore the complete function of the Usherin protein.
  • this application relates to:
  • a method for targeted editing of a target RNA in a cell based on LEAPER technology wherein the target RNA is an RNA containing a G to A mutation in the transcript of the USH2A gene (for example, the human USH2A gene), and the method includes:
  • a construct containing an adenosine deaminase recruiting RNA (arRNA) for editing a target RNA or a construct encoding the arRNA is introduced into the cell, wherein the arRNA includes a complementary RNA sequence that hybridizes with the target RNA , And wherein the arRNA can recruit adenosine deaminase (ADAR) acting on RNA to deaminate the target adenosine in the target RNA.
  • ADAR adenosine deaminase
  • the target RNA is mature mRNA or pre-mRNA (Pre-mRNA).
  • the target RNA is pre-mRNA (Pre-mRNA).
  • the target base that is introduced into the arRNA to pair with the target A in the target sequence is C. In some embodiments, the target base introduced into the arRNA to be paired with the target A in the target sequence is A. In some embodiments, the target base introduced into the arRNA to be paired with target A in the target sequence is U.
  • RNA is a human USH2A gene RNA transcribed with a mutation site of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).
  • arRNA is selected from: SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO : 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • virus is an adeno-associated virus (AAV) or a lentivirus.
  • AAV adeno-associated virus
  • An arRNA for targeted editing of a target RNA containing a G to A mutation in a USH2A gene transcript or its coding sequence comprising a sequence selected from or consisting of a sequence selected from the following: SEQ ID NO : 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • a construct or delivery body comprising the arRNA of item 16.
  • AAV adeno-associated virus
  • the application provides a composition, preparation, kit or biological product comprising the above-mentioned arRNA, or the above-mentioned construct or delivery body.
  • a method for treating Usher type II syndrome in an individual comprising using the method described in any one of items 1-15 to correct G to A mutations related to Usher type II syndrome in cells of the individual.
  • LEAPER technology only requires the introduction of short RNA, which can be limited to 151, 131, 121, and 111 nucleotides, which makes the length of the introduced fragments far less than the upper limit of the delivery length of the adeno-associated virus. Therefore, compared with directly introducing the USH2A gene, this technology is very suitable for the application of adeno-associated virus vectors.
  • the eye is an immune-exempt area, its ability to clear the adeno-associated virus is very poor, which allows the adeno-associated virus to stay in the eye for a long time.
  • the functioning cells are nerve cells. Under normal circumstances, these cells will not divide.
  • Figure 1 shows a schematic diagram of the progression of night blindness in Usher syndrome type II patients 13 .
  • Figure 2 shows the 138 cases of patients with disease-causing mutations Usher profile 7.
  • Figure 3 shows the analysis of USH2A gene mutation types.
  • Figure 7 shows the analysis of USH2A gene mutation types.
  • Figure 4 shows a schematic diagram of the reporting system design reflecting the editing efficiency of the USH2A gene mutation site.
  • Figure 5 shows a schematic diagram of the evaluation index of the editing efficiency of the USH2A gene mutation site.
  • Figure 6 shows the test results of editing efficiency of arRNA of different lengths.
  • Figure 7 shows the FITC channel intensity of false positive cells in the reporter system.
  • Figure 8 shows the relationship LEAPER art arRNA length 10 and editing efficiency.
  • Figure 9 shows the editing efficiency test results of arRNA truncated from 5'and 3'to the middle.
  • Figure 10 shows the editing efficiency test results of fixed arRNA 3'with a length of 25 nt and gradually shortening from 5'.
  • Figure 11 shows the effect of targeted base positions on editing efficiency.
  • Figure 12 shows the comparison of repeated test results of the editing efficiency of the same batch of arRNA.
  • 293T represents the 293T of the uninfected reporting system; the only reporter system is the 293T infected with the reporting system;
  • Opti-DMEM is the blank control of the medium without RNAi MAX during transfection; the delivery vector control is only
  • the RNAi MAX control corresponds to the control (NC) well in the previous test;
  • the random sequence RNA is a 91 nt random RNA sequence that does not match the human genome (uaauccugaauauaucgcgcaauuccccagcagagaacaucgcggugugaacgucccuuuauaccgggcagguauagcugaaaucagcguggc (SEQ ID NO: 35)).
  • Figure 13 shows the comparison of repeated test results of editing efficiency obtained by second-generation sequencing of the same batch of arRNA.
  • Figure 14 shows the editing efficiency of arRNA delivered by AAV2 and AAV5 in vivo when intravitreal injection is used.
  • Figure 15 shows the editing efficiency of arRNA delivered by AAV5 and AAV8 in vivo over time when injected into the subretinal space.
  • this application provides a method based on LEAPER technology for targeted editing of target RNA transcribed in cells containing NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutation site (hereinafter referred to as mutation) Editing method), an arRNA targeting the mutation site of NM_206933.2(USH2A)_c.11864G>A (p.Trp3955Ter), a construct containing the arRNA, a cell prepared by the mutation editing method, A kit for editing target RNA in cells, a method for treating diseases caused by the mutation of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) in an individual (hereinafter referred to as the treatment method), and A preparation used in the mutation editing method or the treatment method.
  • mutation a method based on LEAPER technology for targeted editing of target RNA transcribed in cells containing NM_206933.2(USH2A)_c
  • 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
  • LSAPER 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.
  • arRNA is a type of guide RNA.
  • guide RNA refers to the ability to complementally hybridize with the target RNA to recruit an enzyme that can modify the target base to modify the target base.
  • the guide RNA includes arRNA and gRNA used in other editing systems such as CRISPR.
  • 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.
  • introduction 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 "introduction” includes point transfection, liposome transfection, lipid-nanoparticle delivery and the like.
  • 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. And finally enter the cell nucleus technology.
  • 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_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutation refers to the G to A mutation in the human USH2A gene corresponding to position 11864 of the NM_206933.2 (USH2A) gene transcript.
  • the tryptophan (Trp) coding sequence at position 3955 of the peptide chain that causes the translation of the transcript is converted into a stop codon, so that the final translated amino acid deletes all amino acids after position 3955, thereby losing the protein activity of Usherin.
  • Patients with this mutation will experience degenerative death of cones and rods, eventually leading to blindness.
  • the scheme in this application can restore the activity of Usherin protein by reversing this mutation at the transcription level.
  • the NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutation site is also referred to as the USH2A pathogenic site.
  • target RNA refers to the target RNA to be edited, which is transcribed from the USH2A gene and contains G to A mutations.
  • the target RNA may be mature mRNA or mRNA precursor, and in the present application, mRNA precursor is more preferred.
  • the "target RNA” is an RNA transcribed with a mutation site containing NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).
  • the A base formed by transcription from the G to A mutation site in the "target RNA” is called “target adenosine" or "target A”.
  • targeting base The base opposite to "target A” in arRNA is called “targeting base”.
  • AAV refers to “adeno-associated virus” or “adeno-associated virus vector”.
  • AAV belongs to the Parvoviridae family. It is a non-enveloped single-stranded linear DNA virus.
  • the adeno-associated virus can be used to transfer foreign genes into animal tissues and cells. The transferred foreign gene can exist in a state of free nucleic acid in a relatively stable state outside the genomic gene of the host and be expressed.
  • adeno-associated virus serotypes (12 species), including, for example, AAV1, AAV2, AAV5, AAV8, and AAV9.
  • AAV1, AAV2, AAV5, AAV8, and AAV9 adeno-associated virus serotypes
  • patient or “subject” can be used interchangeably. Unless otherwise specified herein, “patient” or “subject” as used herein refers to the genome with NM_206933.2 (USH2A) _c.11864G>A (p.Trp3955Ter) mutation in human patients.
  • 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.
  • the combination of the biomacromolecule and the substance that encapsulates the biomacromolecule or is connected to the biomacromolecule to promote the biomacromolecule to enter the cell through the cell membrane is referred to as the "delivery body".
  • the delivery body For example, liposomes, LNPs, exosomes, virus particles, etc. packed with nucleic acid molecules.
  • 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 specific nucleic acid sequence may be a DNA sequence or an RNA sequence.
  • the nucleic acid sequence directly performs its function without being transcribed, translated, or expressed.
  • 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.
  • 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.
  • arRNA is modified to make it more stable after being introduced into cells.
  • the length of the target base from the 3'end refers to the number of all bases from the 3'nearest neighbor of the target base to the 3'most terminal base; the target base is 5'from the 5'end. Length refers to the total number of bases from the 5'nearest base to the 5'most terminal base of the targeted base.
  • This application provides a method for targeted editing of target RNA in cells based on LEAPER technology, wherein the target RNA is an RNA containing a G to A mutation in the USH2A gene transcript, and the method includes:
  • a construct containing an adenosine deaminase recruiting RNA (arRNA) for editing a target RNA or a construct encoding the arRNA is introduced into the cell, wherein the arRNA includes a complementary RNA sequence that hybridizes with the target RNA , And wherein the arRNA can recruit adenosine deaminase (ADAR) acting on RNA to deaminate the target adenosine in the target RNA.
  • ADAR adenosine deaminase
  • the target RNA is mRNA or pre-mRNA (Pre-mRNA), preferably, Pre-mRNA.
  • Pre-mRNA pre-mRNA
  • the length of the arRNA is> 51 nt, preferably> 61 nt, more preferably> 65 nt. In some embodiments, 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 arRNA is selected from any natural number of 66nt-131nt, such as 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 78nt, 79nt, 80nt, 83nt , 85nt, 88nt, 91nt, 96nt, 98nt, 100nt, 105nt, 108nt, 100nt, 105nt, 110nt, 115nt, 120nt, 125nt, 130nt, etc. Under the condition that the transfection efficiency can be kept the same, preferably, the length of the arRNA is about 71 nt, for example, any natural number of nt from 66 nt to 76 nt.
  • the length of the target base in the arRNA from the 3'end is> 5 nt, such as ⁇ 7 nt, ⁇ 10 nt, ⁇ 15 nt, such as 16-55 nt, 20-50 nt, 25-45 nt, 25-35 nt, or 25 nt. Any natural number of -30nt.
  • the length of the target base in the arRNA from the 5'end is ⁇ 25 nt, such as ⁇ 30, ⁇ 35, ⁇ 45, such as 46-90 nt, 50-85 nt, 55-80 nt, 60-75 nt, or 65 nt. Any natural number of nt in -70nt.
  • the preferred order of the target base relative to the target base on the target RNA is C, A, U, or G in descending order. That is, under normal circumstances, the length and the sequence other than the target base are the same, and the editing efficiency of the arRNA whose target bases are C, A, U, and G decreases sequentially. Therefore, most preferably, the arRNA The target base of is C, followed by A, then U, and G last.
  • the target RNA is an RNA containing a mutation site transcribed with NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).
  • the arRNA is selected from: SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the arRNA is selected from: SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, more preferably SEQ ID NO: 8 or SEQ ID NO: 9.
  • the construct is selected from linear nucleic acids, viral vectors, or plasmids.
