WO2021212686A1 - RHO-adRP基于基因编辑的方法和组合物 - Google Patents

RHO-adRP基于基因编辑的方法和组合物 Download PDF

Info

Publication number
WO2021212686A1
WO2021212686A1 PCT/CN2020/105881 CN2020105881W WO2021212686A1 WO 2021212686 A1 WO2021212686 A1 WO 2021212686A1 CN 2020105881 W CN2020105881 W CN 2020105881W WO 2021212686 A1 WO2021212686 A1 WO 2021212686A1
Authority
WO
WIPO (PCT)
Prior art keywords
grna
vector
rho
mutation
gene
Prior art date
Application number
PCT/CN2020/105881
Other languages
English (en)
French (fr)
Inventor
杨丽萍
柳小珍
乔静
张凡
张天赋
和赛超
曾露颖
裴红杰
Original Assignee
北京中因科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京中因科技有限公司 filed Critical 北京中因科技有限公司
Priority to CN202080001820.6A priority Critical patent/CN113038971B/zh
Priority to US17/996,786 priority patent/US20230149439A1/en
Publication of WO2021212686A1 publication Critical patent/WO2021212686A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • This application relates to the field of biomedicine, and specifically to a gene editing drug for Chinese RHO-adRP patients based on CRISPR/Cas9 technology and AAV technology.
  • Retinitis Pigmentosa is a group of hereditary blind eye diseases whose main changes are the progressive loss of photoreceptor cell and/or retinal pigment epithelial cell function.
  • RP Retinitis Pigmentosa
  • CRISPR/Cas9 CRISPR/Cas9 technology is convenient and simple to operate. It is currently the most commonly used gene editing technology and one of the main tools for gene therapy of hereditary retinal degeneration.
  • RHO is the earliest discovered RP pathogenic gene. About 30%-40% of autosomal dominant RP (adRP) is caused by this gene and is the most important pathogenic gene for adRP.
  • RHO-adRP Normally, one allele of RHO-adRP patients carries pathogenic mutations, while the other allele is normal; the pathogenic mechanism of RHO mutations is the mechanism of gain-of-function or dominant negative effect.
  • the RHO gene p.Pro23His is a mutation hotspot in the RHO-adRP population in North America.
  • the gene editing therapy based on CRISPR/Cas9 technology for RHO is mostly related to this locus.
  • the hotspots of RHO mutations in Asian populations and they cannot be used for gene editing therapy in Chinese RHO-adRP populations. Therefore, it is necessary to study drug design for the hot spots of gene mutations in the Chinese population.
  • Adeno-associated virus (AAV) vector as a gene transfer vector has the advantages of non-pathogenicity, low immunogenicity, effective transfer of target genes and long-term expression of the therapeutic genes carried, making it the most widely used retinal gene The carrier of treatment.
  • the maximum carrying capacity of AAV vectors is 4.7kb.
  • sgRNA and Cas9 can only be packaged separately. Therefore, in order to improve the efficiency of targeting, it is necessary to select a suitable vector.
  • This application provides a gene editing drug based on CRISPR/Cas9 technology and AAV technology for RHO-adRP patients in China.
  • This application designs mutant allele-specific gRNAs for the mutation hotspots of the Chinese RHO-adRP population, knocks out the mutant alleles, but retains the normal alleles to achieve the purpose of treatment.
  • the mutation site can be located on the gRNA, so that the gRNA and the mutation The alleles are perfectly matched, and there is a difference of one base from the normal alleles.
  • Such gRNAs are mutant allele-specific gRNAs.
  • this application can use the CRISPR/SaCas9 system for gene editing, and the SaCas9 protein and gRNA can be packaged into a single AAV virus.
  • the pX601-SaCas9 plasmid vector can be used, and gRNA and saCas9 can be packaged into an AAV vector (for example, AAV8 vector) to improve targeting efficiency.
  • the vector can be injected into the eyeball of the RHO-adRP patient by subretinal cavity injection for the purpose of treatment.
  • the method and composition described in the application can specifically cut the relevant mutation sites of the RHO gene, have a certain cutting efficiency and safety, have been verified on cells, tissues and animal models, and have great application value.
  • the present application provides a method for treating retinitis pigmentosa, the method comprising the steps of: enabling a subject in need to have a functional RHO gene, wherein the functional RHO gene does not include a RHO gene selected from the group consisting of Group mutation sites: c.C50T and c.C403T.
  • the method includes the step of removing the mutation site of the RHO gene in a subject in need.
  • the removal includes knocking out the mutation site and/or reducing the expression level of the RHO gene containing the mutation site.
  • the removal includes not affecting the expression level and/or function of the wild-type RHO gene in the subject.
  • the removal includes causing a double-strand break in the RHO allele containing the mutation.
  • the removal includes administering to a subject in need at least one vector capable of removing the mutation site.
  • the vector includes a sequence encoding a gRNA that specifically binds to the mutation site.
  • the gRNA specifically binds to at least part of the nucleic acid in the RHO allele that includes the mutation site.
  • the gRNA is specifically complementary to at least part of the nucleic acid sequence of exon 1 of the RHO allele containing the c.C50T mutation.
  • the gRNA that is specifically complementary to at least part of the nucleic acid sequence of exon 1 of the RHO allele containing the c. Amino acid sequence.
  • the sequence encoding the gRNA that is specifically complementary to at least part of the nucleic acid sequence of exon 1 of the RHO allele containing the c.C50T mutation comprises any one of SEQ ID NO. 1-2 The nucleotide sequence shown.
  • the gRNA is specifically complementary to at least part of the nucleic acid sequence of exon 2 of the RHO allele containing the c.C403T mutation.
  • the gRNA that is specifically complementary to at least a part of the nucleic acid sequence of exon 2 of the RHO allele containing the c.C403T mutation comprises the amino acid sequence shown in SEQ ID NO:47.
  • the sequence encoding the gRNA that is specifically complementary to at least part of the nucleic acid sequence of exon 2 of the RHO allele containing the c.C403T mutation comprises the nucleotides shown in SEQ ID NO.4 sequence.
  • the vector includes a nucleic acid encoding a Cas protein.
  • the Cas protein includes a Cas9 protein.
  • sequence encoding the gRNA and the nucleic acid encoding the Cas protein are located in the same vector.
  • the vector includes a viral vector.
  • the vector is an adenovirus-associated vector (AAV).
  • AAV adenovirus-associated vector
  • the vector is AAV8.
  • the subject includes an East Asian.
  • the method is performed under conditions including in vitro, in vivo, or ex vivo.
  • the administration includes injection.
  • the administration includes subretinal cavity injection.
  • the present application provides a method for editing the RHO gene, the method comprising the following steps: removing a mutation site selected from the group consisting of c.C50T and c.C403T in the RHO gene.
  • the removal includes knocking out the mutation site and/or reducing the expression level of the RHO gene containing the mutation site.
  • the removal includes not affecting the expression level and/or function of the wild-type RHO gene in the subject.
  • the removal includes causing a double-strand break in the RHO allele containing the mutation.
  • the removal includes administering at least one vector capable of removing the mutation site.
  • the vector includes a sequence encoding a gRNA that specifically binds to the mutation site.
  • the gRNA specifically binds to at least part of the nucleic acid in the RHO allele that includes the mutation site.
  • the gRNA is specifically complementary to at least part of the nucleic acid sequence of exon 1 of the RHO allele containing the c.C50T mutation.
  • the gRNA-encoding sequence comprises the nucleotide sequence shown in any one of SEQ ID NO. 1-2.
  • the gRNA is specifically complementary to at least part of the nucleic acid sequence of exon 2 of the RHO allele containing the c.C403T mutation.
  • the gRNA-encoding sequence includes the nucleotide sequence shown in SEQ ID NO.4.
  • the vector includes a nucleic acid encoding a Cas protein.
  • the Cas protein includes a Cas9 protein.
  • sequence encoding the gRNA and the nucleic acid encoding the Cas protein are located in the same vector.
  • the vector includes a viral vector.
  • the vector is an adenovirus-associated vector (AAV).
  • AAV adenovirus-associated vector
  • the vector is AAV8.
  • the present application provides a composition for treating retinitis pigmentosa in a subject, which comprises an active ingredient for removing a mutation site of the RHO gene and a pharmaceutically acceptable carrier, wherein the mutation site is selected from the group consisting of Group: c.C50T and c.C403T.
  • the active ingredient includes a sequence encoding a gRNA that specifically binds to the mutation site.
  • the gRNA that specifically binds to the mutation site comprises the nucleotide sequence shown in any one of SEQ ID NOs. 44, 45 and 47.
  • the gRNA-encoding sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs. 1, 2 and 4.
  • the active ingredient includes Cas protein.
  • the Cas protein includes a Cas9 protein.
  • sequence encoding the gRNA and the nucleic acid encoding the Cas protein are located in the same vector.
  • the vector includes a viral vector.
  • the vector is an adenovirus-associated vector (AAV).
  • AAV adenovirus-associated vector
  • the vector is AAV8.
  • Figure 1 shows the structural feature map of the gene editing vector used in this application.
  • Figure 2 shows the three coding gRNA sequences designed for RHO p.Thr17Met.
  • Figure 3 shows two gRNA-encoding sequences designed for RHO p.Arg135Trp.
  • Figure 4 shows the in vitro efficiency test results of RHO17-SgRNA.
  • Figure 5 shows the in vitro efficiency test results of RHO135-SgRNA.
  • Figure 6 shows the editing results of in vitro detection of RHO17-SgRNA1 using the gRNA activity fluorescence detection kit.
  • Figure 7 shows the results of in vitro detection of RHO17-SgRNA2 editing using the gRNA activity fluorescence detection kit.
  • Figure 8 shows the results of in vitro detection of RHO17-SgRNA3 editing using the gRNA activity fluorescence detection kit.
  • Figure 9 shows the editing results of in vitro detection of RHO135-SgRNA1 using the gRNA activity fluorescence detection kit.
  • Figure 10 shows the editing results of in vitro detection of RHO135-SgRNA2 using the gRNA activity fluorescence detection kit.
  • Figure 11 shows the statistics of flow sorting results for in vitro detection of sgRNA editing efficiency using a gRNA activity fluorescence detection kit.
  • Figure 12 shows the results of running gels using 293T cells to verify the safety and specificity of sgRNA in vitro.
  • Figure 13 shows the gene editing efficiency of RHO17-SgRNA1 and RHO17-SgRNA2 only for patient iPSCs.
  • Figure 14 shows the gene editing efficiency of RHO135-SgRNA1 only on patients' iPSCs.
  • Figure 15 shows the gene editing effects of RHO17-SgRNA2 and RHO135-SgRNA1 on the patient's 3D retinal tissue.
  • Figure 16 shows the genotype identification results of RHO humanized mice.
  • Figure 17 shows the gene editing effects of RHO17-SgRNA2 and RHO135-SgRNA1 on the retinal tissue of humanized mice.
  • c.C50T usually refers to the 50th position (from the 5'end to the 3'end, starting from the coding sequence) of the coding sequence of the RHO gene compared with the nucleotide sequence of the wild-type RHO gene.
  • the "A” in the original ATG is the first) base was mutated from cytosine (C) to thymine (T).
  • c usually refers to coding sequence, that is, a coding sequence, which refers to a sequence from the start code ATG to the stop code, and the coding sequence can start and end at any position of the mRNA.
  • c.C50T means that starting from the A of the coding sequence ATG, the 50th nucleotide is mutated from C to T.
  • the mutation of the base can cause the amino acid coded by the RHO gene to change, for example, the amino acid is mutated from threonine (Thr) to methionine (Met).
  • p.Thr17Met generally refers to the mutation of the amino acid at position 17 of the RHO protein from threonine (Thr) to methionine (Met).
  • the term "c.C403T” usually refers to the 403th position (from 5'end to 3'end, the coding sequence starts from the 5'end to the 3'end of the RHO gene coding sequence compared with the nucleotide sequence of the wild-type RHO gene).
  • the "A” in ATG is position 1).
  • the base is mutated from cytosine (C) to thymine (T).
  • the mutation of the base can cause the amino acid coded by the RHO gene to be mutated, for example, the amino acid is changed from the essence.
  • Amino acid (Arg) is mutated to tryptophan (Trp).
  • the term “p.Arg135Trp” generally refers to the mutation of the amino acid at position 135 of the RHO protein from arginine (Arg) to tryptophan (Trp).
  • exon 1 of the RHO allele generally refers to the first exon in the RHO gene.
  • ID of exon 1 of the RHO allele in the Ensembl database is ENSE00001079597, which may include the nucleotide sequence of positions 129,528,639-129,529,094 of Homo sapiens chromosome 3.
  • exon 5 of the RHO allele generally refers to the fifth exon in the RHO gene.
  • ID of exon 5 of the RHO allele in the Ensembl database is ENSE00001079599, which may include the nucleotide sequence of 129,533,608-129,535,344 of Homo sapiens chromosome 3.
  • double-strand breaks generally refers to a phenomenon that occurs when two single strands of a double-stranded DNA molecule are cut at the same position. Double-strand breaks can induce DNA repair and may cause genetic recombination. Cells also have some systems acting on double-strand breaks caused at other times. Double-strand breaks can occur regularly during the normal cell replication cycle, and can also be enhanced under certain circumstances, such as ultraviolet rays, DNA break inducers (for example, various chemical inducers). Many inducers can cause DSB to occur indiscriminately in the genome, and DSB can be regularly induced and repaired in normal cells.
  • the original sequence can be reconstructed with complete fidelity, but, in some cases, small insertions or deletions (called “indels”) will be introduced at the DSB site.
  • double-strand breaks can also be specifically induced at specific locations, which can be used to cause targeted or preferential genetic modifications at selected chromosomal locations.
  • the tendency of homologous sequences to be easily recombined during DNA repair (and replication) can be used, which is the basis for the application of gene editing systems (such as CRISPR).
  • This homology-guided repair is used to insert the target sequence provided by the use of a "donor" polynucleotide into a desired chromosomal location.
  • knockout refers to a change in the nucleic acid sequence of a gene that reduces the biological activity of the polypeptide normally encoded by the gene by at least 80% compared to the unaltered gene.
  • the change can be an insertion, substitution, deletion, frameshift mutation, or missense mutation of one or more nucleotides.
  • nucleic acid e.g., RNA
  • nucleotide sequence e.g., Watson-Crick base pairing
  • it under the conditions of ionic strength of the solution, it "hybridizes” or “complements” to another nucleic acid in a sequence-specific and antiparallel manner (ie, nucleic acid specifically binds to a complementary nucleic acid).
  • standard Watson-Crick base pairing includes: adenine (A) paired with thymidine (T), adenine (A) paired with uracil (U), guanine (G) paired with cytosine (C) Pairing.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids having any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. These terms also cover amino acid polymers that have been modified. These modifications can include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component).
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to a polymeric form of nucleotides of any length, Such as deoxyribonucleotides or ribonucleotides, or their analogs.