  • the linear nucleic acid includes single-stranded nucleic acid, such as single-stranded RNA, single-stranded DNA; and double-stranded nucleic acid, such as double-stranded DNA, and double-stranded RNA.
  • the linear nucleic acid may be chemically modified, such as 2'-O-Me modification and/or phosphorothioate bond modification.
  • the linear nucleic acid is chemically synthesized in vitro.
  • the linear nucleic acid is synthesized by cells or organisms and then obtained by isolation and extraction.
  • the construct is an adeno-associated virus (AAV) vector or a lentiviral vector.
  • AAV adeno-associated virus
  • the AAV vector is packaged into the AAV2, AAV5 or AAV8 capsid and then introduced into the cell by infection.
  • the AAV vector is packaged into the AAV8 capsid and then introduced into the cell by infection.
  • the construct inserts the arRNA coding sequence into the cell genome for long-term expression.
  • the construct allows the arRNA coding sequence to exist and express as free nucleic acid outside the cell genome.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a nerve cell. In some embodiments, the nerve cell is a sensory nerve cell. In some embodiments, the sensory nerve cells are selected from optic nerve cells and auditory nerve cells. In some embodiments, the optic nerve cells are cone cells and/or rod cells.
  • the introduction is electrotransfection, liposome transfection, lipid-nanoparticle delivery, or viral infection.
  • it is preferably a virus infection.
  • a virus infection For example, by wrapping a viral construct encoding arRNA into a viral capsid to form an infective virus particle, such as AAV or lentiviral particle, and introducing the arRNA coding sequence into the cell by the virus particle infecting the cell in.
  • This application also provides an arRNA (hereinafter referred to as functional arRNA), which can be used in the method for targeted editing of target RNA in cells based on LEAPER technology as described above.
  • the method is based on LEAPER technology to target editing a target RNA transcribed in a cell containing a mutation site of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).
  • the arRNA includes a complementary RNA sequence that hybridizes with the target RNA, and wherein the arRNA can recruit adenosine deaminase (ADAR) acting on the RNA to deaminate the target adenosine in the target RNA.
  • ADAR adenosine deaminase
  • the target RNA targeted by the arRNA is selected from mRNA or Pre-mRNA, preferably Pre-mRNA.
  • the length of the arRNA is> 51 nt, preferably> 61 nt, more preferably> 65 nt. In some embodiments, 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 arRNA is selected from any natural number of 66nt-131nt, such as 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 78nt, 79nt, 80nt, 83nt , 85nt, 88nt, 91nt, 96nt, 98nt, 100nt, 105nt, 108nt, 100nt, 105nt, 110nt, 115nt, 120nt, 125nt, 130nt, etc. Under the condition that the transfection efficiency can be kept the same, preferably, the length of the arRNA is about 71 nt, for example, any natural number of nt from 66 nt to 76 nt.
  • the length of the target base in the arRNA from the 3'end is> 5 nt, such as ⁇ 7 nt, ⁇ 10 nt, ⁇ 15 nt, such as 16-55 nt, 20-50 nt, 25-45 nt, 25-35 nt, or 25 nt. Any natural number of -30nt.
  • the length of the target base in the arRNA from the 5'end is ⁇ 25 nt, such as ⁇ 30, ⁇ 35, ⁇ 45, such as 46-90 nt, 50-85 nt, 55-80 nt, 60-75 nt, or 65 nt. Any natural number of nt in -70nt.
  • the preferred order of the target base relative to the target base on the target RNA is C, A, U, or G in descending order. That is, under normal circumstances, the length and the sequence other than the target base are the same, and the editing efficiency of the arRNA whose target bases are C, A, U, and G decreases sequentially. Therefore, most preferably, the arRNA The target base of is C, followed by A, then U, and G last.
  • the arRNA is selected from: SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the arRNA is selected from: SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, more preferably SEQ ID NO: 8 or SEQ ID NO: 9.
  • the target base editing efficiency of the arRNA introduced and matched with the target A in the target sequence is C, A, U, G in descending order, that is, for length and target bases other than The sequences are all the same, and the editing efficiency of arRNAs whose target bases are C, A, U, and G respectively decrease sequentially. Therefore, most preferably, the target base of the arRNA is C, followed by A, and then followed by U, and finally G.
  • This application also provides a construct encoding arRNA (hereinafter referred to as a functional construct), wherein the functional construct can be used in the method for targeted editing of target RNA in cells based on LEAPER technology as described above.
  • the method is based on LEAPER technology to target editing a target RNA transcribed in a cell containing a mutation site of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).
  • the arRNA includes the functional arRNA as described above.
  • the construct is selected from linear nucleic acids, viral vectors, or plasmids.
  • the linear nucleic acid includes single-stranded nucleic acid, such as single-stranded RNA, single-stranded DNA; and double-stranded nucleic acid, such as double-stranded DNA, and double-stranded RNA.
  • the linear nucleic acid may be chemically modified, such as 2'-O-Me modification and/or phosphorothioate bond modification.
  • the linear nucleic acid is chemically synthesized in vitro.
  • the linear nucleic acid is synthesized by cells or organisms and then obtained by isolation and extraction.
  • the construct is an adeno-associated virus (AAV) vector or a lentiviral vector.
  • AAV vector is packaged into AAV2, AAV5, or AAV8 to form a virus particle that can infect cells, and the virus particle introduces the AAV vector into the cell by infecting the cell.
  • the construct is packaged in AAV8.
  • the construct inserts the arRNA coding sequence into the cell genome for long-term expression.
  • the construct allows the arRNA coding sequence to exist and express as free nucleic acid outside the cell genome.
  • This application provides a construct containing the aforementioned functional arRNA, which is selected from linear nucleic acids, plasmids, and viral vectors.
  • the application also provides a delivery body comprising the construct, the delivery body being selected from viral particles, liposomes, lipid nanoparticles and exosomes.
  • the application also provides a cell, which can be edited and prepared by the method for targeted editing of target RNA in a cell based on LEAPER technology as described above.
  • the cell is a cell derived from a patient with a mutation site of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter), and the mutation editing method described above can be used to
  • the target A in the target RNA transcribed at the mutation site is deaminated to form I, which will be recognized as G in the subsequent translation process to restore the complete function of Usherin protein.
  • the patient's cells are stem cells or induced pluripotent stem cells, and the stem cells or differentiated cells thereof can be transplanted to a specific location of the patient.
  • the stem cells are induced to differentiate into healthy cones and/or rods in vitro and then transplanted back to a specific location of the patient's eye to function.
  • the application also provides a kit for editing target RNA in cells, which comprises the aforementioned functional arRNA or the aforementioned functional construct encoding arRNA.
  • the kit contains nucleic acids, plasmids, genomes, cells, etc. that can be used to edit NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutations.
  • the kit further comprises a solvent suitable for dissolving the functional arRNA or the functional construct.
  • 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.
  • This application also provides a method for the treatment of diseases caused by the mutation of NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) in an individual (hereinafter referred to as the treatment method), including the use of the LEAPER-based method as described above.
  • Technology Targeted editing of target RNA containing NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter) mutation site in cells is used to correct individual cells with NM_206933.2(USH2A)_c.11864G>A( p.Trp3955Ter) target RNA mutation related to the mutation site.
  • the disease includes Usher type II syndrome.
  • the treatment method comprises injecting arRNA for editing target RNA or a construct encoding the arRNA into the subretinal space or vitreous of the subject. In some preferred embodiments, the treatment method comprises injecting arRNA for editing target RNA or a construct encoding the arRNA into the subretinal space of the subject. In some embodiments, the treatment requires only one injection of the subject. In some embodiments, the treatment requires injections to the subject at regular intervals. In some specific embodiments, the treatment requires injections to the subject every ⁇ 2 months, for example, injections to the subject every 1 year, 10 years, 20 years, 30 years, etc.
  • the application further provides a preparation comprising the functional arRNA as described above or the functional construct encoding the arRNA as described above or a delivery body thereof.
  • the formulation comprises the functional arRNA or functional construct or delivery body thereof and a transfection reagent.
  • the functional arRNA or functional construct is encapsulated in liposomes.
  • the functional arRNA or functional construct is prepared to form lipid nanoparticles.
  • the functional construct contained in the formulation is a viral vector, such as AAV or lentiviral vector, and the formulation contains live virus or virus lyophilized powder, so the formulation further includes a suitable stabilizer, such as Human albumin, gelatin, sucrose, etc.
  • this application has designed a reporting system as shown in Figure 4.
  • the reporting system uses a lentiviral vector, and the mRNA as shown in Figure 4 is driven by the CMV promoter.
  • This mRNA mainly contains the following parts: 1) mCherry red fluorescent protein sequence, the protein will be stably expressed, and will be translated normally regardless of whether it has been edited or not, so the red fluorescence intensity can be used as an internal reference; 2) USH2A gene mutation site and its two Each side adjacent 100 base pairs, this region is a disease-related sequence; the sequence in (2) is taken from the 12203 to 12403 position of the NM_206933.2 sequence, a total of 201 base pairs, after the USH2A c.11864 The site undergoes a point mutation from G to A, so that there is a TAG stop codon in the reading frame (In-frame) in the sequence, so that translation will stop here during the translation process); 3) GFP green fluorescent protein sequence, the The protein can emit green fluorescence, and
  • the constructed plasmid passes through the second-generation lentivirus packaging system (pCAG-VSVG is a gift from Arthur Nienhuis&Patrick Salmon, Addgene plasma#35616; http://n2t.net/addgene:35616; RRID: Addgene_35616; pCMVR8.74 is a gift from Didier Trono, Addgene Plasmid#22036; http://n2t.net/addgene:22036; RRID: Addgene_22036) was packaged into a lentivirus and infected 293T cells. After 48 hours of cell infection, the mCherry-positive cell population was sorted by flow cytometry, and the cell population was the final reporter system cell.
  • the fluorescence threshold adjusts the fluorescence threshold until the number of cells whose fluorescence intensity is higher than the threshold in uninfected 293T cells or untreated control reporter system is just less than 1% (as close as possible to 1%), at this time the fluorescence intensity is higher than the fluorescence threshold.
  • the cell is considered to be a positive cell.
  • GFP-positive cell ratio abbreviated as %GFP
  • MFI Mean Fluorescent Intensity
  • the proportion of GFP-positive cells refers to the proportion of cells whose GFP intensity exceeds the GFP-positive threshold defined in this application.
  • the level of %GFP indicates the number of edited cells.
  • the test of the editing efficiency of the reporter system further includes MFI. Since cells in each well have similar red fluorescence intensity as an internal control, it is believed that the higher the GFP% and the higher the MFI, the higher the degree of editing of the USH2A pathogenic mutation site in the well cells in the test conditions.
  • SEQ ID NO: 1 Among them, lowercase letter sequences indicate disease-related sequences, and uppercase and lowercase letters of the same base do not represent differences in base types.
  • the arRNA in this example was synthesized in vitro, and its sequence is shown in Table 1 below. Sequences are named in two ways. The "name” is named according to the length or truncated length of the sequence, and the "unified name" is named XCY, where X represents the length of the 5'end, Y represents the length of the 3'end, and C is the target To base C. For example, 111nt named 55-C-55 means that its 5'and 3'short lengths are both 55nt.