  • a polynucleotide can have any three-dimensional structure, and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one loci) defined by linkage analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence Of
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be carried out before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, and is used to transfer the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • Plasmid usually refers to DNA molecules other than chromosomes or nucleoids in bacteria, yeasts and other organisms. They exist in the cytoplasm and have the ability to replicate autonomously, enabling them to maintain a constant copy in the progeny cells. Count and express the genetic information carried. Plasmids are used as gene carriers in genetic engineering research.
  • retroviral vector generally refers to a virus particle that can control and express foreign genes, but cannot self-package into a virus particle that has the ability to proliferate. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains the genes that make up the virus's center and structure; pol, which contains the genes for reverse transcriptase; and env, which contains the genes that make up the virus coat. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome. For example, the CAR molecule can be integrated into the host cell.
  • the term "lentiviral vector” generally refers to a diploid RNA viral vector belonging to retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus. Many of the sequence structures related to the viral activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome skeleton And express the structure, and prepare it into a vector.
  • the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome.
  • the CAR molecule can be integrated into the host cell.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a method for treating retinitis pigmentosa.
  • RP Retinitis Pigmentosa
  • RP Retinitis Pigmentosa
  • RP is characterized by progressive vision loss caused by abnormal function of the receptor cells (cones and rods) of the retina and/or retinal pigment epithelial cells.
  • the clinical manifestations of RP can include night blindness, progressive visual field defect, central vision loss after the macula is involved, and eventually blindness.
  • Electroretinogram shows that the function of rods (Rods) is decreased or even extinct.
  • the main fundus of RP changed into retinal pigment disorder at the equatorial region, and osteocytic-like pigmentation appeared, which gradually developed toward the posterior pole and the jagged edge.
  • RPE photoreceptor cells and choroidal capillary layer gradually atrophy, see the choroidal large blood vessels, the retina is blue-gray, the retinal arteries become thinner, and the optic disc is waxy yellow.
  • retinal vascular stenosis, optic disc color sallow and osteocytic pigmentation are RP The typical triad (Hartong DT et al., 2006).
  • Methods to assess the function and morphology of the retina can include best corrected visual acuity (Best Corrected Visual Acuity, BCVA), fundus autofluorescence, visual field examination, ERG, fundus color photography, optical coherence tomography (optical coherence tomography, OCT), and fluorescein Angiography (fluorescein angiography, FFA), etc.
  • BCVA Best Corrected Visual Acuity
  • OCT optical coherence tomography
  • FFA fluorescein Angiography
  • the methods of evaluating visual function can include methods such as BCVA and visual field.
  • the methods described in this application may include functional RHO genes in subjects in need.
  • the RP described in this application may be caused by mutations in the RHO gene.
  • RHO gene mutations related to RP. These gene mutations can cause the RHO gene to encode a functionally abnormal rhodopsin protein (rhodopsin).
  • the mutations can include but are not limited to gene missense, nonsense, insertion, deletion and other mutations.
  • the mutation site may include a mutation site selected from the group consisting of c.C50T and c.C403T.
  • the mutation site may cause an amino acid change, and the amino acid mutation may include the following changes: p.Thr17Met and/or p.Arg135Trp.
  • the method may include making the subject in need not have a heterozygous mutation site of the RHO gene, and the mutation site may be selected from the group consisting of c. C50T and c. C403T
  • any one or more mutations can be repaired to make the subject in need have a functional RHO gene.
  • the pathological variants c.C50T and/or c.C403T can be removed, restored or corrected.
  • the term "functional RHO gene” generally refers to a gene capable of encoding a functional rhodopsin protein.
  • the term refers to a RHO gene that does not contain a mutation site, for example, the mutation site can be selected from the following group: c.C50T and c.C403T.
  • RHO genes containing mutation sites can be made functional RHO genes.
  • mutation sites e.g., c.C50T and c.C403T
  • mutation sites can be specifically removed, and for example, mutation sites (e.g., , C.C50T and c.C403T) and make RHO genes containing mutation sites (for example, c.C50T and c.C403T) functional RHO genes.
  • Rhodopsin protein is located in the Rods Outer Segment (ROS), which is necessary for normal vision, especially for weak light stimulation. Rods is a scotopic vision of the photoreceptor cells of the retina. Another type of photoreceptor cells in the retina are cone cells (Cones), which are used for vision and color vision. In ROS, rhodopsin usually binds to 11-cis retinal (11cRAL), which is a derivative form of vitamin A.
  • ROS Rods Outer Segment
  • Cons cone cells
  • rhodopsin usually binds to 11-cis retinal (11cRAL), which is a derivative form of vitamin A.
  • ROS absorbs photons to make Rhodopsin active Rhodopsin (R*), and 11cRAL is isomerized to all-trans retinal (atRAL). After atRAL is separated from R*, it is quickly reduced to all-trans retinol. (all-trans retinol, atROL), interphotoreceptor retinoid-binding protein (IRBP) is responsible for transferring atROL into RPE cells.
  • IRBP interphotoreceptor retinoid-binding protein
  • AtROL is derived from lecithin retinol acyltransferase ( lecithin retinol acyltransferase, LRAT) is converted into all-trans retinol ester, and then further converted into 11-cis retinol ester, and then isomerized into 11-cis retinol (11-cis retinol) by hydrolytic isomerase RPE65 retinol, 11cROL), 11cROL is oxidized by RDHs to 11cRAL, combined with IRBP and transported back to the photoreceptor cells to be reused; R* converts the GDP on the G(Gt) ⁇ subunit of the transduction protein in the downstream membrane disc into GTP, making The alpha subunit is separated from the beta gamma subunit, activates cyclic guanosine phosphate-phosphodiesterase 6 (cGMP-PDE6), hydrolyzes cGMP, reduces the cell
  • R* is phosphorylated and combined with inhibitory protein.
  • PDE6 Inhibiting the downstream signal pathway, PDE6 is in an inactive state, and at the same time, cGMP is synthesized in Rods, and the increase of cGMP concentration makes the cation channel open and Ca 2+ inflows, and the cell membrane is depolarized.
  • the nerve impulse triggered by the opening and closing of the cGMP-gated cation channel is transmitted to the visual center of the cerebral cortex through the connection between the synaptic end of the photoreceptor cell and the neurons of the retina and the optic nerve to the visual center of the cerebral cortex to form vision.
  • the human RHO gene is located at position 22.1 (3q22.1) on the long arm of chromosome 3.
  • the molecule is from base pair 129,528,639 to base pair 129,535,344 on chromosome 3 (Homo sapiens, Annotation release) Version 109.20200228, GRCh38.p13, NCBI).
  • nucleotide sequence of the RHO gene please refer to NCBI GenBank Accession No. NG_009115.1.
  • the RHO gene has 5 exons. Table 1 shows the exon identifier of the RHO gene in the Ensembl database and the start/stop site of the exon.
  • the condition that causes the RHO gene to function abnormally may be a gene mutation, and the mutation may include, but is not limited to, nucleotide insertion, deletion, missense, nonsense, frameshift, and/or other mutations.
  • any one or more mutations can be repaired to restore the normal function of the RHO gene.
  • the mutation site of the RHO gene can be removed.
  • the method may include the step of removing the mutation site of the RHO gene in a subject in need.
  • the method may include exon deletion.
  • Targeted deletion of specific exons may be a strategy for treating a large number of patients with a single therapeutic cocktail.
  • Exon deletion can be single exon deletion or multiple exon deletion. Although multi-exon deletions can cover more subjects, for deletions with more nucleotides, the efficiency of deletion will greatly decrease as the size of the nucleotide increases. Therefore, the range of removal can be 40 to 10,000 base pairs (bp). For example, the removal range can be 40-100, 100-300, 300-500, 500-1,000, 1,000-2,000, 2,000-3,000, 3,000-5,000, or 5,000-10,000 base pairs.
  • the RHO gene contains 5 exons. Any one or more of the five exons can contain mutations. It is possible to remove any one or more mutation sites in the 5 mutant exons or abnormal intron splice acceptors or donor sites, so that the functional RHO gene does not contain mutation sites (e.g., affecting RHO Mutations in gene function).
  • the mutation site may be from any one or more of the following groups of the RHO gene: exon 1, exon 2, exon 3, exon 4, and exon 5. Or any combination thereof.
  • the gene mutation may be a mutation selected from the group consisting of c. C50T and c. C403T. The gene mutations can lead to amino acid mutations, and ultimately lead to abnormal functions of the RHO protein. For example, the mutant protein interferes with the function of the normal protein or cannot locate ROS.
  • the method described in the present application may include knocking out the mutation site and/or reducing the expression level of the mutation site.
  • Methods of knocking out genes or reducing gene expression levels may include gene knockout, conditional gene knockout methods (for example, using Cre/LoxP and/or FLP-frt systems), inducible gene knockout methods (for example, using Cre/ Loxp system-based knockout, including tetracycline induction, interferon induction, hormone induction, adenovirus induction, etc.), gene knockout using random insertion mutations (for example, gene trapping), gene knockout using RNAi, zinc Refers to endonuclease (zinc finger nucleases, ZNF) mediated gene editing technology, transcription activator-like effector nucleases (transcription activator-like effector nucleases, TALEN) mediated gene editing technology, clusters of regular and short intervals Gene editing technology and/or NgAgo-gDNA gene editing technology mediated by clustered regularly interspaced
  • the methods described in this application may be based on in vivo cells.
  • the method includes editing the genomic DNA of the subject's cells. For example, it may include editing a mutation in the RHO gene in the subject's cells (e.g., photoreceptor cells and/or retinal progenitor cells).
  • the gene mutation may be a mutation selected from the group consisting of c. C50T and c. C403T.
  • certain cells may be ideal targets for ex vivo methods or ex vivo therapies, the use of effective delivery methods may also allow for the direct delivery of the required agent to such cells in vivo.
  • the method may include targeting and editing to relevant cells. It is also possible to prevent the lysis of other cells by using promoters that are only active in certain cells and/or stages of development.
  • the additional promoter is inducible, so if the nucleic acid molecule is delivered in a plasmid vector, the delivery time can be controlled. The time that the delivered nucleic acid or protein stays in the cell can also be adjusted by changing the half-life. In vivo methods can save some processing steps, but require higher editing efficiency. In vivo treatment can eliminate the problems and losses caused by ex vivo treatment and implantation.
  • In vivo methods can facilitate the production and administration of therapeutic products.
  • the same treatment method or therapy will likely be used to treat more than one subject, for example, many subjects with the same or similar genotypes or alleles.
  • the methods described in this application may include ex vivo methods.
  • subject-specific induced pluripotent stem cells iPSC
  • the genomic DNA of these iPSC cells can be edited using the methods described in this application.
  • the method may include editing in or near the mutation site of the RHO gene of iPSC so that it does not have amino acid mutations of p.Thr17Met and/or p.Arg135Trp, for example, the gene mutation may be selected from the group consisting of The mutations: c.C50T and c.C403T.
  • the gene-edited iPSC can be differentiated into other cells, such as photoreceptor cells or retinal progenitor cells.
  • differentiated cells (such as photoreceptor cells or retinal progenitor cells) can be implanted into the subject.
  • photoreceptor cells or retinal progenitor cells can be isolated from the subject.
  • the method described in this application can be used to edit the genomic DNA of these photoreceptor cells or retinal progenitor cells.
  • the method may include editing in or near the mutation site of the RHO gene of the photoreceptor cell or retinal progenitor cell so that it does not have the amino acid mutation of p.Thr17Met and/or p.Arg135Trp, for example, the gene mutation may be It is a mutation selected from the following group: c.C50T and c.C403T.
  • gene-edited photoreceptor cells or retinal progenitor cells can be implanted into the subject.
  • mesenchymal stem cells can be isolated from the body in other cases, or from bone marrow or peripheral blood in other cases.
  • the genomic DNA of these mesenchymal stem cells can be edited using the method described in this application.
  • the method may include editing in or near the mutation site of the RHO gene of the mesenchymal stem cell so that it does not have the amino acid mutation of p.Thr17Met and/or p.Arg135Trp, for example, the gene mutation may be selected. Mutations from the following group: c.C50T and c.C403T.
  • the gene-edited mesenchymal stem cells can be differentiated into any type of cells, such as photoreceptor cells or retinal progenitor cells.
  • differentiated cells such as photoreceptor cells or retinal progenitor cells, can be implanted into the subject.
  • the method may include a thorough analysis of the therapeutic agent prior to administration. For example, the entire genome of the correction cell is sequenced to ensure that no off-target effects (if any) can be located in the genome location that is associated with minimal risk to the subject.
  • populations of specific cells can be isolated prior to implantation, including clonal cell populations.
  • Using the method described in this application may not affect the expression level and/or function of the wild-type RHO gene in the subject.
  • the method described in the application may include a method of using a site-directed nuclease to cut DNA at a precise target location in the genome, thereby generating single-stranded or double-stranded DNA breaks at a specific location in the genome.
  • breaks can be regularly repaired through endogenous cellular processes, such as HDR and Non-Homologous End Joining (NHEJ).
  • HDR uses homologous sequences or donor sequences as templates to insert specific DNA sequences at breakpoints.
  • Homologous sequences can be in the endogenous genome, such as sister chromatid.
  • the donor may be an exogenous nucleic acid, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, or virus.
  • exogenous nucleic acids may contain regions with high homology to the nuclease cleaved locus, and may also contain additional sequences or sequence changes (including deletions that can incorporate the cleaved target locus).
  • the third repair mechanism can be Microhomology-Mediated End Joining (MMEJ), also known as "Replacement NHEJ (ANHEJ)". Small deletions and insertions may occur at the cutting site.
  • MMEJ Microhomology-Mediated End Joining
  • ANHEJ Replacement NHEJ
  • MMEJ can use several base pairs of homologous sequences on both sides of the DNA break site to drive more favorable DNA end join repair results. In some cases, it is possible to predict possible repair results based on the analysis of the potential microscopic homology of DNA break sites.
  • the method described in this application may include creating one or two DNA breaks in the target locus close to the expected mutation site, and the two DNA breaks may be double-strand breaks or two single-strand breaks. In some cases, the removal may include double-strand breaks in the RHO allele containing the mutation.
  • the cleavage can be achieved by a site-directed polypeptide.
  • Site-directed polypeptides such as DNA endonucleases
  • Double-strand breaks can stimulate the cell's endogenous DNA repair pathways, such as HDR, NHEJ, or MMEJ.
  • NHEJ can repair the cleaved target nucleic acid without the need for a homologous template.
  • homologous recombination can be used to insert an exogenous polynucleotide sequence into a target nucleic acid cleavage site.
  • the exogenous polynucleotide sequence may be referred to as a donor polynucleotide (or donor, or donor sequence, or polynucleotide donor template).
  • the donor polynucleotide, a part of the donor polynucleotide, a copy of the donor polynucleotide, or a part of the copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site.
  • the donor polynucleotide may be an exogenous polynucleotide sequence, that is, a sequence that is not naturally present at the cleavage site of the target nucleic acid.
  • the homologous donor template may comprise at least a part of a wild-type RHO gene or cDNA. At least a part of the wild-type RHO gene or cDNA can be exon 1, exon 2, exon 3, exon 4, exon 5, intron regions, fragments or combinations of the above, or complete The RHO gene or cDNA.
  • the donor template can be a single-stranded or double-stranded polynucleotide.
  • the donor template can be delivered by AAV.
  • the homologous donor template may contain sequences that are homologous to the sequences flanking the target nucleic acid cleavage site. For example, the donor template may have arms that are homologous to the 3q22.1 region.
  • the donor template may also have arms homologous to the pathological variants c.C50T and/or c.C403T.
  • Sister chromatids can be used by cells as repair templates.
  • the repair template can be provided as an exogenous nucleic acid, such as a plasmid, double-stranded oligonucleotide, single-stranded oligonucleotide, or viral nucleic acid.
  • additional nucleic acid sequences such as transgenes
  • modifications such as single-base or multi-base changes or deletions
  • MMEJ can use several base pairs of homologous sequences flanking the cutting site to drive favorable end-joining DNA repair results.
  • the possible repair results can be predicted based on the analysis of potential microhomology in the target region of the nuclease.
  • CRISPR/Cas system or “CRISPR-Cas system” generally refers to a nuclease system composed of clusters of regularly spaced short palindromic repeats (CRISPR) and CRISPR-related proteins (ie Cas proteins), It can cut almost all genomic sequences adjacent to the protospacer-adjacent motif (PAM) in eukaryotic cells.
  • CRISPR regularly spaced short palindromic repeats
  • Cas proteins ie Cas proteins
  • CRISPR/Cas system can be used to collectively refer to the transcripts of CRISPR-related (“Cas") genes, as well as other elements involved in their expression or directing their activities, and can include sequences encoding Cas genes, tracr (transactivation CRISPR) sequences (Such as tracrRNA or its active part), tracr partner sequence (in the context of endogenous CRISPR/Cas system, covering "direct repeats” and processed partial direct repeats), guide sequence (in the context of endogenous CRISPR/Cas system) Also called “spacer”), or other sequences and transcripts from the CRISPR locus.
  • tracr transactivation CRISPR
  • tracr partner sequence in the context of endogenous CRISPR/Cas system, covering "direct repeats” and processed partial direct repeats
  • guide sequence in the context of endogenous CRISPR/Cas system
  • Five types of CRISPR systems have been identified (e.g., type I, type II, type III, U
  • CRISPR protein also referred to as “CRISPR-related protein” generally refers to a class of enzymes complementary to the CRISPR sequence, which can use the CRISPR sequence as a guide to identify and cut a specific DNA strand.
  • Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl4, CsxlO, Csxl4, CsxlO, Csxl6, Csxl6 , Csf2, Csf3, Csf4, and/or their homologues, or their modified forms.
  • the Cas protein is a Cas9 protein.
  • Cas9 protein or “Cas9 nuclease”, also known as Csn1 or Csx12, generally refers to a type of protein in the type II CRISPR/Cas system that is involved in both crRNA biosynthesis and the destruction of invading DNA.
  • Cas9 protein usually includes RuvC nuclease domain and HNH nuclease domain, which cut two different strands of double-stranded DNA molecules respectively. It has been used in different bacterial species such as S.thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.
  • Streptococcus pyogenes S. Pyogenes
  • the Cas9 protein of Streptococcus pyogenes see the SwissProt database accession number Q99ZW2 for its amino acid sequence
  • the Neisseria meningitides Cas9 protein see the UniProt database number A1IQ68 for its amino acid sequence
  • Streptococcus thermophilus Streptococcus thermophilus
  • Cas9 protein its amino acid sequence is in UniProt database number Q03LF7
  • Staphylococcus aureus Staphylococcus aureus
  • Cas9 protein and its amino acid sequence is in UniProt database number J7RUA5.
  • the CRISPR/Cas system can include many short repetitive sequences, called "repeats".
  • the repetitive sequence When expressed, the repetitive sequence may form a secondary structure (e.g., hairpin) and/or comprise an unstructured single-stranded sequence.
  • Repetitive sequences usually appear in clusters and often diverge between species due to evolution.
  • These repetitive sequences are regularly spaced from unique intermediate sequences called "spacers", thereby forming a repeat-spacer-repeat locus structure.
  • the spacer has the same or high homology with the known foreign invader sequence.
  • the spacer-repeat unit encodes crispRNA (crRNA), which is processed into the mature form of the spacer-repeat unit.
  • crRNA crispRNA
  • the crRNA contains a "seed” or spacer sequence (a form naturally occurring in prokaryotes where the spacer sequence targets a foreign invader nucleic acid) that targets a target nucleic acid.
  • the spacer sequence is located at the 5'or 3'end of crRNA.
  • the CRISPR/Cas system may also include a polynucleotide sequence encoding a CRISPR-associated protein (Cas protein).
  • the Cas gene encodes nucleases involved in the biogenesis and interference stages of crRNA functions in prokaryotes. Some Cas genes contain homologous secondary and/or tertiary structures.
  • crRNA biosynthesis in type II CRISPR system requires transactivation of CRISPR RNA (tracrRNA).
  • tracrRNA can be modified by endogenous RNaseIII and then hybridized with the crRNA repeats in pre-crRNA. Endogenous RNaseIII can be recruited to cleave pre-crRNA. The cleaved crRNA can be exonuclease trimmed to produce a mature crRNA form (e.g., 5'end trimming).
  • tracrRNA can remain hybridized to crRNA, and tracrRNA and crRNA are associated with a site-directed polypeptide (for example, Cas9).
  • Cas9 site-directed polypeptide
  • the crRNA in the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid that can hybridize with the crRNA.
  • the hybridization of crRNA and target nucleic acid can activate Cas9 for target nucleic acid cleavage.
  • the target nucleic acid in the type II CRISPR system is called the protospacer adjacent motif (PAM).
  • PAM is essential for promoting the binding of site-directed polypeptides (for example, Cas9) to target nucleic acids.
  • Type II systems also called Nmeni or CASS4 can be further subdivided into Type II-A (CASS4) and Type II-B (CASS4a).
  • the CRISPR/Cas9 system that can be used for RNA programmable gene editing can be found in Jinek et al., Science, 337(6096): 816-82l (2012), International Patent Application Publication No. WO2013/176772 provides that it can be used for site-specific gene editing. Many examples and applications of the CRISPR/Cas endonuclease system.
  • the method of the present application includes providing a genome-targeted nucleic acid, which can direct a related active polypeptide (such as a Cas protein) to a specific target sequence (such as a RHO allele) within the target nucleic acid.
  • the nucleic acid targeted to the genome may be RNA.
  • RNA targeting the genome may be referred to herein as "guide RNA” or "gRNA".
  • the gRNA described in this application may be complementary to the target nucleic acid.
  • the gRNA may be the same as the target nucleic acid (when speaking of the same, the "U” in RNA corresponds to the thymine "T” in DNA due to the difference between the bases encoding RNA and DNA) .
  • the nucleic acid sequence (eg, DNA) encoding the gRNA may be the same or complementary to the target nucleic acid.
  • the terms "target nucleic acid”, “target nucleic acid” and “target region” are used interchangeably, and generally refer to a nucleic acid sequence that can be recognized by gRNA.
  • the target nucleic acid may refer to a double-stranded nucleic acid or Single-stranded nucleic acid.
  • the gRNA can be transcribed or copied from a sequence encoding it.
  • the gRNA can be transcribed from a DNA sequence encoding it.
  • sequence encoding a gRNA generally refers to a DNA sequence that can obtain the gRNA by transcription.
  • sequence encoding gRNA may have the same nucleotide sequence as the target sequence of the gRNA.
  • the gRNA may include at least a spacer sequence and a CRISPR repeat sequence that hybridize to the target nucleic acid sequence of interest.
  • the gRNA also contains a second RNA called the tracrRNA sequence.
  • the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex.
  • gRNA can bind to Cas protein to form a guide RNA-Cas protein complex.
  • the genome-targeted nucleic acid can make the complex target specific due to its association with the Cas protein. Therefore, the nucleic acid targeted to the genome can direct the activity of the Cas protein.
  • the nucleic acid targeted to the genome may be a double-stranded guide RNA.
  • the gRNA may be a single-stranded guide RNA (sgRNA).
  • the double-stranded guide RNA or single-stranded guide RNA may be modified.
  • the double-stranded guide RNA can include two RNA strands.
  • the first strand may include an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence.
  • the second strand may comprise a minimal tracrRNA sequence (complementary to a minimal CRISPR repeat sequence), a 3'tracrRNA sequence and an optional tracrRNA extension sequence.
  • the sgRNA can include an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, 3'in the 5'to 3'direction. tracrRNA sequence and optional tracrRNA extension sequence.
  • the optional tracrRNA extension may include additional functional (e.g., stability) elements that contribute to the guide RNA.
  • the single-molecule guide linker can connect the smallest CRISPR repeat sequence and the smallest tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension can include one or more hairpins.
  • the sgRNA may include a variable-length spacer sequence having 17-30 nucleotides at the 5'end of the sgRNA sequence.
  • the sgRNA may contain a variable-length spacer sequence with 17-24 nucleotides at the 5' end of the sgRNA sequence.
  • the sgRNA may comprise a sequence of 21 nucleotides.
  • the sgRNA may comprise a sequence of 20 nucleotides.
  • the sgRNA may comprise a sequence of 19 nucleotides.
  • the sgRNA may comprise a sequence of 18 nucleotides.
  • the sgRNA may comprise a sequence of 17 nucleotides.
  • the sgRNA may include a sequence of 22 nucleotides.
  • the sgRNA may include a sequence of 23 nucleotides.
  • the sgRNA may comprise a sequence of 24 nucleotides.
  • the sgRNA may be unmodified or modified.
  • the gRNA described in this application can be combined with the sequence in the target nucleic acid.
  • the nucleic acid (or part thereof) targeted to the genome can interact with the target nucleic acid in a sequence-specific manner through hybridization (ie, base pairing).
  • the nucleotide sequence of sgRNA can vary according to the sequence of the target nucleic acid.
  • the gRNA sequence can be designed to hybridize with the target nucleic acid adjacent to the PAM sequence recognizable by the Cas protein used in the system.
  • the gRNA may be a perfect match or mismatch with the target sequence.
  • the Cas protein usually has a specific PAM sequence that can be identified in the target DNA.
  • the Cas9 protein can be derived from S. pyogenes, and the Cas9 protein recognizes the PAM containing the sequence 5'-NRG-3' in the target nucleic acid, wherein R contains A or G, and N can be any Nucleotides.
  • the Cas9 protein can be derived from Staphylococcus aureus, and the Cas9 protein (SaCas9) can recognize the PAM containing the sequence 5'-NNGRR(T)-3' in the target nucleic acid, wherein R contains A or G, where N can be any nucleotide.
  • the PAM sequence recognized by SaCas9 may include 5'-NNGRR-3', where R includes A or G, and N can be any nucleotide.
  • R includes A or G
  • N can be any nucleotide.
  • the PAM described in the present application may include the nucleotide sequence shown in any one of SEQ ID NO: 39-43.
  • the gRNA described in this application can specifically bind to the mutation site.
  • the gRNA may specifically bind to at least part of the nucleic acid in the RHO allele that includes the mutation site.
  • the sequence encoding the gRNA may include the nucleotide sequence shown in any one of SEQ ID NOs. 1, 2 and 4.
  • the gRNA may be specifically complementary to at least part of the nucleic acid sequence of exon 1 of the RHO allele containing the c.C50T mutation.
  • the gRNA that is specifically complementary to at least a part of the nucleic acid sequence of exon 1 of the RHO allele containing the c. C50T mutation may include the nucleotide sequence shown in any one of SEQ ID NO. 44-45 .
  • the sequence encoding the gRNA may include the nucleotide sequence shown in any one of SEQ ID NO. 1-2.
  • the gRNA may be specifically complementary to at least part of the nucleic acid sequence of exon 2 of the RHO allele containing the c.C403T mutation.
  • the gRNA that is specifically complementary to at least a part of the nucleic acid sequence of exon 2 of the RHO allele containing the c. C403T mutation may include the nucleotide sequence shown in SEQ ID NO. 47.
  • the sequence encoding the gRNA may include the nucleotide sequence shown in SEQ ID NO.4.
  • the percentage of complementarity between the gRNA and the target nucleic acid may be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75% , At least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%.
  • the percentage of complementarity between the gRNA and the target nucleic acid can be at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75% , Up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%.
  • the gRNA used in the CRISPR system described in this application can be synthesized by a chemical method, for example, high performance liquid chromatography. For example, linking two or more RNA molecules together. Longer RNAs (such as RNA encoding Cas9) can be obtained through enzymatic reactions. In the art, various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic synthesis of RNA, for example, modifications to enhance stability, reduce innate immune response and/or enhance other properties.
  • RNA or DNA RNA or DNA
  • RNA or DNA polynucleotide
  • RNA or DNA polynucleotide
  • the endonuclease polypeptide can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles.
  • the DNA endonuclease can be delivered as one or more polypeptides alone, or pre-complexed with one or more guide RNA, or one or more crRNA and tracrRNA.
  • Some exemplary non-viral delivery vectors can be found in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011).
  • the vector described in the present application may include the nucleic acid molecule described in the present application (for example, a sequence encoding gRNA and/or gRNA).
  • Polynucleotides can be delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes Things.
  • site-directed polypeptides and genome-targeted nucleic acids can be administered to cells or patients separately.
  • the site-directed polypeptide can be pre-complexed with one or more guide RNA or one or more crRNA and tracrRNA.
  • RNA can form specific interactions with polynucleotides (e.g., RNA or DNA). Although this property is used in many biological processes, it is also accompanied by the risk of promiscuous interactions in the nucleic acid-rich cellular environment.
  • RNP ribonucleoprotein particles
  • RNA can protect RNA from degradation.
  • the nuclease in RNP can be modified or unmodified.
  • gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.
  • nucleotides There are many modifications that are known and can be used in the art. For example, deletion, insertion, translocation, inactivation and/or activation of nucleotides.
  • the modification may include introducing one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, cross-linking, breaking specific nucleotide stretches, and other modifications. Modifications can include the inclusion of at least one non-naturally occurring nucleotide, or one modified nucleotide, or an analog thereof.
  • the nucleotides may be modified at ribose, phosphate and/or base moieties.
  • the vector may also be a polynucleotide vector, for example, a plasmid, cosmid or transposon.
  • Carriers suitable for use have been widely described and are well known in the art.
  • the vector containing the nucleic acid molecule described in the present application may also contain other sequences and elements required for the vector to replicate in prokaryotic and/or eukaryotic cells.
  • the vector described in the present application may include a prokaryotic replicon, that is, a nucleotide sequence that has the ability to guide the host's own replication and maintenance in a prokaryotic host cell (for example, a bacterial host cell).
  • the replicon is well known in the art.
  • the vector may contain shuttle elements that make the vector suitable for replication and integration in prokaryotes and eukaryotes.