  • RNAi MAX reagent Invitrogen 13778150
  • Cell culture uses DMEM (Hyclone SH30243.01) containing 10% FBS (Vistech SE100-011). Use a 12-well plate to culture cells, and the reporter system cells are 15,000 cells/well. The time to plate the cells was recorded as 0 hours.
  • RNAi MAX reagent to transfer 12.5 pmol of arRNA into each well. Refer to the supplier's instructions for the transfection steps.
  • the targeting base of A (target A) of the pathogenic mutation site is determined to be C, so that the sequence lengths of the 5'end and 3'end of the targeted base are the same.
  • the 5'and 3'ends were truncated at the same time to form each test arRNA used in this application.
  • control refers to a control well that does not transfer any arRNA. It can be seen from Figure 6 that there are few GFP-positive cells in the background of the control well (about 0.6%). In contrast, the three arRNAs of 111nt, 91nt and 71nt all cause more than 90% of GFP, while 51nt is only very low. The ratio of GFP positive. It can be seen from the graph of the average fluorescence intensity that there are a small number of false positive cells in the background of the control wells, and although these cells account for a small proportion, their fluorescence intensity is higher, as shown in Figure 7.
  • the "3'-truncated 30" and “55-C-25” in Figure 10 are the same arRNA sequence (SEQ ID NO: 8) as the "-30" of the "3'-truncated” in Figure 9, where:
  • the "3'-Truncation 30" in Figure 10 and the “3' Truncation” in Figure 9 "-30” are synthesized in the same batch, and "55-C-25” are synthesized in different batches. From Figure 10, it is not difficult to see that when the 3'end is fixed at 25 nt and the 5'end is truncated, the editing efficiency basically shows a downward trend with the decrease of the 5'length.
  • 40-C-25 does not meet the above trend , But neither MFI nor %GFP exceeds 55-C-25.
  • the non-targeted base has the highest efficiency when it is in the middle position of arRNA, and before the 3'end length is reduced to 0 (as shown by 85-C-5 and its left column), arRNA has the highest efficiency. Higher editing efficiency.
  • the arRNA with a length of only 71nt such as 55-C-15 in this embodiment not only has higher editing efficiency, but also has lower synthesis cost (see Table 2 for reference prices).
  • RNA extraction Take 1000ng of RNA extracted from each sample, use One-Step gDNA Removal and cDNA Synthesis SuperMix kit (full gold, AT311) performs reverse transcription to synthesize cDNA.
  • PCR was performed with two primers with sequences of gaggtgagtacggtgtgcGGAAGAAAACCCCGGTCCTGCTA (SEQ ID NO: 33) and gagttggatgctggatggAACAGAACTGAATGAGCACTCGTGG (SEQ ID NO: 34) and Q5 hot start enzyme (NEB, M0494L).
  • the PCR products were constructed with Hi-TOM kit (Nuohezhiyuan, REF PT045), and the next-generation sequencing and data analysis were completed according to the following steps.
  • the constructed sequencing library will be used for high-throughput sequencing through the NovaSeq6000 platform in PE150 mode.
  • the raw data obtained by high-throughput sequencing is used for quality control with fastp (v0.19.6) to filter out low-quality, linker-containing sequences, and sequences containing polyG, etc.
  • the high-quality sequencing data obtained is divided into each sample according to the corresponding barcode sequence, and the BWA (v0.7.17-r1188) software is used to compare with the sequence of the amplified target region, and the format is formatted by SAMtools (v1.9) Convert to generate BAM file.
  • Fig. 12 Comparing Fig. 12 with Fig. 13, it is not difficult to see that the editing efficiency obtained by testing of different methods shows basically the same trend. That is to say, the 111nt arRNA designed according to the LEAPER document (Qu et al., 2019) is not the most efficient arRNA, but after we truncated the 3'end of the target base, the 5'end of the target base, and the target base After adjusting the position in the sequence, it was finally found that the 55-C-15 (SEQ ID NO: 9) sequence not only showed higher editing efficiency in the reading of GFP, but also can be further confirmed by second-generation sequencing.
  • the editing efficiency to G can reach about 50%, which means that 50% of the target base sites on the mRNA in the cells edited by 55-C-15 arRNA have been edited from A to G.
  • Example 2 the pathogenic sites of USH2A were edited through in vitro experiments.
  • in vitro experiments can achieve good editing efficiency, the environment of in vitro cell culture cannot simulate many factors such as human internal circulation, immune system, and intraocular injection. These still need more in vivo experiments to verify.
  • Adeno-Associated Virus AAV
  • AAV vector AAV vector as the delivery method of arRNA for mouse eyes.
  • SEQ ID NO:32 151nt arRNA
  • the arRNA hybridizes with the target RNA, an AC mismatch (mismatch) can be formed at the target A.
  • the 5'end and 3'end of the arRNA targeting base C are respectively 75 nt base sequences that are completely complementary to the target sequence. Because the mouse Ush2A gene is different from the human USH2A gene sequence, and the test mouse does not carry Usher syndrome-related mutations, the 151nt arRNA used in this example is an arRNA targeting the mouse Ush2A-related site, not human The arRNA of the USH2A gene sequence.
  • This example uses SPF C57 male mice aged 6-8 weeks.
  • the test group (AAV2 and AAV5 group) and the control group (PBS) each have 8 mice, a total of 24 mice.
  • 2 ⁇ L of AAV or PBS was injected into the right eyes of the experimental group and the control group mice through the vitreous body.
  • 4 mice of each group were killed everywhere, and their right eyes were taken out and cut with scissors to 50 ⁇ Small pieces of 100mg. After grinding with liquid nitrogen, the samples were collected with 800 ⁇ L TRIzol (Invitrogen, 15596018).
  • the Direct-zol RNA Miniprep kit (Zymo Resaerch, R2052) was used for RNA extraction. Take 1000ng of extracted RNA for each sample One-Step gDNA Removal and cDNA Synthesis SuperMix kit (full gold, AT311) performs reverse transcription to synthesize cDNA. 1 ⁇ L of the reverse transcription product was taken from each mouse, and two primers with the sequence of gaggtgagtacggtgtgcCCCATCTCTTTGGCTTGGAACCAT (SEQ ID NO: 22) and gagttggatgctggatggCTTTTCTCTGCTCCACTGTGAAGTT (SEQ ID NO: 24) and Q5 hot start enzyme (NEBPCR, M0494L) were used. The PCR products were constructed with Hi-TOM kit (Nuohezhiyuan, REF PT045) to complete the second-generation sequencing and data analysis (the sequencing and data analysis methods are the same as in Example 2).
  • mice used were 6-8 weeks old SPF grade C57 male mice, the test group (AAV5, AAV8 group) each had 20 mice, and the control group (NaCl) 12 mice.
  • the subretinal cavity injection method is as follows: 1 to 2 drops of compound tropikamide eye drops are applied to the eyes of the animal to dilate pupils, and the intramuscular injection (50 mg/kg, 50 mg/mL) of sultai 50 is used to perform general anesthesia on the animal.
  • the anesthetized mouse was placed on the operating table on its side.
  • Povidone-iodine cotton balls were used to disinfect the periorbital skin of mice. Clamp the conjunctiva on one side of the eyeball with toothed microsurgery forceps, and cut the subconjunctival fascia with microscissors to expose part of the sclera.
  • RNA was extracted from a pre-cooled 400 ⁇ L TRIzol (Thermo, 15596018) sterile EP tube. Then mix it with a tissue homogenizer. Use Direct-zol RNA Microprep kit (Zymo Resaerch, R2062) to extract RNA.
  • RNA extracted from each sample Take 1000ng of total RNA extracted from each sample and use Quan Jin Reverse Transcription Kit (Full Gold, AH341-01) for reverse transcription, and take 5 ⁇ L of reverse transcribed cDNA with the sequence gaggtgagtacggtgtgcCCCATCTCTTTGGCTTGGAACCAT (SEQ ID NO: 22 ) And two primers of gagttggatgctggatggCTTTTCTCTCTGCTCCACTGTGAAGTT (SEQ ID NO: 2) and Q5 hot start enzyme (NEB, M0494L) for PCR.
  • the PCR products were constructed with Hi-TOM kit (Nuohezhiyuan, REF PT045) to complete the second-generation sequencing and data analysis (the sequencing and data analysis methods are the same as in Example 2).