  • the vector may also include a gene capable of expressing a detectable marker (for example, a drug resistance gene).
  • the vector may also have a reporter gene, for example, a gene that can encode fluorescence or other detectable proteins.
  • the vector may include viral vectors, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus.
  • viral vectors for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus.
  • Methods for generating viral vectors containing nucleic acid molecules (for example, the isolated nucleic acid molecules described in this application) as part of the vector genome are well known in the art, and those skilled in the art can perform them without undue experimentation.
  • the vector may be a recombinant AAV virus particle packaging the nucleic acid molecule described in the present application.
  • the method for producing recombinant AAV may include introducing the nucleic acid molecules described in the present application into a packaging cell line, generating AAV infection, AAV cap and rep gene auxiliary functions, and recovering the recombinant AAV from the supernatant of the packaging cell line.
  • a packaging cell line Various types of cells can be used as packaging cell lines.
  • packaging cell lines that can be used include but are not limited to HEK 293 cells, HeLa cells and Vero cells.
  • the vector may be an adenovirus-associated vector (AAV).
  • AAV adenovirus-associated vector
  • the AAV may include different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variants or mixtures.
  • ITR inverted terminal repeats
  • AAV inverted terminal repeat
  • the technology for producing AAV vectors is a standard technology in the art, which includes the provision of polynucleotides to be delivered, rep and cap genes, and helper virus functions to be packaged AAV genomes to cells.
  • the production of AAV vectors usually requires the following components in a single cell (referred to herein as packaging cells): rAAV genome, AAV rep and cap genes separated from (for example, not in) rAAV genome, and helper virus.
  • the AAV rep and cap genes can be from any AAV serotype, or from an AAV serotype different from the AAV gene group ITR, including but not limited to the AAV serotype described herein.
  • the AAV vector in this application may include gRNA targeting the mutation site of the RHO gene.
  • the sequence encoding gRNA may include the nucleotide sequence shown in any one of SEQ ID NOs. 1, 2 and 4.
  • sequence encoding the gRNA and the nucleic acid encoding the Cas9 protein may be located in the same vector. In other cases, the sequence encoding the gRNA and the nucleic acid encoding the Cas9 protein may be located in different vectors.
  • the AAV vector of the present application can be from a variety of species.
  • the AAV may be avian AAV, bovine AAV or goat AAV.
  • the vector is AAV8.
  • the method of the present application may include the production of packaging cells, that is, the production of cell lines that can be used to stably express all the necessary components of AAV.
  • packaging cells that is, the production of cell lines that can be used to stably express all the necessary components of AAV.
  • the AAV genome lacking AAV rep and cap genes, the AAV rep and cap genes isolated from the AAV genome, and a plasmid (or multiple plasmids) with a selection marker such as a neomycin resistance gene are integrated into the genome of the cell middle. It has been possible to introduce AAV genomes into bacterial plasmids by methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. ETSA, 79: 2077-2081).
  • the packaging cell line can then be infected with a helper virus (e.g. adenovirus).
  • a helper virus e.g. adenovirus
  • adenovirus or baculovirus can also be used to introduce AAV genome and/or rep and cap genes into packaging cells.
  • the term "subject" generally refers to any subject for whom diagnosis, treatment, or treatment is desired.
  • the RHO gene of a subject in need may contain a mutation site selected from the group consisting of c.C50T and c.C403T.
  • the subject may include a mammal.
  • the subject may include a human.
  • the subject may include an East Asian.
  • the present application provides a composition for treating retinitis pigmentosa in a subject
  • the composition may include an active ingredient for removing the mutation site of the RHO gene and a pharmaceutically acceptable carrier, wherein the mutation site
  • the points are selected from the following group: c.C50T and c.C403T.
  • the composition may include a physiologically tolerable carrier and a cellular composition, and optionally at least one biologically active agent, which is dissolved or dispersed in the therapeutic composition as an active ingredient.
  • the carrier described herein can be administered in the form of a suspension with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may contain buffers, compounds, cryopreservatives, preservatives, or other agents, and will not interfere with the delivery of the carrier to be delivered.
  • the composition may also comprise a cell preparation, for example, an osmotic buffer, which allows maintaining the integrity of the cell membrane, and optionally, a nutrient solution, which maintains cell viability or enhances implantation when administered.
  • a cell preparation for example, an osmotic buffer, which allows maintaining the integrity of the cell membrane, and optionally, a nutrient solution, which maintains cell viability or enhances implantation when administered.
  • the formulations and suspensions described are known to those skilled in the art, or routine experiments can be used to make them suitable for use with the vectors and/or cells of the present application.
  • the term "administration" can introduce cells and/or vectors into a subject, or a certain desired site of the subject, through a method or route.
  • the cell and/or vector can express the nucleic acid molecule of the present application (for example, the sequence encoding gRNA and/or gRNA) at a desired site (for example, a site of damage or repair), thereby producing a desired effect.
  • the cells (or their differentiated progeny) and/or the vector can be administered by any suitable route, which can deliver the cells (or their differentiated progeny) and/or the vector to the subject as desired Site, and at least a part of the implanted cells (or cell components) and/or carriers remain viable.
  • the survival period of the cells can be as short as a few hours, such as twenty-four hours, several days, or as long as several years, even consistent with the lifespan of the patient.
  • the administration includes injection.
  • the vector can be administered by systemic administration routes such as intraperitoneal or intravenous routes.
  • the administration may include subretinal cavity injection.
  • the cleavage efficiency of the gRNA and the vector described in the present application on the mutation site of the RHO allele can reach about 50% or more, for example, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75%. % Or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more, as detected in an in vitro digestion reaction.
  • the gRNA and vector described in this application can cleave the target nucleic acid with a certain cutting efficiency and specificity.
  • the gRNA and vector of the present application do not affect the survival of host cells and have certain safety.
  • Example 1 Collecting information about the patient's genetic diagnosis
  • the targeting vector used in this patent is: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA; U6::BsaI-sgRNA, the map is shown in Figure 1 (for vector information, please refer to https://www. addgene.org/61591/).
  • DNA sequence mixture encoding gRNA 7 ⁇ l 10 ⁇ T4 connection buffer 1 ⁇ l T4 PNK 2 ⁇ l Total 10 ⁇ l
  • connection system 200 ⁇ l PCR tube
  • the constructed pX601-SgRNA plasmid vector is named as follows:
  • the SaCas9-sgRNA target efficiency detection kit detects the in vitro editing efficiency of each sgRNA of RHO
  • the PCR reaction system is as follows:
  • the PCR reaction procedure is as follows:
  • the genomic DNA extraction steps are as follows:
  • each cutting site requires two sets of dsDNA, one is dsDNA containing mutation sites, including RHO17-M-dsDNA and RHO135-M-dsDNA.
  • the PCR process uses patient iPSCs gDNA as the template; the other group contains no The dsDNA at the mutation site includes RHO17-C-dsDNA and RHO135-C-dsDNA.
  • the PCR process uses normal human iPSCs gDNA as a template.
  • the primers used are as shown in Table 12:
  • the PCR reaction program is as follows:
  • the reaction system is as follows:
  • RHO17-SgRNA in vitro efficiency detection are shown in Figure 4.
  • RHO17-SgRNA1 and SgRNA2 both cut the in vitro templates of the experimental group.
  • the cutting efficiency of SgRNA1 is more than 50%, and the cutting efficiency of SgRNA2 is close to 100%.
  • the in vitro template is not cut, which proves that the cutting efficiency and specificity of the two SgRNAs are good, and can be used in subsequent cell experiments and in vivo experiments; while RHO17-SgRNA3 cuts both the experimental group and the control template, and the specificity is poor. unavailable.
  • M experimental group
  • C control group (wild-type dsDNA).
  • RHO135-SgRNA in vitro efficiency test results are shown in Figure 5.
  • RHO135-SgRNA1 cuts the in vitro template of the experimental group, and its cutting efficiency is nearly 50%, and it does not cut the in vitro template of the control group, which proves the cutting efficiency and specificity of the sgRNA It can be used in subsequent cell experiments and in vivo experiments.
  • RHO135-SgRNA2 cuts the template of the experimental group and the control group, which has poor specificity and cannot be used.
  • M experimental group
  • C control group (wild-type dsDNA).
  • Example 4 Using gRNA activity fluorescence detection kit to detect gRNA editing efficiency in vitro
  • the fluorescent reporter gene mKate in the fluorescent reporter plasmid in the kit is terminated early by the stop codon. This truncated mKate is inactive.
  • the target site recognized by Cas9/gRNA can be inserted into the stop codon Then, under the action of Cas9 and gRNA, the double-stranded DNA at the target site is cleaved to form DSB, and the cells form active fluorescent protein through homologous recombination effect.
  • Use a fluorescence microscope or flow cytometer to detect whether the fluorescent protein is active or not. Increase to determine the activity and knockout efficiency of gRNA.
  • the primer sequences are shown in Table 15.
  • Fluorescence signal detection by fluorescence microscope 48h after transfection, it can be observed by flow cytometer. Compared with the negative control group: if the experimental group detects a stronger fluorescence signal, it indicates that the gRNA activity is higher. If the fluorescent signal is weakened or no fluorescent signal is detected in the experimental group, it indicates that the gRNA activity is weak or inactive.
  • the constructed plasmid containing the target sequence and the plasmid vector containing gRNA and Cas9 were co-transfected into the target cells, and a negative control group was set up. The results of each group were repeated three times for statistical analysis (two-tailed t test, p ⁇ 0.05).
  • Figure 6 shows the in vitro detection of RHO17-SgRNA1 editing efficiency using a gRNA activity fluorescence detection kit.
  • Picture A is the experimental group.
  • the co-transfected plasmids are RHO17-mkate-mut-sgRNA1 and pX601-R17-sg1.
  • pX601-R17-sg1 cuts the target site of the RHO17-mkate-mut-sgRNA1 plasmid;
  • B The picture shows the experimental control group RHO17-mkate-mut-sgRNA1 and pX601 empty plasmids, without cutting;
  • picture C control group, the co-transfected plasmids are RHO17-mkate-wt-sgRNA1 and pX601-R17-sg1, pX601-R17-sg1
  • the target site of RHO17-mkate-wt-sgRNA1 plasmid is not cut.
  • the above results prove that the sgRNA has both cutting efficiency and specificity, which are the same as the results of the above-mentioned in vitro cutting experiment, and can be applied to subsequent cell experiments and in vivo experiments.
  • Figure 7 shows the in vitro detection of RHO17-SgRNA2 editing efficiency using a gRNA activity fluorescence detection kit.
  • Picture A is the experimental group.
  • the co-transfected plasmids are RHO17-mkate-mut-sgRNA2 and pX601-R17-sg2.
  • pX601-R17-sg2 cuts the target site of the RHO17-mkate-mut-sgRNA2 plasmid;
  • B The picture shows the experimental control group RHO17-mkate-mut-sgRNA2 and pX601 empty plasmids, without cutting;
  • the C picture control group, the co-transfected plasmids are RHO17-mkate-wt-sgRNA2 and pX601-R17-sg2, pX601-R17-sg2
  • the target site of RHO17-mkate-wt-sgRNA2 plasmid is not cut.
  • the above results prove that the cleavage efficiency and specificity of the SgRNA are both, which are the same as the results of the above-mentioned in vitro cleavage experiment, and can be applied to subsequent cell experiments and in vivo experiments.
  • Figure 8 shows the in vitro detection of RHO17-SgRNA3 editing efficiency using a gRNA activity fluorescence detection kit.
  • Picture A is the experimental group.
  • the co-transfected plasmids are RHO17-mkate-mut-sgRNA3 and pX601-R17-sg3.
  • pX601-R17-sg3 cuts the target site of the RHO17-mkate-mut-sgRNA3 plasmid;
  • B The picture shows the experimental control group RHO17-mkate-mut-sgRNA3 and pX601 empty plasmids, without cutting;
  • the C picture control group, the co-transfected plasmids are RHO17-mkate-wt-sgRNA3 and pX601-R17-sg3, pX601-R17-sg3 Cut the target site of RHO17-mkate-wt-sgRNA3 plasmid.
  • the above results prove that the SgRNA has poor cleavage specificity, which is the same as the result of the above-mentioned in vitro cleavage experiment, and cannot be applied to subsequent cell experiments and in vivo experiments.
  • Figure 9 shows the in vitro detection of RHO135-SgRNA1 editing efficiency using a gRNA activity fluorescence detection kit.
  • Picture A is the experimental group.
  • the co-transfected plasmids are RHO135-mkate-mut-sgRNA1 and pX601-R135-sg1.
  • pX601-R135-sg1 cuts the target site of the RHO135-mkate-mut-sgRNA2 plasmid;
  • B The picture shows the experimental control group RHO135-mkate-mut-sgRNA1 and pX601 empty plasmids without cutting;
  • the C picture control group, the co-transfected plasmids are RHO135-mkate-wt-sgRNA1 and pX601-R135-sg1, pX601-R135-sg1
  • the target site of RHO135-mkate-wt-sgRNA1 plasmid is not cut.
  • the above results prove that the cleavage efficiency and specificity of the SgRNA are both, which are the same as the results of the above-mentioned in vitro cleavage experiment, and can be applied to subsequent cell experiments and in vivo experiments.
  • Figure 10 shows the in vitro detection of RHO135-SgRNA2 editing efficiency using a gRNA activity fluorescence detection kit.
  • Picture A is the experimental group.
  • the co-transfected plasmids are RHO135-mkate-mut-sgRNA2 and pX601-R135-sg2.
  • pX601-R135-sg2 cuts the target site of the RHO135-mkate-mut-sgRNA2 plasmid;
  • B The picture shows the experimental control group RHO135-mkate-mut-sgRNA2 and pX601 empty plasmids, without cutting;
  • the C picture control group, the co-transfected plasmids are RHO135-mkate-wt-sgRNA2 and pX601-R135-sg2, pX601-R135-sg2 Cut the target site of RHO135-mkate-wt-sgRNA2 plasmid.
  • the above results prove that the SgRNA has poor cleavage specificity, which is the same as the result of the above-mentioned in vitro cleavage experiment, and cannot be applied to subsequent cell experiments and in vivo experiments.
  • Figure 11 shows the statistics of flow sorting results for in vitro detection of sgRNA editing efficiency using a gRNA activity fluorescence detection kit. It can be seen from Figure A that the cutting efficiency of the experimental group (RHO17-m1) and the control group (RHO17-W1) are statistically different; from Figure B, it can be seen that the experimental group (RHO17-m2) and the control group (RHO17- The cutting efficiency of W2) is statistically different; as can be seen from Figure C, the cutting efficiency of the experimental group (RHO135-m1) and the control group (RHO135-W1) are statistically different, which can be used for subsequent cell and in vivo experiments.
  • the experimental group and the control group each set three repeating groups for flow sorting.
  • Example 5 Using 293T cells to verify the safety and specificity of gRNA in vitro
  • the medium used for 293T cells is high-sugar DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin double antibody, cultured at 37°C with 5% CO 2.