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Abstract

一种基于LEAPER技术靶向编辑USH2A基因转录本中含有G到A突变的靶标RNA的方法，包括将用于编辑靶标RNA的腺苷脱氨酶招募RNA(arRNA)或编码所述arRNA的构建体导入所述细胞中，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能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基，安全有效地进行RNA上由A到I碱基的体内编辑，修复致病的突变位点，实现治疗疾病例如Usher综合征的目的。

Description

一种治疗Usher综合征的方法和其组合物 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Usher综合征Ⅱ型(Usher Syndrome Type Ⅱ)相关基因突变的方法。
背景技术
Usher综合征，又称遗传性耳聋-色素性视网膜炎综合征，是1914年由Charles Howard Usher描述并命名的 12，是一种由基因突变引起、导致病患先天或逐渐丧失视觉和听觉能力的常染色体隐性遗传性罕见病。Usher综合征发病率在德国约为1/12500 8，在挪威中为1/28000 4，在美国为1/23000，据估计在美国16000盲聋人中，一半以上患有Usher综合征 1。全球有数万甚至数十万病人亟待治疗。
1977年，Davenport等人研究了Usher综合征，并根据其严重程度将其分为Ⅰ、Ⅱ、Ⅲ、Ⅳ型 3。其中，Ⅰ型为重度先天性耳聋，10岁左右出现严重视觉障碍，Ⅱ型为中度至重度先天性耳聋，约在10～50岁之间逐渐丧失视力。Ⅲ、Ⅳ型相对罕见，并且病情较轻，听觉表现为进行性感音神经性聋，而视觉丧失时间不确定。由于Ⅰ型发病早，介入窗口较小，而Ⅲ、Ⅳ型相对病情较轻。Ⅱ型病人的视觉丧失会从10～20岁起逐渐开始，发病初期为夜盲症，并最终丧失视力(图1)，这使得我们有较长的治疗介入窗口，并有希望终止病人视力的逐渐丧失。
Neuhaus等人2017年通过高通量测序对138例Usher综合征病人进行了研究，其结果显示在这138例中有82例为USH2A基因突变，而这82例中，又有80例为Usher综合征Ⅱ型(图2) 7。由此，当只考虑Usher综合征Ⅱ型病人时，USH2A基因突变的占比超过了90％(图2) 7，而针对USH2A基因的治疗方案，很有可能使得这些病人受益。
USH2A基因编码一种被称为Usherin的蛋白，该蛋白在视觉中发挥了重要的功能，主要是在感光的视锥细胞和视杆细胞中，起到连接内节(Inner Segment)与外节(Outer Segment)的重要作用。当USH2A突变后，会导致视锥 细胞和视杆细胞的退行性死亡 5。Neuhaus等人2017年的研究表明，在其测序的USH2A基因突变案例中，有13％为NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)，而这一种突变类型为所有突变类型中占比最多的单一突变类型(图3) 7。
由于USH2A基因突变而失去Usherin正常功能是导致USHER综合征Ⅱ型的重要原因，针对USH2A基因的治疗成为了目前Usher II型治疗的主要研发方向。
目前通过基因疗法治疗USHER综合征Ⅱ型的研发方向主要有两个，一是通过病毒等方式，介导完整的USH2A基因在眼部重新表达。但是，USH2A基因的蛋白序列非常长，大于6000个氨基酸，这使得其对应的编码区序列大于18000个碱基对。而通常的病毒载体的递送长度有限。通常情况下，慢病毒递送不超过10000个碱基对，而腺相关病毒递送不超过4500个碱基对。这使得通过病毒载体递送全长USH2A基因较难实现。因此，医疗和科研工作者们只能选择递送截短的USH2A基因。这样的方式，虽然可以一定程度缓解病情的恶化，但由于患者依然缺乏正常的全长Usherin，其无法完全实现Usherin原有的正常生理功能。
另外一种常见的针对USH2A基因的治疗研发方向是通过外显子跨越(Exon Skipping)实现。由于一部分USH2A基因的突变方式是某个外显子的移码(Frame Shift)或者无义突变(Nonsense Mutation)导致的，因此只要能在RNA剪接的过程中，特异性地跳过该外显子，就能使得该外显子之后的序列正常翻译。常见的做法，是导入一小段反义核苷酸(Anti-Sense Oligo,ASO)，从而在剪接过程中特异性地跳过所述翻译核苷酸靶向的外显子。这种方法与上一种方法类似，由于跳过了出现突变的外显子，最终仍然不能得到全长的Usherin。
近年来，以CRISPR(Clustered regularly interspaced short palindromic repeats)为首的基因组编辑技术正在飞速发展，并对生物以及医学诸多领域产生了深远的影响。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。2019年9月，北京大学邓宏魁教授与其合作者发表文章 11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试验结果，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，需要外源表达的核酸酶通常具有较大的分子量，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，使这种方法存在潜在的核酸酶脱靶可能，这将导致其应用中的潜在致癌风险。最后，外源表达的Cas9等类似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，影响治疗效果。
2017年，麻省理工学院张锋教授及其课题组报道了一种名为REPAIR(RNA Editing for Programmable A to I Replacement)的RNA编辑技术，通过外源表达Cas13-ADAR融合蛋白及单向导RNA(single guide RNA,sgRNA)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课题组报道了名为RESTORE(recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing,Merkle et al.,2019)的RNA单碱基编辑技术。RESTORE能够摆脱对外源蛋白的依赖，但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，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。在这些化学修饰中，有一部分是非天然的修饰，使得该寡核苷酸可能存在毒性或免疫原性；还有一部分修饰会导致相同碱基链的不同构象，使得对于相同的RNA序列，可能有数十种不同的构象组合，这增加了其向细胞内递送的难度。
由于上述三种技术均存在一定的缺陷，并且鉴于USH2A基因本身的特殊性，因此上述三种技术均难以用于治疗USHER综合征Ⅱ型。
2019年7月，北京大学生命科学学院教授魏文胜课题组在Nature Biotechnology上发表文章，“Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs”，首次报道了新的核酸编辑技术：LEAPER(Leveraging Endogenous ADAR for Programmable Editing of RNA,Qu et al.,2019)。一方面与CRISPR技术相比，该技术从原理上摆脱了对外源核酸酶过表达的依赖，而仅需要一段RNA作为向导，以将内源的核酸酶招募至所需编辑的位点，使得该技术在向医学领域转化的过程中，有更大的优势；另一方面该技术只在转录水平上实现了腺苷A到肌酐I的编辑(肌酐I在蛋白质翻译过程中会被识别为鸟酐G)，而不改变基因组中的序列，因此更加安全方便。LEAPER技术不仅可以通过化学合成RNA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。
发明内容
本申请涉及基于LEAPER技术靶向编辑USH2A基因转录本中含有G到A突变的靶标RNA的方法，通过将用于编辑靶标RNA的腺苷脱氨酶招募RNA(arRNA)或编码所述arRNA的构建体导入含有靶标RNA的细胞中，使所述arRNA能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基。本申请通过对LEAPER技术的优化和改进，创造性地将其应用于USH2A基因转录本中含有G到A突变的靶标RNA，例如具有NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变的USHER综合征Ⅱ相关靶标RAN，通过对所述突变位点进行逆转修复，达到治疗USHER综合征Ⅱ的目的。
本申请的目的是针对Usher综合征Ⅱ型致病基因，例如针对人USH2A中占比最高的突变型NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)致病基因，提供一种全新的技术方案，无需导入过大的核酸或外源蛋白分子，便能使其精准地在靶标RNA上编辑突变位点，恢复Usherin蛋白的完整功能。
具体地，本申请涉及：
1.一种基于LEAPER技术靶向编辑细胞中靶标RNA的方法，其中所述靶标RNA为USH2A基因(例如人USH2A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的腺苷脱氨酶招募RNA(arRNA)的构建体或编码所述arRNA的构建体导入所述细胞中，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能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基。
2.如项1中所述的方法，其中所述靶标RNA为成熟mRNA或mRNA前体(Pre-mRNA)。在一些实施方案中，所述靶标RNA为mRNA前体(Pre-mRNA)。
3.如项1或2中所述的方法，其中所述arRNA长约151-61nt、131-66nt、121-66nt、111-66nt、91-66nt或81-66nt。本申请涵盖该数据范围内的任何自然数。
4.如项3中所述的方法，其中所述arRNA长度选自66nt-131nt中的任意自然数。
5.如项1-4中任一项所述的方法，其中所述arRNA中靶向碱基距离3’端的长度≥7nt，≥10nt、≥15nt，优选16-55nt、20-50nt、25-45nt、25-35nt或25-30nt。本申请涵盖该数据范围内的任何自然数。
6.如项1-5中任一项所述的方法，其中所述arRNA中靶向碱基距离5’端的长度≥25nt，≥30、≥35、≥45，优选46-90nt、50-85nt、55-80nt、60-75nt或65-70nt。本申请涵盖该数据范围内的任何自然数。
7.项1-6中任一项的方法，其中向所述arRNA引入与靶标序列中的靶标A配对的靶向碱基，该靶向碱基的优选次序从高到低依次为C、A、U或G。
在一些实施方案中，向所述arRNA引入与靶标序列中的靶标A配对的靶向碱基为C。在一些实施方案中，向所述arRNA引入与靶标序列中的靶标A配对的靶向碱基为A。在一些实施方案中，向所述arRNA引入与靶标序列中的靶标A配对的靶向碱基为U。
8.如项1-7中任一项的方法，其中所述靶标RNA为包含转录有NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的人USH2A基因RNA。
9.如项1-8中任一项所述的方法，其中所述arRNA选自：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4。
10.如项1-9中任一项所述的方法，其中所述arRNA选自：SEQ ID NO：7、SEQ ID NO：8或SEQ ID NO：9。
11.如项1-10中任一项所述的方法，其中所述构建体为线性核酸、病毒载体、或质粒。
12.如项11中所述的方法，其中所述病毒为腺相关病毒(AAV)或慢病毒。
13.如项12中所述的方法，其中所述AAV载体包装入AAV2、AAV5或AAV8衣壳后通过侵染导入所述细胞中。
14.如项13中所述的方法，所述AAV载体包装入AAV8衣壳后通过侵染导入所述细胞中。
15.如权力要求1-14中任一项所述的方法，其中所述细胞为哺乳动物视神经细胞或听神经细胞。
16.一种用于靶向编辑USH2A基因转录本中含有G到A突变的靶标RNA的arRNA或其编码序列，所述arRNA包含选自如下的序列或由选自如下的序列组成：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3、或SEQ ID NO：4。
17.包含项16的arRNA的构建体或递送体。
18.如项17的构建体或递送体，其为质粒、病毒、脂质体、或脂质纳米颗粒。
19.如项18的构建体或递送体，其为腺相关病毒(AAV)或慢病毒。
20.如项19的构建体或递送体，其所述病毒为AAV2、AAV5或AAV8。
在一些实施方案中，本申请提供了包含上述arRNA、或包含上述构建体或递送体的组合物、制剂、试剂盒或生物制品。
21.通过项1-15中任一项的方法编辑获得的细胞。
22.一种治疗个体中Usher II型综合征的方法，包括使用项1-15中任一项所述的方法校正所述个体的细胞中与Usher II型综合征有关的G到A的突变。