  • Protein changes Plasmid name c.50C>T p.Thr17Met pX601-R17-sg1 To To pX601-R17-sg2 To To pX601-R17-sg3 c.403C>T p.Arg135Trp pX601-R135-sg1 To To pX601-R135-sg2
  • the cells in the negative control wells are completely dead, but the cells in the experimental group and the control group are viable (indicating successful transfection), stop the antibiotic selection, switch to normal medium, and wait until the cells in the 6-well plate grow to 80-90% After the confluence, the cells are cultured in a 6cm petri dish. After the cells have grown to 80-90% confluence, the cells are harvested and ready to extract genomic DNA. The whole process takes about 7-10 days.
  • patient A carries the RHO c.50C>T mutation
  • patient B carries the RHO c.403C>T
  • normal person C does not have the disease and does not carry any gene mutation sites.
  • kidney epithelial cell isolation and culture kit provided by Beijing Saibei Company.
  • the experimental steps are as follows:
  • Urine collection wear gloves, disinfect, preferably mid-section urine, and seal with PARAFILM.
  • UrinEasy separation complete medium to resuspend the cell cluster: one well for male and two wells for female, denoted as D0.
  • D2 Supplemental separation medium-female: 500ul/well; male: 250ul/well;
  • Digestion and passage can be carried out when the confluence of somatic cells reaches 70-90%.
  • the cells are seeded in a 96-well plate; the seeding density is controlled at 5000-15000 cells/well, and 3 density gradients can be set according to the cell conditions, each gradient Set 3 multiple holes.
  • the day of cell seeding was recorded as day -1.
  • Day 1-2 Observe under a microscope and take photos to record the morphological changes of the cells. If the cell morphology changes significantly, the reprogramming medium B can be removed and replaced with reprogramming medium A to continue the culture; if the morphological change is not obvious, the medium may not be changed.
  • Day 3 If the cell morphology has undergone significant deformation in the first two days, and the cell growth rate is relatively fast, trypsinization and passage can be carried out. Transfer the cells to 2-6 wells of the six-well plate according to the cell state and cell volume, and add reprogramming medium C to form single-cell attachment as much as possible. Please configure the reprogramming medium C according to the following table:
  • Reprogramming medium C volume Reprogramming Medium A 9.8mL (remaining above) Reprogramming additives III 5 ⁇ L
  • Day 9-20 Observe under a microscope and take photos to record the changes in cell morphology. Replace the fresh PSCeasy human pluripotent stem cell medium that has been equilibrated at room temperature every day.
  • the clones are inoculated with PSCeasy human pluripotent stem cell recovery medium. After the cells adhere to the wall, they can be replaced with PSCeasy human pluripotent stem cell medium and continue to be cultured to the required number of generations.
  • the transformed plasmids are named pX601-R17-puro-sg1, pX601-R17-puro-sg2 and pX601-R135 -puro-sg1, the blank control plasmid is pX601-GFP-puro plasmid.
  • the method is the same as before.
  • the primers used are shown in Table 24, and the method is the same as before.
  • A-GFP indicates that the iPSCs of patient A were only transfected with pX601-GFP-puro blank control plasmid;
  • A-17sgRNA1 or A-17sgRNA2 indicates that the iPSCs of patient A were transfected with pX601 -R17-puro-sg1 or pX601-R17-puro-sg2 plasmid;
  • C-17sgRNA1 or C-17sgRNA2 means that normal human iPSCs have been transfected with pX601-R17-puro-sg1 or pX601-R17-puro-sg2 plasmid, T7E1
  • RHO17-SgRNA1 and RHO17-SgRNA2 only had an editing effect on the RHO gene mutation site of patient A's iPSCs, but had no editing effect on the corresponding gene position of normal people. It is consistent with the results of the above in vitro experiments.
  • the experimental results of patient B are shown in Figure 14.
  • the Sanger sequencing result in the left image shows that patient B carries the heterozygous mutation of RHO c.403C>T, but normal people do not; the right image shows that RHO135-SgRNA1 is in patient B and normal people.
  • B-GFP means that patient B’s iPSCs were only transfected with pX601-GFP-puro blank control plasmid
  • B-135sgRNA1 means that patient B’s iPSCs were transfected with pX601-R135-puro-sg1 plasmid
  • C -135sgRNA1 means that the iPSCs of normal people have been transfected with the pX601-R135-puro-sg1 plasmid.
  • Example 7 Using 3D retinal tissue to verify gRNA gene editing efficiency in vitro
  • RHO17-SgRNA1 and RHO17-SgRNA2 edit the same site.
  • the latter has a stronger cutting effect than the former.
  • Both 3D retina experiments and mouse experiments use RHO17-SgRNA2 for experiments.
  • the above-mentioned pX601 plasmid was modified to carry GFP fluorescent protein for subsequent screening of GFP+ cells.
  • the modified plasmids were named pX601-R17-GFP-sg2 and pX601-R135-GFP-sg1, and the blank control plasmid was pX601- GFP plasmid.
  • Plasmid amplification The constructed AAV vector, packaging plasmid and helper plasmid need to undergo a large amount of endotoxin-free extraction, and the Qiagen large extraction kit is used to carry out a large amount of plasmid extraction. The steps are the same as before.
  • virus particles exist in both packaging cells and culture supernatant. Both the cells and the culture supernatant can be collected to obtain the best yield.
  • the primers used are as follows in Table 26, and the method is the same as before.
  • the method is the same as before.
  • Figure 15A shows the gene editing effect of RHO-SgRNA2 in the 3D retinal tissues of patient A and normal C.
  • A-GFP indicates that the 3D retina of patient A is only infected with pX601-GFP blank control virus;
  • A-17sgRNA2 indicates that of patient A The 3D retina was infected with the pX601-R17-GFP-sg2 virus;
  • C-17sgRNA2 indicates that the 3D retina of a normal person was transfected with the pX601-R17-GFP-sg2 virus.
  • T7E1 showed that RHO-17SgRNA2 only affects the 3D retina of patient A.
  • RHO gene mutation site has editing effect, but no editing effect on the corresponding gene position of normal people
  • Figure 15B shows the gene editing effect of RHO135-SgRNA1 in the 3D retina tissue of patient B and normal person C, B-GFP represents patient B's
  • the 3D retina was only infected with the pX601-GFP blank control plasmid; B-135sgRNA1 indicated that the iPSCs of patient B were infected with the pX601-R135-GFP-sg1 virus; C-135sgRNA1 indicated that the normal 3D retina was infected with the pX601-R135-GFP-sg1 virus
  • T7E1 show that RHO135-SgRNA1 only has an editing effect on the RHO gene mutation site of the 3D retina of patient B, but has no editing effect on the corresponding gene position in normal people. Consistent with the results of the above in vitro experiments.
  • 6Mice fertilized eggs are injected with sgRNA/Cas9 mRNA and targeting vectors;
  • 6Mice fertilized eggs are injected with sgRNA/Cas9 mRNA and targeting vectors;
  • the primer pair WT-F/WT-R is designed in the wild gene sequence.
  • this pair of primers When used for PCR, it cannot amplify the product of the mutant allele, but can only amplify the product of the wild-type allele, while the primer /Mut-R is designed in the mouse humanized RHO gene sequence.
  • the primer /Mut-R When using primers to perform PCR on WT-F/Mut-R, the wild-type allele cannot be amplified, but only the mutant type. The product of an allele.
  • the primer sequences are as follows in Table 27:
  • the WT-F/WT-R pair of primers are mainly used to identify the presence of wild-type alleles, and combined with the PCR results of the WT-F/Mut-R pair of primers to determine the specific genotype of the animal: homozygous/heterozygous/ Wild type.
  • Y PCR product of expected length detected by gel electrophoresis
  • N PCR product of expected length not detected by gel electrophoresis
  • H/H homozygous genotype
  • H/+ heterozygous genotype