23.如项22中所述的方法，其包括将如项16中所述的arRNA或如项17-20中任一项所述的构建体或递送体导入受试者。
24.项23的方法，其中将如项16中所述的arRNA或如项17-20中任一项所述的构建体或递送体导入受试者视网膜下腔中。
本申请提供的基于LEAPER技术靶向编辑细胞中转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的靶标RNA的方法，在治疗USHER综合征Ⅱ型方面具有明显的优势：
首先，LEAPER技术只需要导入短RNA，可以限制在151、131、121、111核苷酸以内，这使得其对导入片段长度的要求远远小于腺相关病毒的递送长度上限。因此，与直接通过导入USH2A基因相比，该技术非常适合应用腺相关病毒载体。而由于眼部为免疫豁免区域，对腺相关病毒的清除能力很差，这使得腺相关病毒可以在眼部停留很长时间。此外，发挥功能的细胞均为神经细胞，正常情况下这些细胞也不会分裂，这使得腺相关病毒可以长期停留在这些细胞中，即不被免疫清除，也不会因为细胞分裂导致病毒被稀释。更关键的是，相比于此前的常用手段，本申请的技术方案既不需要人为截短Usherin，也不需要跨过突变的外显子，从而可以得到全长的正常的Usherin。因此，理论上，病患通过该技术治疗后，可以得到与常人无异的正常Usherin。由于USHER综合征Ⅱ型是隐性遗传病，这说明，通常只要有部分正常蛋白可以发挥功能，即可获得正常的功能。本申请通过实验数据证明，本申请的技术方案可以校正USHER综合征Ⅱ型相关基因转录本(例如含有 NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的人USH2A基因转录本)中的G>A突变，实现对USHER综合征Ⅱ型的治疗目的。
附图说明
图1显示了Usher综合征Ⅱ型病人夜盲症病情进展示意图 13。
图2显示了138例Usher病人致病基因突变分布图 7。
图3显示了USH2A基因突变类型分析图 7。
图4显示了反映USH2A基因突变位点的编辑效率的报告体系设计示意图。
图5显示了USH2A基因突变位点的编辑效率的评判指标示意图。
图6显示了不同长度的arRNA编辑效率的测试结果。
图7显示了报告体系中假阳性细胞的FITC通道强度。
图8显示了LEAPER技术中arRNA长度与编辑效率的关系 10。
图9显示了arRNA从5’以及3’向中间截短后的编辑效率测试结果。
图10显示了固定arRNA 3’长度为25nt并从5’逐渐截短后的编辑效率测试结果。
图11显示了靶向碱基位置对编辑效率的影响。
图12显示了同批次arRNA编辑效率的重复测试结果的比较。图中293T表示未侵染报告体系的293T；仅报告子体系为侵染有报告体系的293T；Opti-DMEM为在转染中不加入RNAi MAX的培养基空白对照；仅递送载体对照为仅有RNAi MAX的对照，对应此前测试中的对照(NC)孔；随机序列RNA为不与人类基因组匹配的91nt随机RNA序列(uaauccugaauaucgcgcaauuccccagcagagaacaucgcggugugaacgucccuuuauaccgggcagguauagcugaaaucagcguggc(SEQ ID NO：35))。
图13显示了同批次arRNA通过二代测序获得的编辑效率重复测试结果的比较。
图14显示采用玻璃体内注射时，通过AAV2和AAV5递送的arRNA在体内的编辑效率。
图15显示采用视网膜下腔注射时，通过AAV5和AAV8递送的arRNA在体内随时间变化的编辑效率。
发明详述
本申请针对Usher综合征Ⅱ型致病基因USH2A中占比最高的突变型NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)，以创造一种全新的技术方案，在不导入过大的核酸或外源蛋白分子的条件下，通过AAV精准地在靶标RNA上编辑突变位点，恢复Usherin蛋白的完整功能，并在相当长的一段时间内持续保持Usherin蛋白的功能。
为满足上述目的，本申请提供了一种基于LEAPER技术靶向编辑细胞中转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的靶标RNA的方法(下文简称突变编辑方法)、一种靶向NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的arRNA、包含所述arRNA的构建体、一种经过所述突变编辑方法制备的细胞、一种用于编辑细胞中的靶标RNA的试剂盒、一种治疗个体中由于NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变导致的疾病的方法(下文简称治疗方法)、以及一种用于所述突变编辑方法或所述治疗方法的制剂。
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。arRNA是向导RNA的一种，在本申请中“向导RNA” 是指可通过与靶标RNA互补杂交，招募可修饰靶标碱基的酶对靶标碱基进行修饰。所述向导RNA包含arRNA以及用于CRISPR等其他编辑系统的gRNA。
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％，99％或100％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“导入”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“导入”包括点转染、脂质体转染、脂质-纳米颗粒递送等方式。
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等递送至细胞并最终进入细胞核的技术。
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_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变是指人USH2A基因中对应于NM_206933.2(USH2A)基因转录本11864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3955位色氨酸(Trp)编码序列转变为终止密码子，使得最终翻译的氨基酸缺失了3955位之后的全部氨基酸，从而丧失Usherin的蛋白活性。发生这一突变的患者会出现视锥细胞和视杆细胞的退行性死亡，最终导致失明。而本申请中的方案可通过在转录水平逆转这一突变，恢复Usherin蛋白的活性。在本申请的一些实施方案中NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点又被称为USH2A致病位点。
如本文所用，术语“靶标RNA”是指是指待编辑的目标RNA，其由USH2A基因转录而成，并包含G至A的突变。靶标RNA可以是成熟mRNA或mRNA前体，在本申请中，更优选mRNA前体。在一些实施方案中，所述“靶标RNA”为转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的RNA。所述“靶标RNA”中由G至A的突变位点转录形成的A碱基被称为“靶标腺苷”或“靶标A”。arRNA中与“靶标A”相对的碱基称为“靶向碱基”。
如本文所使用，术语“AAV”是指“腺相关病毒”或“腺相关病毒载体”，在本文中，如非特别说明，“AAV”、“腺相关病毒”及“腺相关病毒载体”可以互换使用。AAV属于微小病毒科，为无包膜的单链线状DNA病毒，利用腺相关病毒可以将外源基因转入动物组织和细胞中。所述转入的外源基因可以游 离核酸的状态较为稳定地存在于宿主的基因组基因之外并进行表达。腺相关病毒血清型众多(12种)，包括例如：AAV1、AAV2、AAV5、AAV8和AAV9等。如本文所用，“患者”或“受试者”可互换使用，在本文中如非特别指出，本文所述“患者”或“受试者”均指基因组中带有NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变的人类患者。
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。本文中，将所述生物大分子及包裹所述生物大分子或与所述生物大分子连接以促进所述生物大分子通过细胞膜进入细胞内的物质的结合体称为“递送体”。例如包装了核酸分子的脂质体、LNP、外泌体、病毒颗粒等。
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。所述特定的核酸序列可以是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。
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。在本申请的一些实施方案中，通过对arRNA的修饰，使其在导入细胞后更稳定。在本文中，靶向碱基距离3’端的长度是指靶向碱基的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。
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。
突变编辑方法
本申请提供了一种基于LEAPER技术靶向编辑细胞中靶标RNA的方法，其中所述靶标RNA为USH2A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的腺苷脱氨酶招募RNA(arRNA)的构建体或编码所述arRNA的构建体导入所述细胞中，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能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基。
在一些实施方案中，所述靶标RNA为mRNA或mRNA前体(Pre-mRNA)，优选地，为Pre-mRNA。
在一些实施方案中，其中所述arRNA长度＞51nt，优选地＞61nt，更优选地≥65nt。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长度选自66nt-131nt中的任意自然数，例如66nt、67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、78nt、79nt、80nt、83nt、85nt、88nt、91nt、96nt、98nt、100nt、105nt、108nt、100nt、105nt、110nt、115nt、120nt、125nt、130nt等。在可保持转染效率相同的情况下，优选地，所述arRNA的长度为约71nt，例如：66nt-76nt中的任一自然数个nt。
在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度＞5nt，例如≥7nt、≥10nt、≥15nt，例如16-55nt、20-50nt、25-45nt、25-35nt或25-30nt中的任意自然数个nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度≥25nt，例如≥30、≥35、≥45，例如46-90nt、50-85nt、55-80nt、60-75nt或65-70nt中的任意自然数个nt。
在一些实施方案中，当所述arRNA与靶标RNA互补杂交时，所述arRNA与靶标RNA上的靶标碱基相对的靶向碱基优选次序从高到低依次为C、A、U或G。即在通常情况下，对于长度及靶向碱基以外的序列均相同，而靶向碱基分别为C、A、U、G的arRNA，其编辑效率依次降低，因此最优选地，所述arRNA的靶向碱基为C，其次为A，再其次为U，最次为G。
在一些实施方案中，所述靶标RNA为包含转录有NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的RNA。在一 些实施方案中，所述arRNA选自：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4。优选地，在一些实施方案中，所述arRNA选自：SEQ ID NO：7、SEQ ID NO：8或SEQ ID NO：9，更优选地为SEQ ID NO：8或SEQ ID NO：9。
在一些实施方案中，所述构建体选自线性核酸、病毒载体、或质粒。所述线性核酸包括单链核酸，例如单链RNA、单链DNA；以及双链核酸，例如双链DNA、双链RNA。在一些实施方案中，所述线性核酸可以经过化学修饰，例如2’-O-Me修饰和/或硫代磷酸酯键修饰。在一些特定的实施方案中，所述线性核酸由体外化学合成。在一些特定的实施方案中，所述线性核酸由细胞或机体合成后经分离提取获得。在一些实施方案中，所述构建体为腺相关病毒(AAV)载体或慢病毒载体。在一些实施方案中，所述其中所述AAV载体包装入AAV2、AAV5或AAV8衣壳后通过侵染导入所述细胞中。在一些实施方案中，所述AAV载体包装入AAV8衣壳后通过侵染导入所述细胞中。在一些实施方案中，所述构建体将所述arRNA编码序列插入细胞基因组中长期表达。在一些实施方案中，所述构建体使所述arRNA编码序列作为游离核酸存在于细胞基因组外并进行表达。
在一些实施方案中，所述细胞为真核细胞。在一些实施方案中，所述细胞为哺乳动物细胞。在一些实施方案中，所述细胞为神经细胞。在一些实施方案中，所述神经细胞为感觉神经细胞。在一些实施方案中，所述感觉神经细胞选自视神经细胞及听神经细胞。在一些实施方案中，所述视神经细胞为视锥细胞和/或视杆细胞。
在一些实施方案中，所述导入为电转染、脂质体转染、脂质-纳米颗粒递送或病毒侵染。在本申请的实施方案中，优选地为病毒侵染。例如通过将编码arRNA的病毒构建体包裹入病毒衣壳形成具有侵染性的病毒颗粒，例如AAV或慢病毒颗粒，并通过所述病毒颗粒对细胞的侵染将所述arRNA的编码序列导入细胞中。
功能arRNA
本申请同时还提供了一种arRNA(下文简称为功能arRNA)，所述arRNA可用于如前所述的基于LEAPER技术靶向编辑细胞中靶标RNA的方法。在一些实施方案中，所述方法为基于LEAPER技术靶向编辑细胞中转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的靶标RNA的方法。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能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基。在一些实施方案中，所述arRNA靶向的靶标RNA选自mRNA或Pre-mRNA，优选地为Pre-mRNA。
在一些实施方案中，所述arRNA长度＞51nt，优选地＞61nt，更优选地＞65nt。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长度选自66nt-131nt中的任意自然数，例如66nt、67nt、68nt、69nt、70nt、71nt、72nt、73nt、74nt、75nt、76nt、78nt、79nt、80nt、83nt、85nt、88nt、91nt、96nt、98nt、100nt、105nt、108nt、100nt、105nt、110nt、115nt、120nt、125nt、130nt等。在可保持转染效率相同的情况下，优选地，所述arRNA的长度为约71nt，例如：66nt-76nt中的任一自然数个nt。
在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度＞5nt，例如≥7nt、≥10nt、≥15nt，例如16-55nt、20-50nt、25-45nt、25-35nt或25-30nt中的任意自然数个nt。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度≥25nt，例如≥30、≥35、≥45，例如46-90nt、50-85nt、55-80nt、60-75nt或65-70nt中的任意自然数个nt。
在一些实施方案中，当所述arRNA与靶标RNA互补杂交时，所述arRNA与靶标RNA上的靶标碱基相对的靶向碱基优选次序从高到低依次为C、A、U或G。即在通常情况下，对于长度及靶向碱基以外的序列均相同，而靶向碱基分别为C、A、U、G的arRNA，其编辑效率依次降低，因此最优选地，所述arRNA的靶向碱基为C，其次为A，再其次为U，最次为G。
在一些实施方案中，所述arRNA选自：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO： 8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4。优选地，在一些实施方案中，所述arRNA选自：SEQ ID NO：7、SEQ ID NO：8或SEQ ID NO：9，更优选地为SEQ ID NO：8或SEQ ID NO：9。在一些实施方案中，所述arRNA引入与靶标序列中的靶标A配对的靶向碱基编辑效率从大到小依次为C、A、U、G，即：对于长度及靶向碱基以外的序列均相同，而靶向碱基分别为C、A、U、G的arRNA，其编辑效率依次降低，因此最优选地，所述arRNA的靶向碱基为C，其次为A，再其次为U，最后为G。
功能构建体
本申请还提供了一种编码arRNA的构建体(下文简称为功能构建体)，其中所述功能构建体可以用于如前所述的基于LEAPER技术靶向编辑细胞中靶标RNA的方法。在一些实施方案中，所述方法为基于LEAPER技术靶向编辑细胞中转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的靶标RNA的方法。其中，所述arRNA包含如前所述的功能arRNA。
在一些实施方案中，所述构建体选自线性核酸、病毒载体、或质粒。所述线性核酸包括单链核酸，例如单链RNA、单链DNA；以及双链核酸，例如双链DNA、双链RNA。在一些实施方案中，所述线性核酸可以经过化学修饰，例如2’-O-Me修饰和/或硫代磷酸酯键修饰。在一些特定的实施方案中，所述线性核酸由体外化学合成。在一些特定的实施方案中，所述线性核酸由细胞或机体合成后经分离提取获得。在一些实施方案中，所述构建体为腺相关病毒(AAV)载体或慢病毒载体。在一些实施方案中，所述AAV载体包装入AAV2、AAV5或AAV8以形成可侵染细胞的病毒颗粒，所述病毒颗粒通过侵染细胞将所述AAV载体导入细胞中。优选地，在一些实施方案中，所述构建体包装入AAV8。在一些实施方案中，所述构建体将所述arRNA编码序列插入细胞基因组中长期表达。在一些实施方案中，所述构建体使所述arRNA编码序列作为游离核酸存在于细胞基因组外并进行表达。
本申请提供了包含如前所述的功能arRNA的构建体，选自线性核酸、质粒、病毒载体。本申请还提供了包含所述构建体的递送体，所述递送体选自病毒颗粒、脂质体、脂质纳米颗粒和外泌体。
细胞
本申请还提供了一种细胞，其可通过如前所述的基于LEAPER技术靶向编辑细胞中靶标RNA的方法进行编辑和制备。在一些实施方案中，所述细胞为源自具有NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的患者的细胞，通过如前所述的突变编辑方法可将所述突变位点转录的靶标RNA中的靶标A脱氨基形成I，在后续的翻译过程中将被识别为G而恢复Usherin蛋白完整的功能。在一些实施方案中，所述患者的细胞为干细胞或诱导多能干细胞，所述干细胞或其诱导分化的细胞可以移植至患者特定位置。在一些实施方案中，所述干细胞在体外通过诱导分化为健康的视锥和/或视杆细胞后再移植回患者眼部特定位置发挥功能。
试剂盒
本申请还提供了一种用于编辑细胞中的靶标RNA的试剂盒，其包含如前所述的功能arRNA或如前所述所述的编码arRNA的功能构建体。在一些实施方案中，所述试剂盒包含可以用于编辑NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变的核酸、质粒、基因组、细胞等。在一些实施方案中，所述试剂盒还进一步包含适于溶解所述功能arRNA或所述功能构建体的溶剂。在一些实施方案中，所述试剂盒还进一步包含使用说明，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。
治疗方法
本申请同时还提供了一种治疗个体中由于NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变导致的疾病的方法(下文简称治疗方法)，包括使用如前所述的基于LEAPER技术靶向编辑细胞中转录有包含NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的靶标RNA的方法校正个体细胞中与NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点相关的靶标RNA突变。在一些实施方案中，所述疾病包括Usher II型综合征。
在一些实施方案中，所述治疗方法包含将用于编辑靶标RNA的arRNA或编码所述arRNA的构建体注射入受试者视网膜下腔或玻璃体。在一些优选的实施方案中，所述治疗方法包含将用于编辑靶标RNA的arRNA或编码 所述arRNA的构建体注射入受试者视网膜下腔。在一些实施方案中，所述治疗仅需对受试者进行一次注射。在一些实施方案中，所述治疗需要每隔一段时间对受试者进行一次注射。在一些特定的实施方案中，所述治疗需要每隔≥2个月对受试者进行一次注射，例如每隔1年、10年、20年、30年等对受试者进行一次注射。
制剂
本申请还进一步提供了一种制剂，其包含如前所述的功能arRNA或如前所述所述的编码所述arRNA的功能构建体或其递送体。在一些实施方案中，所述制剂包含所述功能arRNA或功能构建体或其递送体以及转染试剂。在一些特定的实施方案中，所述功能arRNA或功能构建体包裹在脂质体中。在一些特定的实施方案中，所述功能arRNA或功能构建体经制备形成脂质纳米颗粒。在一些实施方案中，所述制剂包含的功能构建体为病毒载体，例如AAV或慢病毒载体，所述制剂包含活体病毒或病毒冻干粉，因此所述制剂还进一步包括适宜的稳定剂，例如人血白蛋白、明胶、蔗糖等。
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。
实施例
实施例1：报告体系的构建
本申请主要研究LEAPER技术对NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)突变位点的修复。因此，需要一个简单可用的，可以反应该位点编辑效率高低的报告体系。由于USH2A基因致病突变位点NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter)导致的原TGG密码子至TAG密码子的突变为无义突变，突变后的mRNA序列在翻译时，会停止在 新形成的TAG终止密码子处，导致其后序列无法继续翻译成蛋白。利用以上特点，本申请设计了如图4所示的报告体系。所述报告体系使用慢病毒载体，由CMV启动子驱动如图4所示的mRNA。该段mRNA主要包含以下部分：1)mCherry红色荧光蛋白序列，该蛋白将稳定表达，无论是否经过编辑均会正常翻译，因此红色荧光强度可以作为内部参照；2)USH2A基因突变位点以及其两侧相邻的各100个碱基对，该区域为疾病相关序列；(2)中的序列取自NM_206933.2序列的第12203到12403位，共201个碱基对，经过对USH2A c.11864位点进行G到A的点突变，使得该序列中存在阅读框(In-frame)内的TAG终止密码子，从而在翻译过程中会在此停止翻译)；3)GFP绿色荧光蛋白序列，该蛋白可以发绿色荧光，并在流式细胞仪中可以经FITC通道检测荧光强度(由于GFP序列与USH2A相关序列位于同一阅读框且位于其下游，若USH2A相关序列中的TAG终止密码子未通过LEAPER技术进行编辑，则其中的TAG终止密码子会导致后续GFP无法被正常翻译，而不会出现绿色荧光；若USH2A相关序列中的TAG成功通过LEAPER技术进行编辑，形成TIG密码子，则该终止密码子消失，后续GFP翻译继续，从而出现绿色荧光；4)在mCherry下游以及GFP上游插入P2A、T2A序列，在翻译时，这两个序列会由于空间位阻导致其中的一个肽键无法正常形成，因而会在翻译过程中将mCherry、中间的USH2A相关序列的201个碱基对以及GFP三个部分分开，从而使得中间插入的USH2A相关序列不会对mCherry以及GFP功能产生影响。
以上序列全序列通过本领域常规技术进行体外合成，具体序列为如SEQ ID NO：1所示。该序列通过CMV启动子后的多克隆位点克隆至pCDH-CMV载体载体。pCDH-CMV载体质粒由Kazuhiro Oka惠赠(Addgene plasmid#72265；http://n2t.net/addgene:72265；RRID:Addgene_72265)。构建好的质粒通过二代慢病毒包装体系(pCAG-VSVG由Arthur Nienhuis&Patrick Salmon惠赠，Addgene plasmid#35616；http://n2t.net/addgene:35616；RRID:Addgene_35616；pCMVR8.74由Didier Trono惠赠，Addgene plasmid#22036；http://n2t.net/addgene:22036；RRID:Addgene_22036)包装成慢病毒并侵染293T细胞。细胞侵染48h后使用流式细胞仪分选mCherry阳性的细胞群，该细胞群为最终的报告体系细胞。其中，调节荧光阈值直至未侵染的293T 细胞或未处理的对照报告体系中荧光强度高于阈值的细胞数刚好小于1％(尽量接近1％)时为止，此时荧光强度高于荧光阈值的细胞即被认为是阳性细胞。对于报告体系的测试，如图5所示通常有两个评价指标：1.GFP阳性细胞比例(缩略为％GFP)；2.GFP+细胞平均荧光强度(MFI，Mean Fluorescent Intensity)。其中，GFP阳性细胞比例，即：GFP强度超过本申请所定义的GFP阳性阈值的细胞的比例。％GFP的高低表示发生编辑的细胞数量。而在GFP阳性的细胞中，由于不同细胞的荧光强度不同，因此对报告体系编辑效率的测试还进一步包括了MFI。由于每孔细胞均有近似的作为内参的红色荧光强度，因此认为GFP％越高以及MFI越高，则代表该孔细胞的USH2A致病突变位点在测试条件中被编辑的程度越高。
SEQ ID NO：1：其中，小写字母序列表示疾病相关序列，同一种碱基的大小写字母不代表碱基类型的差异。
Figure PCTCN2020141501-appb-000001
Figure PCTCN2020141501-appb-000002
实施例2：针对致病突变位点的arRNA优化
由于LEAPER技术无需向细胞内导入任何外源蛋白，本实施例在测试LEAPER对致病位点的编辑时，只需向报告体系细胞中导入arRNA。本实施例中的arRNA由体外合成，其序列见下表1。序列以两种方式命名，其中“名称”根据序列的长短或截短长度进行命名，“统一化名称”以X-C-Y进行命名，其中X代表5’端的长度，Y代表3’端的长度，C为靶向碱基C。例如111nt命名为55-C-55则表示其5’及3’短的长度均为55nt。