  • +/+ Wild type.
  • Example 3 for the PCR reaction system and procedures.
  • the PCR products are sent for sequencing to detect whether the humanized mouse RHO gene includes the mutation site that is expected to be knocked in.
  • mice The AAV virus injected by mice is the virus used for 3D retinal tissue infection.
  • mice Observe whether the mice are abnormal after operation, and give neomycin ointment to prevent infection.
  • the primers used are as shown in Table 29, and the method is the same as before.
  • the method is the same as before.
  • Figure 16A shows the results of PCR running gel for genotype identification of RHO humanized mice.
  • Mouse numbered 20 is a humanized mouse carrying heterozygous mutations, and mice numbered 21 and 22 are humanized mice carrying WT.
  • the mouse number 23 is a humanized mouse carrying a homozygous mutation;
  • Figure 16B shows a humanized mouse carrying a RHO c.50C>T homozygous mutation, and
  • Figure 16C shows a humanized mouse carrying a RHO c.403C> Humanized mice with homozygous mutations of T.
  • Figure 17A shows the gene editing efficiency of RHO17-SgRNA2 on humanized mice.
  • M-17sgRNA2 indicates that humanized mice carrying the c.50C>T homozygous mutation were injected with AAV-R17-sg2-SaCas9 virus, and C-17sgRNA2 means that humanized mice carrying WT are injected with AAV-R17-sg2-SaCas9 virus.
  • T7E1 results show that RHO-17SgRNA2 only has an editing effect on humanized mice carrying mutations, but it has an editing effect on humanized mice carrying WT. The corresponding site of the mouse has no editing effect;
  • Figure 17B shows the gene editing efficiency of RHO135-SgRNA1 on humanized mice.
  • M-135sgRNA1 indicates that humanized mice carrying the c.403C>T homozygous mutation were injected AAV-R135-sg1-SaCas9 virus
  • C-135sgRNA1 means that humanized mice carrying WT are injected with AAV-R135-sg1-SaCas9 virus
  • T7E1 results show that RHO-135SgRNA1 only edits humanized mice carrying mutations There is no editing effect on the corresponding sites of humanized mice carrying WT.
  • the results of the in vivo experiment are consistent with the results of the above in vitro experiment.

Abstract

一种治疗视网膜色素变性的方法，所述方法包括以下步骤：使有需要的受试者具有功能性的RHO基因，其中所述功能性的RHO基因不包含选自下组的突变位点：c.C50T和c.C403T。还涉及一种编辑RHO基因的方法，以及用于治疗受试者视网膜色素变性的组合物。

Description

RHO-adRP基于基因编辑的方法和组合物 技术领域
本申请涉及生物医药领域，具体的涉及一种针对中国RHO-adRP患者基于CRISPR/Cas9技术和AAV技术的基因编辑药物。
背景技术
目前，视网膜色素变性(Retinitis Pigmentosa，RP)是一组以感光细胞和/或视网膜色素上皮细胞功能渐进性丧失为主要改变的遗传性致盲性眼病。目前尚无有效治疗方法(传统药物和手术治疗基本无效)。近年来，CRISPR/Cas9技术的快速发展为RP的基因治疗带来了曙光，CRISPR/Cas9技术操作方便简洁，是目前最为常用的基因编辑技术，也是遗传性视网膜变性基因治疗主要的工具之一。RHO是最早发现的RP致病基因，约30％-40％的常染色体显性遗传(autosomal dominant RP，adRP)由该基因所致，是adRP最主要的致病基因。
通常情况下，RHO-adRP患者的一个等位基因携带致病突变，而另一个等位基因正常；RHO突变的致病机制是功能获得性的或显性负向效应的机制。文献报道RHO基因p.Pro23His是北美地区RHO-adRP人群的突变热点，目前关于RHO基于CRISPR/Cas9技术的基因编辑治疗多与该位点有关。而针对亚洲人群RHO突变热点的研究几乎没有，无法用于中国RHO-adRP人群的基因编辑治疗。因此，需要研究针对中国人群的基因的突变热点进行药物设计。
腺相关病毒(adeno-associated virus，AAV)载体作为基因转移载体具有无致病性、免疫原性低、能有效转移目的基因和携带的治疗基因能长期表达等优势而成为最广泛使用的视网膜基因治疗的载体。但AAV载体的载运能力，最大限度为4.7kb，通常情况下，只能将sgRNA和Cas9分开包装，因此，为了提高打靶效率，需要选择合适的载体。
发明内容
本申请提供了一种针对中国RHO-adRP患者基于CRISPR/Cas9技术和AAV技术的基因编辑药物。
本申请针对中国RHO-adRP人群的突变热点设计突变等位基因特异性的gRNA，敲除突变等位基因，但保留正常等位基因而达到治疗目的。鉴于导致adRP的RHO基因突变多为错义突变(突变等位基因和正常等位基因之间只有一个碱基的差别)，设计gRNA时，可使突变位点位于gRNA上，使该gRNA和突变等位基因完全匹配，而和正常等位基因有一个碱基的差别，这样的gRNA即为突变等位基因特异性的gRNA。在某些实施方式中，本申请可使用 CRISPR/SaCas9体系进行基因编辑，可将SaCas9蛋白和gRNA包入单一的AAV病毒。例如，可使用pX601-SaCas9质粒载体，并将gRNA和saCas9包入一个AAV载体(例如，AAV8载体)，以提高打靶效率。在某些实施方式中，可通过视网膜下腔注射的方法将载体注入到RHO-adRP患者的眼球以达到治疗目的。本申请所述的方法和组合物能够特异性切割RHO基因的相关突变位点，具有一定的切割效率和安全性，在细胞、组织和动物模型上均得到了验证，具有巨大的应用价值。
一方面，本申请提供了一种治疗视网膜色素变性的方法，所述方法包括以下步骤：使有需要的受试者具有功能性的RHO基因，其中所述功能性的RHO基因不包含选自下组的突变位点：c.C50T和c.C403T。
在某些实施方式中，所述方法包括以下步骤：去除有需要的受试者中RHO基因的所述突变位点。
在某些实施方式中，所述去除包括敲除所述突变位点和/或降低包含所述突变位点的RHO基因的表达水平。
在某些实施方式中，所述去除包括不影响所述受试者中野生型RHO基因的表达水平和/或功能。
在某些实施方式中，所述去除包括使包含所述突变的RHO等位基因发生双链断裂。
在某些实施方式中，所述去除包括向有需要的受试者施用至少一个能够去除所述突变位点的载体。
在某些实施方式中，所述载体包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
在某些实施方式中，所述gRNA与包含所述突变位点的RHO等位基因中的至少部分核酸特异性结合。
在某些实施方式中，所述gRNA与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补。
在某些实施方式中，所述与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补的gRNA包含SEQ ID NO:44-45中任一项所示的氨基酸序列。
在某些实施方式中，所述编码与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补的gRNA的序列包含SEQ ID NO.1-2中任一项所示的核苷酸序列。
在某些实施方式中，所述gRNA与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补。
在某些实施方式中，所述与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补的gRNA包含SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述编码与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补的gRNA的序列包含SEQ ID NO.4所示的核苷酸序列。
在某些实施方式中，所述载体包括编码Cas蛋白的核酸。
在某些实施方式中，所述Cas蛋白包括Cas9蛋白。
在某些实施方式中，所述编码gRNA的序列与所述编码Cas蛋白的核酸位于同一个所述载体中。
在某些实施方式中，所述载体包括病毒载体。
在某些实施方式中，所述载体为腺病毒相关载体(AAV)。
在某些实施方式中，所述载体为AAV8。
在某些实施方式中，所述受试者包括东亚人。
在某些实施方式中，所述方法在包括体外、体内或离体的条件下进行。
在某些实施方式中，所述施用包括注射。
在某些实施方式中，所述施用包括视网膜下腔注射。
另一方面，本申请提供了一种编辑RHO基因的方法，所述方法包括以下步骤：去除所述RHO基因中选自下组的突变位点：c.C50T和c.C403T。
在某些实施方式中，所述去除包括敲除所述突变位点和/或降低包含所述突变位点的RHO基因的表达水平。
在某些实施方式中，所述去除包括不影响所述受试者中野生型RHO基因的表达水平和/或功能。
在某些实施方式中，所述去除包括使包含所述突变的RHO等位基因发生双链断裂。
在某些实施方式中，所述去除包括施用至少一个能够去除所述突变位点的载体。
在某些实施方式中，所述载体包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
在某些实施方式中，所述gRNA与包含所述突变位点的RHO等位基因中的至少部分核酸特异性结合。
在某些实施方式中，所述gRNA与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补。
在某些实施方式中，所述编码gRNA的序列包含SEQ ID NO.1-2中任一项所示的核苷酸 序列。
在某些实施方式中，所述gRNA与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补。
在某些实施方式中，所述编码gRNA的序列包含SEQ ID NO.4所示的核苷酸序列。
在某些实施方式中，所述载体包括编码Cas蛋白的核酸。
在某些实施方式中，所述Cas蛋白包括Cas9蛋白。
在某些实施方式中，所述编码gRNA的序列与所述编码Cas蛋白的核酸位于同一个所述载体中。
在某些实施方式中，所述载体包括病毒载体。
在某些实施方式中，所述载体为腺病毒相关载体(AAV)。
在某些实施方式中，所述载体为AAV8。
另一方面，本申请提供了用于治疗受试者视网膜色素变性的组合物，其包括去除RHO基因的突变位点的活性成分和药学上的可接受载体，其中所述突变位点选自下组：c.C50T和c.C403T。
在某些实施方式中，所述活性成分包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
在某些实施方式中，所述与所述突变位点特异性结合的gRNA包含SEQ ID NO.44、45和47中任一项所示的核苷酸序列。
在某些实施方式中，所述编码gRNA的序列包含SEQ ID NO.1、2和4中任一项所示的核苷酸序列。
在某些实施方式中，所述活性成分包括Cas蛋白。
在某些实施方式中，所述Cas蛋白包括Cas9蛋白。
在某些实施方式中，所述编码gRNA的序列与编码所述Cas蛋白的核酸位于同一个载体中。
在某些实施方式中，所述载体包括病毒载体。
在某些实施方式中，所述载体为腺病毒相关载体(AAV)。
在某些实施方式中，所述载体为AAV8。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉 及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述使用的基因编辑载体的结构特征图谱。
图2显示的是针对RHO p.Thr17Met设计的三条编码gRNA的序列。
图3显示的是针对RHO p.Arg135Trp设计的两条编码gRNA的序列。
图4显示的是RHO17-SgRNA体外效率检测结果。
图5显示的是RHO135-SgRNA体外效率检测结果。
图6显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA1编辑结果。
图7显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA2编辑结果。
图8显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA3编辑结果。
图9显示的是使用gRNA活性荧光检测试剂盒体外检测RHO135-SgRNA1编辑结果。
图10显示的是使用gRNA活性荧光检测试剂盒体外检测RHO135-SgRNA2编辑结果。
图11显示的是使用gRNA活性荧光检测试剂盒体外检测sgRNA编辑效率的流式分选结果统计。
图12显示的是使用293T细胞体外验证sgRNA安全性和特异性的跑胶结果。
图13显示的是RHO17-SgRNA1和RHO17-SgRNA2仅对患者iPSCs的基因编辑效率。
图14显示的RHO135-SgRNA1仅对患者的iPSCs的基因编辑效率。
图15显示的是RHO17-SgRNA2和RHO135-SgRNA1对患者的3D视网膜组织的基因编辑效果。
图16显示的是RHO人源化小鼠基因型鉴定结果。
图17显示的是RHO17-SgRNA2和RHO135-SgRNA1对人源化小鼠的视网膜组织的基因编辑效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“c.C50T”通常是指与野生型的RHO基因的核苷酸序列相比，RHO基因的编码序列的第50位(从5’端至3’端，编码序列起始ATG中的“A”为第1位)的碱基由胞嘧啶(C)突变为胸腺嘧啶(T)。在本申请中，“c.”通常是指coding sequence，即编码序列，是指起始密码ATG到终止密码的一段序列，编码序列可以从mRNA的任何位置开始和结束。因此，“c.C50T”表示从编码序列ATG的A开始，第50个核苷酸由C突变为T。所述碱基的突变可导致RHO基因编码的氨基酸发生改变，例如，使得氨基酸由苏氨酸(Thr)突变为甲硫氨酸(Met)。在本申请中，术语“p.Thr17Met”通常是指RHO蛋白的第17位的氨基酸由苏氨酸(Thr)突变为甲硫氨酸(Met)。
在本申请中，术语“c.C403T”通常是指与野生型的RHO基因的核苷酸序列相比，RHO基因编码序列的第403位(从5’端至3’端，编码序列起始ATG中的“A”为第1位)的碱基由胞嘧啶(C)突变为胸腺嘧啶(T)，所述碱基的突变可导致RHO基因编码的氨基酸发生突变，例如，使得氨基酸由精氨酸(Arg)突变为色氨酸(Trp)。在本申请中，术语“p.Arg135Trp”通常是指RHO蛋白的第135位的氨基酸由精氨酸(Arg)突变为色氨酸(Trp)。
在本申请中，术语“RHO等位基因的外显子1”通常是指RHO基因中的第1个外显子。例如，在Ensembl数据库中RHO等位基因的外显子1的ID为ENSE00001079597，其可包括智人3号染色体的第129,528,639-129,529,094位的核苷酸序列。
在本申请中，术语“RHO等位基因的外显子5”通常是指RHO基因中的第5个外显子。例如，在Ensembl数据库中RHO等位基因的外显子5的ID为ENSE00001079599，其可包括智人3号染色体的第129,533,608-129,535,344位的核苷酸序列。
在本申请中，术语“双链断裂(double-strand breaks，DSB)”通常是指双股DNA分子的两条单链在同一位置被切割时发生的现象。双股断裂可诱发DNA修复，可能造成遗传重组，细胞也有一些系统作用于其他时候造成的双股断裂。双链断裂可在正常细胞复制周期中定期发生，也可以在某些情况下增强，例如紫外线、DNA断裂的诱导剂(例如，各种化学诱导剂)。许多诱导剂可以导致DSB在基因组中无差别地发生，并且DSB可以在正常细胞中有规律地诱导和修复。在修复过程中，可以完全保真地重建原始序列，但是，在某些情况下，会在DSB站点引入小的插入或缺失(称为“indels”)。在某些情形中，还可以在特定的位置特异性诱导双链断裂，其可以用于在选定的染色体位置引起定向或优先的基因修饰。在许多情况下，可以利用DNA修复(以及复制)过程中同源序列易于重组的趋势，这是基 因编辑系统(如CRISPR)应用的基础。该同源性指导的修复用于将通过使用“供体”多核苷酸提供的目的序列插入所需的染色体位置。