表1.实施例2所用arRNA序列汇总
Figure PCTCN2020141501-appb-000003
Figure PCTCN2020141501-appb-000004
Figure PCTCN2020141501-appb-000005
注：RNA序列中大写字母仅用于区分靶向碱基，同一种碱基的大小写字母不代表碱基类型的差异。
本实施例中所有测试均使用RNAi MAX试剂(Invitrogen 13778150)将arRNA转染入细胞，具体步骤如下：
1.细胞培养使用含有10％FBS(Vistech SE100-011)的DMEM(Hyclone SH30243.01)。使用12孔板培养细胞，报告体系细胞为15000个细胞/孔。铺入细胞的时间记为0小时。
2.细胞传代后24小时，用RNAi MAX试剂将12.5pmol的arRNA转入各孔。转染步骤参照供应商说明书。
3.细胞传代后72小时，用胰酶(Invitrogen 25300054)消化整孔细胞，并用流式细胞仪在FITC通道分析细胞荧光强度。
首先，参照LEAPER参考文献中的arRNA的设计原则(Leveraging Endogenous ADAR for Programmable Editing of RNA,Qu et al.,2019，该文献中体外合成的arRNA仅采用了111nt长度的arRNA进行测试)设计arRNA，即将致病突变位点的A(靶标A)的对位靶向碱基确定为C，使所述靶向碱基的5’端以及3’端的序列长度一致。从总长111nt(55-C-55)的arRNA(与参考文献一致)为基础，同时对5’及3’端进行截短，以形成本申请所使用的各测试arRNA。由于体外合成RNA的限制，目前阶段更长的RNA长度难以在体外合成，因此本实施例仅测试了111nt、91nt、71nt和51nt这四种长度的arRNA，编辑效率的结果见图6。
其中，“对照”表示不转入任何arRNA的对照孔。从图6中可以看出，对照孔本底的GFP阳性细胞很少(约0.6％)，相比之下111nt、91nt和71nt的三条arRNA导致的％GFP均超过90％，而51nt只有很低的GFP阳性比例。从平均荧光强度的图中可以看出，对照孔本底有少量假阳性细胞，且虽然这些细胞占比很少，但其荧光强度较高，如图7所示。
令人意外的是，与上述LEAPER参考文献报道不同，本申请发现51nt～111nt的arRNA可以取得较好的编辑效果，然而并非越长的arRNA编辑效率越高。如图8所示，根据上述LEAPER参考文献(同Qu et al.,2019图2d)中的报道，在其报告体系中，比较了51nt～111nt的arRNA。其中，可见71nt的GFP+细胞比例比111nt要低，其长度与编辑效率的关系从51nt～111nt的范围内基本呈现逐渐增高的趋势。而在实施例1的报告体系测试中，与 111nt相比，71nt导致的GFP阳性细胞比例差异不大，而平均荧光强度却更高。
其次，由于越长的arRNA合成成本越高，并且较长的arRNA可能会导致更大的脱靶可能性，因此，本申请中测试了通过各种方式截短的arRNA的编辑效率。如图9所示，以靶向碱基C为中点，以111nt的arRNA序列(SEQ ID NO：2)为基础，从5’端上游或3’端下游以10nt为步长，逐步向中间截短，以获得用于测试的arRNA。
从图9中不难看出，从5’端截短时，与LEAPER参考文献中的报道相符，即相对更长的RNA编辑效率更高，然而不同的是，从3’端截短时，编辑效率并非逐渐下降，反而在3’端截短30nt时表现出最高的编辑效率。由于从3’端截短30nt时编辑效率最高，而此时的arRNA长度为：55-C-25，因此，在后续试验中，我们将3’端的长度为25nt，从5’端继续进行截短以获得测试arRNA，结果如图10所示。
图10中的“3’-截短30”以及“55-C-25”与图9中“3’截短”的“-30”为相同的arRNA序列(SEQ ID NO：8)，其中，图10中的“3’-截短30”与图9中“3’截短”的“-30”为同批次合成，与“55-C-25”为不同批次合成。从图10中，不难看出，当3’端固定为25nt而从5’端截短时，编辑效率基本呈现随5’长度下降而下降的趋势，其中40-C-25虽然不符合以上趋势，但无论其MFI还是％GFP均未超过55-C-25。
最后，除了长度以外，靶向碱基的位置也对编辑效率有影响。因此随后我们将arRNA长度固定为91nt，通过对靶向碱基位置的移动获得测试arRNA，结果如图11所示。
从图11中可以看出，并非靶向碱基在arRNA中间位置时效率最高，且在3’端长度减少到0之前(如85-C-5及其左侧柱子所示)，arRNA均有较高编辑效率。
最后，我们在同一批次中合成了以上所有arRNA，并对其进行了再次测试，以便排除不同批次导致的测试误差。结果如图12所示。从图12中不难看出，效率最高的3条arRNA为55-C-35(SEQ ID NO：7)、55-C-25(SEQ ID NO：8)、55-C-15(SEQ ID NO：9)。重复测试中，可能由于所用的arRNA经过冻融，整体编辑效率相比第一次测试略有下降。对于两端对称的arRNA， 71nt(35-C-35)与111nt(55-C-55)相当。此外，本批次中最长的111nt arRNA编辑效率并非最高，相比之下，55-C-15、55-C-25、55-C-35这3条长度分别为71nt、81nt、91nt的arRNA拥有相对更高的编辑效率。
并且，相较于现有技术中优选的111nt arRNA，本实施例中55-C-15等长度仅71nt的arRNA不仅有更高的编辑效率，而且合成成本更低(参考价格见表2)。此外，可以预期，更短的arRNA可能会有更低的脱靶风险。
表2.arRNA合成长度及价格
Figure PCTCN2020141501-appb-000006
通过对GFP信号的读取，我们可以快速粗略地判断不同arRNA的编辑效率，但如果更准确地确认编辑效率，则需通过二代测序(Next Generation Sequencing,NGS)，来最终确认mRNA中有多少比例的A被编辑成I(G)。
按照本实施例中上述相同步骤1、2进行细胞铺板和转染，并于细胞传代后72h用800μL TRIzol(Invitrogen,15596018)收样，采用Direct-zol RNA Miniprep试剂盒(Zymo Resaerch,R2052)进行RNA提取。每个样品取1000ng提取的RNA，用
Figure PCTCN2020141501-appb-000007
One-Step gDNA Removal and cDNA Synthesis SuperMix试剂盒(全式金，AT311)进行反转录，以合成cDNA。之后，取1μL反转录产物，用序列为ggagtgagtacggtgtgcGGAAGAAAACCCCGGTCCTGCTA(SEQ ID NO：33)和gagttggatgctggatggAACAGAACTGAATGAGCACTCGTGG(SEQ ID NO：34)两个引物以及Q5热启动酶(NEB,M0494L)进行PCR。PCR产物用Hi-TOM试剂盒(诺禾致源，REF PT045)进行建库并按照以下步骤完成二代测序和数据分析。
i.Illumina测序
将构建好的测序文库，通过NovaSeq6000平台以PE150方式进行高通量测序。
ii.测序数据处理
将高通量测序得到的原始数据用fastp(v0.19.6)进行质控，过滤掉低质量、带有接头的序列、以及含有polyG等的序列。将得到的高质量测序数据按照相应的barcode序列拆分到每个样本，使用BWA(v0.7.17-r1188)软件与扩增的目标区域的序列进行比对，通过SAMtools(v1.9)进行格式转换以生成BAM文件。统计比对信息并重新排序和建立索引。
iii.编辑效率分析
使用JACUSA(v1.3.0)软件检测所有潜在的RNA突变位点，所用参数为：call-1 -a B,R,D,I,Y,M:4-C ACGT -c 2 -p 1 -P UNSTRANDED-R -u DirMult-CE。过滤掉同时在对照和处理样本中出现的高频点突变之后，以A->G突变位点之外的平均突变频率的三倍作为阈值，将靶标碱基A->G突变频率在阈值之上的部分作为真实的靶标A突变为G的频率。
采用如上步骤，我们最终对图12中全部样品进行了二代测序，以靶标碱基为G的Reads数占其为ATCG的全部Reads数的百分比作为编辑效率，并以此作图，结果如图13所示。
对比图12与图13，不难看出，通过不同方法测试得到的编辑效率呈现基本相同的趋势。即按照LEAPER文献(Qu et al.,2019)中设计的111nt的arRNA并非编辑效率最高的arRNA，而经过我们对靶向碱基3’端的截短、5’端的截短以及对靶向碱基在序列中位置的调整，最终发现55-C-15(SEQ ID NO：9)的序列不仅在GFP的读值上体现出较高的编辑效率，并且通过二代测序也可以进一步证实，其A到G的编辑效率可达到50％左右，这意味着经55-C-15的arRNA编辑后的细胞中有50％的mRNA上的靶标碱基位点发生了从A到G的编辑。
通过以上试验，我们证实，按照本申请所述技术方案设计的arRNA相较于现有技术可以达到更高的编辑效率，并且成本更低。同时，可以预知，由于本申请的arRNA所需长度更短，因此在转染拷贝数相同的情况下，本申请中的arRNA转染入细胞的质量更低，这将有益于降低过量RNA的转入对细胞造成的毒性，同时也减少了arRNA与非靶标RNA序列的结合，从而提高了体内编辑的安全性。
实施例3：小鼠眼部Ush2A基因位点的体内编辑
实施例2中，通过体外试验对USH2A致病位点进行了编辑。虽然体外 试验可以取得好的编辑效率，但体外细胞培养的环境无法模拟人体内循环、免疫系统以及眼球内注射等多方面因素，这些仍需更多体内试验验证。考虑到后续与实际治疗对接，而目前对于眼部的小RNA递送，通常选用腺病毒相关病毒(Adeno-Associated Virus，AAV)，我们最终选择了使用AAV载体作为arRNA的递送方式进行小鼠眼部Ush2A基因位点的体内编辑。我们采取了151nt arRNA(SEQ ID NO：32)进行本试验。即本实施例所使用的测试arRNA中包含了与靶标A对位靶向碱基C，当该arRNA与靶标RNA杂交时，即可在靶标A处形成A-C不匹配(错配)。同时，该arRNA靶向碱基C的5’端和3’端则分别为与靶标序列完全互补的75nt碱基序列。由于小鼠Ush2A基因与人USH2A基因序列不同，且测试小鼠并不携带Usher综合征相关突变，本实施例中使用的151nt arRNA是靶向小鼠Ush2A相关位点的arRNA，而非靶向人USH2A基因序列的arRNA。我们将该arRNA编码序列插入至AAV载体U6启动子起始位点之后，并将插入arRNA序列的AAV载体包装成1×10 13GC/mL的AAV2和AAV5。其中，AAV载体的构建(构建后的载体见SEQ ID NO：16,其中arRNA编码序列以大写表示，同一字母的大小不代表碱基差异)和AAV病毒的包装均由广州派真生物技术有限公司完成。
本实施例采用的是6～8周龄的SPF级别C57雄性小鼠，试验组(AAV2及AAV5组)与对照组(PBS)每组各8只小鼠，一共24只。通过玻璃体向试验组及对照组小鼠右眼分别注入2μL AAV或PBS。在注射后第7天(后标记为第1周)、第14天(后标记为第2周)，将每组小鼠各处死4只，并取出其右眼，用剪刀剪碎至50～100mg的小块。加液氮研磨后用800μL TRIzol(Invitrogen,15596018)收样。采用Direct-zol RNA Miniprep试剂盒(Zymo Resaerch,R2052)进行RNA提取。每个样品取1000ng提取的RNA用
Figure PCTCN2020141501-appb-000008
One-Step gDNA Removal and cDNA Synthesis SuperMix试剂盒(全式金，AT311)进行反转录以合成cDNA。每只小鼠各取1μL反转录产物，用序列为ggagtgagtacggtgtgcCCCATCTCTTTGGCTTGGAACCAT(SEQ ID NO：22)和gagttggatgctggatggCTTTTCTCTCTGCTCCACTGTGAAGTT(SEQ ID NO：24)的两个引物以及Q5热启动酶(NEB,M0494L)进行PCR。PCR产物用Hi-TOM试剂盒(诺禾致源，REF PT045)进行建库并完成二代测序和数据分析(测序和数据分析方法同实施例2)。
结果如图14所示。从图中可以看出，在小鼠体内试验中，通过小鼠玻璃体注射时，小鼠眼球细胞中的AA5编辑效率较AAV2更高。但是从图中可以明显的看出，无论是AAV2还是AAV5，编辑效率均较低。例如，在第7天，AAV2的平均编辑效率为0.5％，与PBS组无明显差异。而AAV5的平均效率虽然为1％左右，但4个平行试验呈现明显不一致的编辑效率，其中一个编辑效率为2％以上，另外3个为约0.5％。而到了第14天，AAV2组也有了将近1％的编辑效率，而AAV5组有平均2％的编辑效率，并且其中有一个样品有接近5％的编辑效率。通过该试验，我们发现如下几个问题：首先，在小鼠眼球中似乎AAV5比AAV2的编辑效率更高；其次，不同小鼠间数据差异较大，这可能预示着我们玻璃体注射的条件并不稳定，导致每次注射的差异较大；最后，从第7天到第14天编辑效率有上升的趋势，这意味着如果延后检测时间，可能会检测到更高的编辑效率。