术语“敲除”是指与未改变的基因相比，将通常由基因编码的多肽的生物活性降低至少80％的基因的核酸序列改变。例如，改变可以是一个或多个核苷酸的插入、取代、缺失、移码突变或错义突变。
在本申请中，术语“互补”通常是指核酸(例如RNA)包含使其能够非共价结合的核苷酸序列(例如，Watson-Crick碱基配对)，在适当的体外和/或体内温度和溶液离子强度条件下，以序列特异性、反平行的方式(即核酸特异性结合互补核酸)“杂交”或“互补”至另一个核酸。如本领域所知，标准的Watson-Crick碱基配对包括：腺嘌呤(A)与胸苷(T)配对，腺嘌呤(A)与尿嘧啶(U)配对，鸟嘌呤(G)与胞嘧啶(C)配对。
在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。
在本申请中，所述“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是 当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“质粒”通常是指细菌、酵母菌等生物中染色体或拟核以外的DNA分子，存在于细胞质中，具有自主复制能力，使其能够在子代细胞中保持恒定的拷贝数，并表达所携带的遗传信息。质粒在遗传工程研究中被用作基因的载体。
在本申请中，术语“逆转录病毒载体”通常是指可以可控并表达外源基因，但不能自我包装成有增殖能力的病毒颗粒。此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因；和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构，并将之制备成载体。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
RP和RHO基因
一方面，本申请提供了一种治疗视网膜色素变性的方法。
在本申请中，术语“视网膜色素变性(Retinitis Pigmentosa，RP)”通常是指一种引起视网膜变性的遗传疾病。已鉴定出的与RP相关的基因约有80多个，且这些基因涉及常染色体隐性遗传(50-60％)、常染色体显性遗传(autosomal dominant：AD，30-40％)，以及X连锁遗传(5-15％)。RP的特征在于视网膜的感受器细胞(视锥和视杆细胞)和/或视网膜色素上皮细胞的功能异常而导致的进行性视力丧失。RP的临床表现可包括夜盲、进行性视野缺损，黄斑受累后出现中心视力减退，最终可致盲，视网膜电图(Electroretinogram，ERG)显示视杆细胞(Rods)功能下降甚至呈熄灭型。RP主要眼底改 变为赤道部视网膜色素紊乱，出现骨细胞样色素沉着，逐渐向后极部和锯齿缘方向发展。RPE、感光细胞和脉络膜毛细血管层逐渐萎缩，透见脉络膜大血管，视网膜呈青灰色，视网膜动脉变细，视盘蜡黄色萎缩，其中，视网膜血管狭窄、视盘颜色蜡黄及骨细胞样色素沉着是RP的典型三联征(Hartong DT et al.,2006)。评估视网膜功能和形态的方法可包括通过最佳矫正视力(Best Corrected Visual Acuity，BCVA)、眼底自发荧光、视野检查、ERG、眼底彩色照相、光学相干断层扫描(optical coherence tomography，OCT)和荧光素血管造影术(fluorescein angiography，FFA)等。其中评估视力功能性的方法可包括BCVA、视野等的方法。
本申请所述的方法可包括使有需要的受试者具有功能性的RHO基因。
在某些情形中，本申请所述的RP可以是由RHO基因的突变引起的。与RP相关的RHO基因突变有多种，这些基因突变可导致RHO基因编码功能异常的视紫红质蛋白(rhodopsin)，突变可包括但不限于基因的错义、无义、插入、缺失等突变。例如，突变位点可以包含选自下组的突变位点：c.C50T和c.C403T。又例如，所述突变位点可引起氨基酸改变，所述氨基酸突变可包含如下改变：p.Thr17Met和/或p.Arg135Trp。在本申请中，所述方法可以包括使有需要的受试者不具有杂合突变位点的RHO基因，所述突变位点可以选自下组：c.C50T和c.C403T
可以修复任何一个或多个突变使有需要的受试者具有功能性的RHO基因。例如，可以去除、恢复或纠正病理变异c.C50T和/或c.C403T。
在本申请中，术语“功能性的RHO基因”通常是指能够编码功能正常的视紫红质蛋白的基因。在本申请中，该术语表示不包含突变位点的RHO基因，例如，所述突变位点可以选自下组：c.C50T和c.C403T。在某些情形中，可以使包含突变位点(例如，c.C50T和c.C403T)的RHO基因成为功能性的RHO基因。例如，可以特异性去除突变位点(例如，c.C50T和c.C403T)，又例如，可以通过基因敲除结合同源依赖性修复(Homology Directed Repair，HDR)的方法除去突变位点(例如，c.C50T和c.C403T)并使包含突变位点(例如，c.C50T和c.C403T)的RHO基因成为功能性的RHO基因。
术语“RHO基因”也可称为视紫红质2(Rhodopsin 2)、Opsin-2、Opsin 2、OPN2、CSNBAD1，或RP4。视紫红质蛋白定位于视杆细胞外节(Rods Outer Segment，ROS)，对于正常视力特别是感受弱光刺激是必须的，Rods是视网膜的光感受器细胞的一种司暗视觉。视网膜中的另一种光感受器细胞是视锥细胞(Cones)，司明视觉和色觉。在ROS，rhodopsin通常与11-顺式视黄醛(11-cis retinal，11cRAL)结合，该分子是维生素A的一种 衍生物形式。ROS吸收光子使Rhodopsin成为活性Rhodopsin(R*)，使11cRAL异构为全反式视黄醛(all-trans retinal，atRAL)，atRAL与R*分离后很快被还原为全反式视黄醇(all-trans retinol，atROL)，感光细胞间维生素A类结合蛋白(interphotoreceptor retinoid-binding protein,IRBP)负责将atROL转入RPE细胞，在RPE细胞中，atROL由卵磷脂视黄醇酰基转移酶(lecithin retinol acyltransferase，LRAT)转化为全反式视黄醇酯，再进一步转化为11-顺式视黄醇酯，然后由水解异构酶RPE65异构为11-顺式视黄醇(11-cis retinol，11cROL)，11cROL被RDHs氧化成11cRAL，和IRBP结合后转运回感光细胞内被重新利用；R*将下游膜盘中转导蛋白G(Gt)α亚基上的GDP转化为GTP，使α亚基与βγ亚基分离，激活环磷酸鸟苷-磷酸二酯酶6(cGMP-PDE6)，水解cGMP，细胞cGMP浓度降低关闭OS的cGMP门控性阳离子通道，使感光细胞内Ca 2+浓度降低，细胞膜超极化，使光信号转变为视觉电信号；光传导终止后感光细胞经过一系列的化学反应回到非光照时的状态，此时R*被磷酸化并和抑制蛋白结合并抑制下游信号通路，PDE6呈非激活状态，同时Rods内合成cGMP，cGMP浓度升高使阳离子通道开放进而Ca 2+内流，细胞膜去极化。这种由cGMP门控性阳离子通道的开放与关闭引发的神经冲动，经感光细胞的突触末端与视网膜各级神经元的连接及视神经传导至大脑皮层的视觉中枢，形成视觉。
人的RHO基因位于染色体3的长臂的位置22.1处(3q22.1)，分子在3号染色体上第129,528,639碱基对至第129,535,344碱基对(智人更新注释发行(Homo sapiens，Annotation release)第109.20200228版，GRCh38.p13，NCBI)。RHO基因的核苷酸序列可参见NCBI GenBank Accession No.NG_009115.1。RHO基因具有5个外显子。表1显示了Ensembl数据库中RHO基因的外显子标识符以及外显子的起始/终止位点。
表1 RHO基因的外显子
外显子 外显子ID 起始/终止位点(3号染色体)
1 ENSE00001079597 129,528,639-129,529,094
2 ENSE00001152211 129,530,876-129,531,044
3 ENSE00001152205 129,532,251-129,532,416
4 ENSE00001152199 129,532,533-129,532,772
5 ENSE00001079599 129,533,608-129,535,344
使RHO基因功能异常的情况可以是基因突变，突变可包括但不限于核苷酸的插入、缺失、错义、无义、移码和/或其他突变。在某些情形中，可以修复任何一个或多个突变以恢复RHO基因的正常功能。例如，可以去除RHO基因的突变位点。
在某些情形中，所述方法可包括以下步骤：去除有需要的受试者中RHO基因的所述突变位点。所述方法可包括外显子缺失。特定外显子的靶向缺失可能是一种用单一治疗性鸡尾酒治疗大量患者的策略。外显子缺失可以是单外显子缺失或多外显子缺失。尽管多外显子缺失可以覆盖更多的受试者，但对于较多核苷酸的缺失，缺失的效率会随着核苷酸大小的增加而大大降低。因此，去除的范围可以是40到10,000个碱基对(bp)。例如，去除范围可以是40-100、100-300、300-500、500-1,000、1,000-2,000、2,000-3,000、3,000-5,000，或5,000-10,000个碱基对。
如前所述，RHO基因含有5个外显子。5个外显子中的任何一个碱基或多个碱基都可以包含突变。可以去除5个突变外显子或异常的内含子剪接受体或供体位点中的任何一个或多个突变位点，使所述功能性的RHO基因不包含突变位点(如，影响RHO基因功能的突变)。在某些情形中，所述突变位点可以来自RHO基因的以下组中的任意一个或多个：外显子1、外显子2、外显子3、外显子4、外显子5或其任何组合。例如，所述基因突变可以是选自下组的突变：c.C50T和c.C403T。所述基因突变可以导致氨基酸突变，最终导致RHO蛋白功能异常，如突变蛋白会干扰正常蛋白的功能或者不能定位到ROS等。
体内或体外方法
本申请所述方法可包括敲除所述突变位点和/或降低所述突变位点的表达水平。敲除基因或降低基因的表达水平的方法可包括进行基因敲除、条件性基因敲除法(例如，利用Cre/LoxP和/或FLP-frt系统)、诱导性基因敲除法(例如，以Cre/loxp系统为基础的敲除，包括四环素诱导、干扰素诱导、激素诱导、腺病毒诱导等)、利用随机插入突变进行基因敲除(例如，基因捕获法)、利用RNAi引起的基因敲除、锌指核酸内切酶(zinc finger nucleases，ZNF)介导的基因编辑技术、转录激活子样效应物核酸酶(transcription activator-like effector nucleases，TALEN)介导的基因编辑技术、成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats，CRISPR)/CRISPR相关蛋白(CRISPR associated proteins，Cas)(CRISPR/Cas)系统介导的基因编辑技术和/或NgAgo-gDNA基因编辑技术。对于任何基因组编辑策略，可以通过测序或PCR分析来确认基因编辑。
在某些情形中，本申请所述的方法可以是基于体内细胞的方法。在某些情形中，所述方法包括编辑受试者的细胞的基因组DNA。例如，可包括编辑受试者的细胞(例如，感光细胞和/或视网膜祖细胞)中的RHO基因中的突变。例如，所述基因突变可以是选自下组的突变：c.C50T和c.C403T。尽管某些细胞可以是离体方法或离体疗法的理想靶标，但是使用有效的递送方法也可允许直接在体内将所需要的试剂递送至此类细胞。在某些情形中，所述方法可 以包括靶向和编辑至相关细胞。也可通过使用仅在某些细胞和/或发育阶段具有活性的启动子来防止其他细胞的裂解。
外加的启动子是可诱导的，因此，如果将核酸分子在质粒载体中递送，则可以控制递送时间。递送的核酸或蛋白质在细胞内留存的时间也可通过改变半衰期的方法进行调整。体内方法可以节省某些处理步骤，但是要求较高的编辑效率。体内治疗可消除离体治疗和植入带来的问题和损失。
体内方法可以易于治疗性产品的产生和给药。相同的治疗方法或疗法将有可能用于治疗不止一名受试者，例如，许多具有相同或相似基因型或等位基因的受试者。
本申请所述的方法可包括离体的方法。在某些情形中，可以获得受试者特异性的诱导多能干细胞(iPSC)。然后，可以使用本申请所述的方法编辑这些iPSC细胞的基因组DNA。例如，该方法可以包括在iPSC的RHO基因的突变位点内或附近进行编辑，使得其不具有p.Thr17Met和/或p.Arg135Trp的氨基酸突变，例如，所述基因突变可以是选自下组的突变：c.C50T和c.C403T。接下来，可以将经基因编辑的iPSC分化为其他细胞，例如感光细胞或视网膜祖细胞。最后，可以将分化的细胞(例如感光细胞或视网膜祖细胞)植入受试者体内。
在另一些情形中，可以从受试者中分离出感光细胞或视网膜祖细胞。接下来，可以使用本申请所述的方法编辑这些感光细胞或视网膜祖细胞的基因组DNA。例如，该方法可以包括在感光细胞或视网膜祖细胞的RHO基因的突变位点内或附近进行编辑，使得其不具有p.Thr17Met和/或p.Arg135Trp的氨基酸突变，例如，所述基因突变可以是选自下组的突变：c.C50T和c.C403T。最后，可以将经基因编辑的感光细胞或视网膜祖细胞植入受试者体内。
在另一些情形中，可以从在另一些情形中体内分离间充质干细胞，也可以从在另一些情形中的骨髓或外周血中分离出来。接下来，可以使用本申请所述的方法编辑这些间充质干细胞的基因组DNA。例如，该方法可以包括在间充质干细胞的RHO基因的突变位点内或附近进行编辑，使得其不具有p.Thr17Met和/或p.Arg135Trp的氨基酸突变，例如，所述基因突变可以是选自下组的突变：c.C50T和c.C403T。接下来，可以将经基因编辑的间充质干细胞分化为任何类型的细胞，例如感光细胞或视网膜祖细胞。最后，可以将分化的细胞，例如感光细胞或视网膜祖细胞植入受试者体内。
所述方法可包括在给药前对治疗剂进行全面分析。例如，对校正细胞的整个基因组进行测序，以确保没有脱靶效应(如果有的话)可以处于与对受试者的最小风险相关的基因组位置。此外，可以在植入之前分离特定细胞的群，包括克隆细胞群。
使用本申请所述的方法可以不影响所述受试者中野生型RHO基因的表达水平和/或功能。
基因编辑
本申请所述的方法本文所述的方法可包括使用定点核酸酶在基因组中精确的靶标位置切割DNA，从而在基因组内特定位置产生单链或双链DNA断裂的方法。此类断裂可以通过内源性细胞过程进行定期修复，例如HDR和非同源末端连接(Non-Homologous End Joining，NHEJ)。这两个主要的DNA修复过程由一系列替代途径组成。NHEJ直接连接双链断裂所导致的DNA末端，有时会丢失或添加核苷酸序列，这可能会破坏或增强基因表达。HDR利用同源序列或供体序列作为模板，在断点处插入特定的DNA序列。同源序列可以在内源基因组中，例如姐妹染色单体(sister chromatid)。或者，所述供体可以是外源核酸，例如质粒、单链寡核苷酸、双链寡核苷酸、或病毒。这些外源核酸可以包含与核酸酶切割的基因座具有高度同源性的区域，此外还可包含额外的序列或序列变化(包括可掺入切割的靶基因座的缺失)。第三种修复机制可以是微同源介导的末端连接(Microhomology-Mediated End Joining，MMEJ)，也称为“替代NHEJ(ANHEJ)”，在切割位点可能发生小的缺失和插入，其遗传结果与NHEJ相似。MMEJ可以利用位于DNA断裂位点两侧的几个碱基对的同源序列来驱动更有利的DNA末端连接修复结果。在某些情况下，有可能基于对DNA断裂位点潜在的微观同源性的分析来预测可能的修复结果。
这些基因编辑机制均可用于本申请所需的基因突变位点的去除。本申请所述的方法可包括在目标基因座中靠近预期突变位点的位置创建一个或两个DNA断裂，两个DNA断裂可以为双链断裂或两个单链断裂。在某些情形中，所述去除可包括使包含所述突变的RHO等位基因发生双链断裂。所述断裂可通过定点(site-directed)多肽来实现。定点多肽(例如DNA内切核酸酶)可以在核酸(例如基因组DNA)中引入双链断裂或单链断裂。双链断裂可以刺激细胞的内源性DNA修复途径，例如，HDR、NHEJ，或MMEJ。NHEJ可以修复裂解的靶核酸，而无需同源模板。
在某些情况下，可以使用同源重组将外源多核苷酸序列插入靶核酸切割位点。外源多核苷酸序列可以被称为供体多核苷酸(或供体，或供体序列，或多核苷酸供体模板)。可将供体多核苷酸，供体多核苷酸的一部分，供体多核苷酸的拷贝或供体多核苷酸的拷贝的一部分插入靶核酸切割位点。供体多核苷酸可以是外源多核苷酸序列，即不是天然存在于靶核酸切割位点的序列。
当同源修复模板或供体可用时，会发生HDR。同源供体模板可包含野生型RHO基因或cDNA的至少一部分。野生型RHO基因或cDNA的至少一部分可以是外显子1、外显子2、外显子3、外显子4、外显子5、内含子区域、上述这些的片段或组合，或完整的RHO基因 或cDNA。供体模板可以是单链或双链多核苷酸。供体模板可以由AAV递送。同源供体模板可以包含与靶核酸切割位点侧翼的序列同源的序列。例如，供体模板可具有与3q22.l区域同源的臂。供体模板也可以具有与病理变异c.C50T和/或c.C403T同源的臂。姐妹染色单体可以被细胞用作修复模板。但是，出于基因编辑的目的，修复模板可以作为外源核酸提供，例如质粒、双链寡核苷酸、单链寡核苷酸或病毒核酸。利用外源供体模板，可以在同源的侧翼区域之间引入另外的核酸序列(例如转基因)或修饰(例如单碱基或多碱基改变或缺失)，从而也可以将另外的或改变的核酸序列纳入目标基因座。MMEJ可以利用位于切割位点两侧的几个碱基对的同源序列来驱动有利的末端连接DNA修复结果。在某些情况下，可以基于对核酸酶靶区域中潜在的微同源性的分析来预测可能的修复结果。
CRISPR/Cas系统
在本申请中，术语“CRISPR/Cas系统”或“CRISPR-Cas系统”通常是指由成簇规律间隔短回文重复序列(CRISPR)和CRISPR相关蛋白(即Cas蛋白)组成的核酸酶系统，能够对真核细胞中几乎所有与前间区序列邻近基序(protospacer-adjacent motif,PAM)相邻的基因组序列进行切割。“CRISPR/Cas系统”可用来统称涉及CRISPR相关(“Cas”)基因的转录物，以及涉及其表达或指导其活性的其他元件，可包括编码Cas基因的序列、tracr(反式激活CRISPR)序列(例如tracrRNA或其活性部分)、tracr配偶序列(在内源CRISPR/Cas系统背景下，涵盖“同向重复”和加工的部分同向重复)、指导序列(在内源CRISPR/Cas系统背景下也称为“spacer”)、或来自CRISPR座位的其他序列和转录物。已经鉴定出五种类型的CRISPR系统(例如，I型、II型、III型、U型和V型)。