因此，我们重新进行了小鼠眼球注射的试验，并对试验体系进行了如下的优化：1)由于AAV5比AAV2的编辑效率高，此次试验我们保留了AAV5，并且测试了另一血清型的AAV病毒：AAV8；2)针对玻璃体注射编辑效果不稳定的问题，我们将注射条件改为视网膜下腔注射，并在注射后通过光学相干断层扫描(Optical Coherence Tomography，OCT)确认小鼠视网膜下腔注射成功；3)检测时间修改为第14(第2周)、28(第4周)、42(第6周)、56(第8周)；4)由于Usher综合征是由于视网膜中视锥细胞和视杆细胞的退行性病变导致的，因此本次试验仅检测了视网膜中Ush2A的编辑效率，相较于检测整个眼球的编辑效率，仅检测视网膜中Ush2A的编辑效率对未来治疗更具指导意义。
在本次试验中，采用的小鼠为6～8周龄的SPF级别C57雄性小鼠，试验组(AAV5、AAV8组)每组各20只，对照组(NaCl)12只。
视网膜下腔注射方法如下：向动物双眼各滴复方托吡卡胺滴眼液1～2滴散瞳，用舒泰50进行肌肉注射(50mg/kg，50mg/mL)以对动物进行全身麻醉。使麻醉后的小鼠侧卧于操作台上。使用聚维酮碘棉球对小鼠眶周皮肤进行消毒。用有齿显微镊夹住眼球一侧的结膜，用显微剪剪开球结膜下筋膜，暴露部分巩膜。保持显微镊不动，将盖玻片覆盖在角膜上，用卡波姆滴眼液排除其间空气，后用一次性胰岛素针与巩膜成一个小角度，行巩膜、脉 络膜穿刺，直达视网膜上皮与视网膜交界处，形成通道。继续保持显微镊不动，退出胰岛素针，并将其更换为33G显微注射器。将33G显微注射器从通道口扎至视网膜下，缓慢给药1μL后，停针至少3-5s。在动物麻醉状况良好的状态下继续将显微注射器保持在通道内30s左右(以尽量减少退针时的漏液体积)。在每只小鼠给药后需清洗针头，以确保无交叉污染。双眼注射结束后，使用OCT扫描确认注射位置为视网膜下腔。若双眼均成功注射至视网膜下腔则判定该动物注射成功。注射后于第14、28、42、56天分别安乐死5只(试验组)或3只(对照组)小鼠。取出小鼠左眼，用手术刀沿角巩膜缘360°切开角膜。去掉虹膜和晶状体，取视网膜-脉络膜-巩膜复合体，将最外侧巩膜剔除后，剪碎并放入装有经预冷的400μL TRIzol(Thermo,15596018)的无菌EP管中。然后用组织匀浆机混匀。用Direct-zol RNA Microprep试剂盒(Zymo Resaerch,R2062)提取RNA。每个样品取1000ng提取的总RNA用全式金反转录试剂盒(全式金，AH341-01)反转录，并取5μL反转录后的cDNA用序列为ggagtgagtacggtgtgcCCCATCTCTTTGGCTTGGAACCAT(SEQ ID NO：22)和gagttggatgctggatggCTTTTCTCTCTGCTCCACTGTGAAGTT(SEQ ID NO：2)的两个引物以及Q5热启动酶(NEB,M0494L)进行PCR。PCR产物用Hi-TOM试剂盒(诺禾致源，REF PT045)进行建库并完成二代测序和数据分析(测序和数据分析方法同实施例2)。
结果如图15所示。从图中可以明显的看出，相比于首次试验最高平均2％的编辑效率(图14)，本次试验最高可达超过5％的编辑效率。另外，由于本次试验剥离了小鼠视网膜进行收样测序，而非将小鼠整个眼球剪碎研磨，因此更能反应实际治疗时产生的实际治疗效应。通过本试验可以证明，LEAPER技术可以在小鼠体内试验中编辑视网膜细胞中Ush2A内源基因位点，并且这种编辑可以在小鼠体内持续至少2个月。
综合以上实施例结果，本申请充分证明了使用本申请公开的技术方案治疗Usher综合征的可行性。
SEQ ID NO：32：
Figure PCTCN2020141501-appb-000009
Figure PCTCN2020141501-appb-000010
SEQ ID NO： 16：
Figure PCTCN2020141501-appb-000011
Figure PCTCN2020141501-appb-000012
参考文献：
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2.Cox,D.B.,Gootenberg,J.S.,Abudayyeh,O.O.,Franklin,B.,Kellner,M.J.,Joung,J.,&Zhang,F.(2017).RNA editing with CRISPR-Cas13.Science,358(6366),1019-1027.
3.Davenport,S.L.H.,&Omenn,G.S.(1977).The heterogeneity of Usher syndrome.Vth Int.Conf.Birth Defects,Montreal.
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Figure PCTCN2020141501-appb-000013
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5.Liu,X.,Bulgakov,O.V.,Darrow,K.N.,Pawlyk,B.,Adamian,M.,Liberman,M.C.,&Li,T.(2007).Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells.Proceedings of the National Academy of Sciences,104(11),4413-4418.
6.Merkle,T.,Merz,S.,Reautschnig,P.,Blaha,A.,Li,Q.,Vogel,P.,...&Stafforst,T.(2019).Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.Nature biotechnology,37(2),133.
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8.Otterstedde,C.R.,Spandau,U.,Blankenagel,A.,Kimberling,W.J.,&Reisser,C.(2001).A new clinical classification for Usher's syndrome based on a new subtype of Usher's syndrome type I.The Laryngoscope,111(1),84-86.
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Claims (25)

  1. 一种基于LEAPER技术靶向编辑细胞中靶标RNA的方法，其中所述靶标RNA为USH2A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的腺苷脱氨酶招募RNA(arRNA)的构建体或编码所述arRNA的构建体导入所述细胞中，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能够招募作用于RNA的腺苷脱氨酶(ADAR)以使靶标RNA中的靶标腺苷脱氨基。
  2. 如权利要求1中所述的方法，其中所述靶标RNA为mRNA前体(Pre-mRNA)。
  3. 如权利要求1或2中所述的方法，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长度选自66nt-131nt中的任意自然数。
  5. 如权利要求1-4中任一项所述的方法，其中所述arRNA中靶向碱基距离3’端的长度≥7nt，≥10nt、≥15nt，优选16-55nt、20-50nt、25-45nt、25-35nt或25-30nt。
  6. 如权利要求1-5中任一项所述的方法，其中所述arRNA中靶向碱基距离5’端的长度≥25nt，≥30、≥35、≥45，优选46-90nt、50-85nt、55-80nt、60-75nt或65-70nt。
  7. 权利要求1-6中任一项的方法，其中向所述arRNA引入与靶标序列中的靶标A配对的靶向碱基，该靶向碱基的优选次序从高到低依次为C、A、U或G。
  8. 如权利要求1-7中任一项的方法，其中所述靶标RNA为包含转录有人USH2A基因NM_206933.2(USH2A)_c.11864 G>A(p.Trp3955Ter)突变位点的RNA。
  9. 如权利要求1-8中任一项所述的方法，其中所述arRNA选自：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ  ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4。
  10. 如权利要求1-9中任一项所述的方法，其中所述arRNA选自：SEQ ID NO：7、SEQ ID NO：8或SEQ ID NO：9。
  11. 如权利要求1-10中任一项所述的方法，其中所述构建体为线性核酸、病毒载体、或质粒。
  12. 如权利要求11中所述的方法，其中所述病毒载体为腺相关病毒(AAV)载体或慢病毒载体。
  13. 如权利要求12中所述的方法，其中所述AAV载体包装入AAV2、AAV5或AAV8衣壳后通过侵染导入所述细胞中。
  14. 如权利要求13中所述的方法，所述AAV载体包装入AAV8衣壳后通过侵染导入所述细胞中。
  15. 如权力要求1-14中任一项所述的方法，其中所述细胞为哺乳动物视神经细胞或听神经细胞。
  16. 一种用于靶向编辑USH2A基因转录本中含有G到A突变的靶标RNA的arRNA，包含选自如下的序列或由选自如下的序列组成：SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：2、SEQ ID NO：3、或SEQ ID NO：4。
  17. 包含权利要求16的arRNA的构建体或递送体。
  18. 如权利要求17的构建体或递送体，其为质粒、病毒、脂质体、或脂质纳米颗粒。
  19. 如权利要求18的构建体或递送体，其为腺相关病毒(AAV)或慢病毒。
  20. 如权利要求19的构建体或递送体，其所述病毒为AAV2、AAV5或AAV8。
  21. 通过权利要求1-15中任一项的方法编辑获得的细胞。
  22. 一种治疗个体中Usher II型综合征的方法，包括使用权利要求1-15 中任一项所述的方法校正所述个体的细胞中与Usher II型综合征有关的G到A的突变。
  23. 如权利要求22中所述的方法，其包括将如权利要求16中所述的arRNA或如权利要求17-20中任一项所述的构建体或递送体导入受试者。
  24. 权利要求23的方法，其中将如权利要求16中所述的arRNA或如权利要求17-20中任一项所述的构建体或递送体导入受试者视网膜下腔中。
  25. 如权利要求16中所述的arRNA或如权利要求17-20中任一项所述的构建体或递送体在制备治疗Usher II型综合征的药物中的用途。
PCT/CN2020/141501 2019-12-30 2020-12-30 一种治疗Usher综合征的方法和其组合物 Ceased WO2021136404A1 (zh)

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JP2022540983A JP2023509177A (ja) 2019-12-30 2020-12-30 Ｕｓｈｅｒ症候群を治療する方法及びその組成物
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CR20220366A CR20220366A (es) 2019-12-30 2020-12-30 Método para el tratamiento del síndrome de usher y composición del mismo
PH1/2022/551594A PH12022551594A1 (en) 2019-12-30 2020-12-30 Method for treating usher syndrome and composition thereof
US17/790,484 US20230067480A1 (en) 2019-12-30 2020-12-30 Method for treating usher syndrome and composition thereof
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BR112022012921A BR112022012921A2 (pt) 2019-12-30 2020-12-30 Métodos para editar de forma alvejada ácido ribonucleico e para tratar a síndrome de usher tipo ii, ácido ribonucleico de recrutamento da adenosina desaminase, construto ou vetor de entrega, célula, e, uso do ácido ribonucleico de recrutamento da adenosina desaminase ou do construto ou vetor de entrega
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