在本申请中，术语“Cas蛋白”也称为“CRISPR相关蛋白”通常是指与CRISPR序列互补的一类酶，能够使用CRISPR序列作为指导(guide)，从而识别和切割特定的DNA链。Cas蛋白的非限制性实例包括：Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csnl和Csxl2)、CaslO、Csyl、Csy2、Csy3、Csel、Cse2、Cscl、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csbl、Csb2、Csb3、Csxl7、Csxl4、CsxlO、Csxl6、CsaX、Csx3、Csxl、Csxl5、Csf1、Csf2、Csf3、Csf4，和/或他们的同系物、或其修饰形式。在一些实施例中，该Cas蛋白是Cas9蛋白。
在本申请中，术语“Cas9蛋白”或“Cas9核酸酶”，也称为Csn1或Csx12，通常是指II型CRISPR/Cas系统中一类既参与crRNA生物合成又参与摧毁入侵DNA的蛋白质。Cas9蛋白通常包括RuvC核酸酶结构域和HNH核酸酶结构域，分别切割双链DNA分子的两条不同的链。已经在不同的细菌物种如嗜热链球菌(S.thermophiles)、无害利斯特氏菌(Listeria innocua) (Gasiunas，Barrangou et al.2012；Jinek，Chylinski et al.2012)和化脓性链球菌(S.Pyogenes)(Deltcheva，Chylinski et al.2011)中描述了Cas9蛋白。例如，化脓链球菌(Streptococcus pyogenes)Cas9蛋白，其氨基酸序列参见SwissProt数据库登录号Q99ZW2；脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白，其氨基酸序列见UniProt数据库编号A1IQ68；嗜热链球菌(Streptococcus thermophilus)Cas9蛋白，其氨基酸序列见UniProt数据库编号Q03LF7；金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白，其氨基酸序列见UniProt数据库编号J7RUA5。
CRISPR/Cas系统可包括许多短重复序列，称为“重复(repeat)”。当表达时，重复序列可以形成二级结构(例如发夹)和/或包含非结构化的单链序列。重复序列通常成簇出现，并且经常在物种之间由于演变而不同(diverge)。这些重复序列与称为“间隔子(spacer)”的独特的中间序列有规律地间隔，从而形成重复-间隔子-重复(repeat-spacer-repeat)基因座结构。间隔子与已知的外来入侵者序列相同或具有高度同源性。间隔重复(spacer-repeat)单元编码crispRNA(crRNA)，其被加工成间隔重复单元的成熟形式。crRNA包含靶向靶核酸的“种子(seed)”或间隔子序列(原核生物中天然存在的形式，间隔子序列靶向外来入侵者核酸)。间隔区序列位于crRNA的5'或3'端。
CRISPR/Cas系统还可包括编码CRISPR相关蛋白(Cas蛋白)的多核苷酸序列。Cas基因编码参与原核生物中crRNA功能的生物合成(biogenesis)和干扰阶段的核酸酶。一些Cas基因包含同源的二级和/或三级结构。
自然界中，II型CRISPR系统中的crRNA生物合成需要反式激活CRISPR RNA(tracrRNA)。tracrRNA可以通过内源性RNaseIII修饰，然后与pre-crRNA中的crRNA重复序列杂交。可以募集内源RNaseIII以切割pre-crRNA。切开的crRNA可以进行外切核酸酶修整以产生成熟的crRNA形式(例如5’端修整)。tracrRNA可以保持与crRNA杂交，并且tracrRNA和crRNA与定点多肽(例如，Cas9)缔合。crRNA-tracrRNA-Cas9复合物中的crRNA可以将该复合物引导至可以与crRNA杂交的靶核酸。crRNA与靶核酸的杂交可以激活Cas9进行靶核酸切割。II型CRISPR系统中的靶核酸称为原间隔子相邻基序(PAM)。实际上，PAM对于促进定点多肽(例如，Cas9)与靶核酸的结合是必不可少的。II型系统(也称为Nmeni或CASS4)可以进一步细分为II-A型(CASS4)和II-B型(CASS4a)。可用于RNA可编程基因编辑的CRISPR/Cas9系统可参见Jinek等人，Science，337(6096)：8l6-82l(2012)，国际专利申请公开号WO2013/176772提供了可用于位点特异性基因编辑的CRISPR/Cas核酸内切酶系统的众多示例和应用。
gRNA
本申请的方法包括提供靶向基因组的核酸，其可以将相关活性多肽(例如Cas蛋白)引导至靶核酸内的特定靶序列(例如，RHO等位基因)。靶向基因组的核酸可以是RNA。靶向基因组的RNA在本文中可以被称为“指导RNA”或“gRNA”。在某些情形中，本申请所述的gRNA可以与靶核酸互补。在另一些情形中，所述的gRNA可以与靶核酸相同(当说到相同时，由于编码RNA和DNA的碱基的区别，RNA中的“U”对应于DNA中的胸腺嘧啶“T”)。在另一些情形中，编码所述gRNA的核酸序列(例如，DNA)可以与靶核酸相同或互补。在本申请中，术语“靶核酸”、“靶核酸”和“靶区域”可以互换的使用，通常是指可以被gRNA识别的核酸序列，所述靶核酸可以指双链核酸，也可以指单链核酸。所述gRNA可以由编码其的序列进行转录或复制得到，例如，所述gRNA可通过编码其的DNA序列转录得到。在本申请中，术语“编码gRNA的序列”通常是指可通过转录得到所述gRNA的DNA序列。在本申请中，所述“编码gRNA的序列”可以与gRNA的靶序列具有相同的核苷酸序列。
在某些情形中，gRNA可以至少包含与感兴趣的靶核酸序列杂交的间隔子序列和CRISPR重复序列。在II型系统中，gRNA还包含称为tracrRNA序列的第二个RNA。在II型CRISPR系统中，CRISPR重复序列和tracrRNA序列彼此杂交形成双链体。gRNA可以结合Cas蛋白，从而形成指导RNA-Cas蛋白复合物。靶向基因组的核酸由于其与Cas蛋白的缔合而可以使所述复合物具有靶标特异性。因此，靶向基因组的核酸可以指导Cas蛋白的活性。在某些情形中，靶向基因组的核酸可以是双链指导RNA。在某些情形中，gRNA可以是单链指导RNA(sgRNA)。所述双链指导RNA或单链指导RNA可以被修饰。
在某些情形中，双链指导RNA可以包含两条RNA链。第一链可包含任选的间隔子延伸序列、间隔子序列和最小的CRISPR重复序列。第二链可包含最小tracrRNA序列(与最小CRISPR重复序列互补)，3’tracrRNA序列和任选的tracrRNA延伸序列。
在某些情形中，sgRNA可以在5’至3’方向上包含一个可选的间隔子延伸序列、一个间隔子序列、一个最小的CRISPR重复序列、一个单分子指导接头、最小tracrRNA序列、3’tracrRNA序列和任选的tracrRNA延伸序列。任选的tracrRNA延伸可以包含对指导RNA有贡献的附加功能(例如，稳定性)的元件。单分子引导接头可以连接最小的CRISPR重复序列和最小的tracrRNA序列，以形成发夹结构。任选的tracrRNA延伸可以包含一个或多个发夹。
例如，所述sgRNA可以包含一个可变长度的间隔子序列，该序列在sgRNA序列的5’端具有17-30个核苷酸。在其他情形中，sgRNA可以包含可变长度的间隔区序列，其在sgRNA 序列的5’末端具有17-24个核苷酸。例如，所述sgRNA可以包含21个核苷酸的序列。例如，所述sgRNA可以包含20个核苷酸的序列。例如，所述sgRNA可以包含19个核苷酸的序列。例如，所述sgRNA可以包含18个核苷酸的序列。例如，所述sgRNA可以包含17个核苷酸的序列。例如，所述sgRNA可以包含22个核苷酸的序列。例如，所述sgRNA可以包含23个核苷酸的序列。例如，所述sgRNA可以包含24个核苷酸的序列。所述sgRNA可以是未修饰的或修饰的。
本申请所述的gRNA可以与目标靶核酸中的序列结合。靶向基因组的核酸(或其部分)可以通过杂交(即碱基配对)以序列特异性的方式与靶核酸相互作用。sgRNA的核苷酸序列可以根据目标靶核酸的序列而变化。
在本申请的CRISPR/Cas系统中，gRNA序列可以设计成与所述系统中使用的Cas蛋白可识别的PAM序列临近处的靶核酸杂交。所述gRNA可以与靶序列完全匹配或不匹配。Cas蛋白通常都有一个可以在目标DNA中识别的特定的PAM序列。
例如，所述Cas9蛋白可以来自化脓性链球菌(S.pyogenes)，该Cas9蛋白在靶核酸中识别包含序列5’-NRG-3’的PAM，其中R包含A或G，其中N可以是任意核苷酸。又例如，所述Cas9蛋白可以来自金黄色葡萄球菌(Staphylococcus aureus)，该Cas9蛋白(SaCas9)在靶核酸中可识别包含序列5’-NNGRR(T)-3’的PAM，其中R包含A或G，其中N可以是任意核苷酸。在一些更具体的情况下，SaCas9识别的PAM序列可包含5’-NNGRR-3’，其中R包含A或G，其中N可以是任意核苷酸。例如，本申请所述的PAM可包含SEQ ID NO:39-43中任一项所示的核苷酸序列。
本申请所述的gRNA可以与所述突变位点特异性结合。
在某些情形中，所述gRNA可以与包含所述突变位点的RHO等位基因中的至少部分核酸特异性结合。例如，编码所述gRNA的序列可以包含SEQ ID NO.1、2和4中任一项所示的核苷酸序列。
在某些情形中，所述gRNA可以与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补。例如，所述与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补的gRNA可以包含SEQ ID NO.44-45中任一项所示的核苷酸序列。例如，编码所述gRNA的序列可以包含SEQ ID NO.1-2中任一项所示的核苷酸序列。
在某些情形中，所述gRNA可以与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补。例如，所述与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补的gRNA可以包含SEQ ID NO.47所示的核苷酸序列。例如， 编码所述gRNA的序列可以包含SEQ ID NO.4所示的核苷酸序列。
在一些情形中，gRNA与靶核酸之间的互补百分比可为至少约30％、至少约40％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约97％、至少约98％、至少约99％，或100％。在一些情形中，gRNA与靶核酸之间的互补百分比可以为至多约30％、至多约40％、至多约50％、至多约60％、至多约65％、至多约70％、至多约75％、至多约80％、至多约85％、至多约90％、至多约95％、至多约97％、至多约98％、至多约99％，或100％。
本申请所述用于CRISPR系统的gRNA可以通过化学方法合成，例如，高效液相色谱法。例如，将两个或两个以上的RNA分子连接在一起。长度较长的RNA(例如编码Cas9的RNA)可以通过酶促反应得到。在本领域中，可以在RNA的化学合成和/或酶促合成期间或之后引入各种类型的RNA修饰，例如，为增强稳定性，降低先天免疫应答和/或增强其他性质的修饰。
载体
本申请提供了载体。指导RNA的多核苷酸(RNA或DNA)和/或编码核酸内切酶的多核苷酸(RNA或DNA)可以通过本领域已知的病毒或非病毒递送载体来递送。或者，所述核酸内切酶多肽可以通过本领域已知的病毒或非病毒递送载体来递送，例如电穿孔或脂质纳米颗粒。在其他方面，DNA核酸内切酶可以作为一种或多种多肽单独地递送，或者，与一种或多种引导RNA，或一种或多种crRNA以及tracrRNA，一起预复合地递送。一些示例性非病毒递送载体可参见Peer和Lieberman，Gene Therapy，18：1127-1133(2011)。
本申请所述的载体可包含所述本申请的核酸分子(例如，编码gRNA的序列和/或gRNA)。多核苷酸可以通过非病毒递送载体递送，包括但不限于纳米颗粒、脂质体、核糖核蛋白、带正电荷的肽、小分子RNA缀合物、适体-RNA嵌合体和RNA融合蛋白复合物。如前所述，定点多肽和靶向基因组的核酸可各自分别给予细胞或患者。另一方面，定点多肽可以与一个或多个引导RNA或一个或多个crRNA以及tracrRNA预复合。然后可以将预复合材料施用于细胞或患者。这种预复合材料被称为核糖核蛋白颗粒(RNP)。RNA能够与多核苷酸(例如，RNA或DNA)形成特异性相互作用。尽管在许多生物过程中都利用了这种特性，但它也伴随着在富含核酸的细胞环境中发生混杂相互作用的风险。解决该问题的一种方法是形成核糖核蛋白颗粒(RNP)，其中RNA与核酸酶预复合。RNP可保护RNA免受降解。RNP中的核酸酶可以被修饰或未被修饰。同样，gRNA、crRNA、tracrRNA或sgRNA可以被修饰或未被修饰。本领域中存在许多已知并可被使用的修饰。例如，核苷酸的缺失、插入、转位、失活和/ 或激活。所述修饰可包括引入一个或多个突变(包括单个或多个碱基对改变)、增加发夹的数目、交联、断开具体的核苷酸段以及其他修饰。修饰可以包括包含至少一个非天然存在的核苷酸、或一个经修饰的核苷酸、或其类似物。所述核苷酸可以在核糖、磷酸和/或碱基部分处被修饰。
所述载体也可以是多核苷酸载体，例如，质粒、cosmid或转座子。适合使用的载体已被广泛描述并且是本领域公知的。本领域技术人员将理解，包含本申请所述的核酸分子的载体还可包含可用于载体在原核和/或真核细胞中复制所需的其他序列和元件。例如，本申请所述载体可包括原核复制子，即，具有在原核宿主细胞(例如，细菌宿主细胞)中引导宿主自身复制和维持的能力的核苷酸序列。所述复制子在本领域是公知的。在某些情形中，所述载体可包含穿梭元件，其使载体适于在原核生物和真核生物中复制和整合。此外，所述载体还可包括能够表达可检测标记(例如，药物抗性基因)的基因。所述载体还可具有报告基因，如，可编码荧光或其他可检测蛋白的基因。
在某些情形中，所述载体可包括病毒载体，例如，AAV、慢病毒、逆转录病毒、腺病毒、疱疹病毒和肝炎病毒。用于产生包含核酸分子(例如，本申请所述分离的核酸分子)作为载体基因组一部分的病毒载体的方法是本领域公知的，并且本领域技术人员可无需进行过多的实验进行。在另一些情形中，所属载体可以是包装了本申请所述核酸分子的重组AAV病毒粒子。产生重组AAV的方法可包括将本申请所述的核酸分子引入包装细胞系，产生AAV感染、AAV cap和rep基因的辅助功能，以及，从包装细胞系的上清液中回收重组的AAV。可以使用各种类型的细胞作为包装细胞系。例如，可以使用的包装细胞系包括但不限于HEK 293细胞，HeLa细胞和Vero细胞。
在某些情形中，所述载体可以为腺病毒相关载体(AAV)。在本申请中，术语“腺病毒相关载体”通常指来源于天然存在且可用的腺相关病毒以及人工AAV的载体。所述AAV可包括不同的血清型AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12或AAV13，以及任何AAV变体或混合物。AAV基因组两端通常有末端反向重复序列(ITR)，术语“ITR”或“末端反向重复”是指存在于AAV和/或重组AAV中的核酸序列段，其可形成完成AAV溶解和潜伏生命周期所需的T形回文结构。产生AAV载体的技术是本领域的标准技术，其中包括将要递送的多核苷酸、rep和cap基因以及辅助病毒功能的待包装AAV基因组提供给细胞。生产AAV载体通常需要在单个细胞(此处称为包装细胞)内存在以下成分：rAAV基因组，与rAAV基因组分离(例如不在其中)的AAV rep和cap基因以及辅助病毒。AAV rep和cap基因可以来自任何地AAV血清型，也可以来自与AAV基 因组ITR不同的AAV血清型，包括但不限于本文所述的AAV血清型。本申请中的AAV载体可包含靶向RHO基因的突变位点的gRNA。例如，所述编码gRNA的序列可包含SEQ ID NO.1、2和4中任一项所示的核苷酸序列。
在某些情形中，所述编码gRNA的序列可与所述编码Cas9蛋白的核酸可以位于同一个所述载体中。在另一些情形中，所述编码gRNA的序列可与所述编码Cas9蛋白的核酸可以位于不同的所述载体中。
本申请的AAV载体可以来自多种物种。例如，所述AAV可以是禽类AAV、牛AAV或山羊AAV。在某些实施方式中，所述载体为AAV8。
本申请的方法可包括产生包装细胞，即产生可用于稳定表达AAV的所有必需组分的细胞系。例如，将缺少AAV rep和cap基因的AAV基因组，从AAV基因组分离的AAV rep和cap基因，以及带有选择标记如新霉素抗性基因的质粒(或多个质粒)整合到该细胞的基因组中。已经可通过诸如GC拖尾的方法(Samulski等，1982，Proc.Natl.Acad.S6.ETSA，79：2077-2081)将AAV基因组引入细菌质粒中。然后可以用辅助病毒(例如腺病毒)感染包装细胞系。除了质粒，还可采用腺病毒或杆状病毒，来将AAV基因组和/或rep和cap基因引入包装细胞。
在本申请中，术语“受试者”通常是指是希望对其进行诊断、治疗或治疗的任何受试者。例如，在本申请中，有需要的受试者可的RHO基因包含选自下组的突变位点：c.C50T和c.C403T。在某些情形中，所述受试者可以包括哺乳动物。在某些情形中，受试者可以包括人。在某些情形中，所述受试者可包括东亚人。
另一方面，本申请提供了用于治疗受试者视网膜色素变性的组合物，所述组合物可包括去除RHO基因的突变位点的活性成分和药学上的可接受载体，其中所述突变位点选自下组：c.C50T和c.C403T。所述组合物可包含生理上可耐受的载体以及细胞组合物，以及任选地至少一种生物活性剂，其溶解或分散在治疗组合物中作为活性成分。通常，本文所述的载体可以与药学上可接受的载体的悬浮液形式施用。本领域技术人员将认识到，可使用的药学上可接受的载体可包含缓冲剂、化合物、冷冻保存剂、防腐剂或其他试剂，且不会干扰待载体的递送。所述组合物还可包含细胞制剂，例如，渗透性缓冲液，其允许维持细胞膜的完整性，以及任选地，营养液，其维持细胞生存力或在施用时增强植入。所述的制剂和悬浮液是本领域技术人员已知的，或者可以使用常规实验使其适于与本申请的载体和/或细胞一起使用。
在本申请中，术语“施用”可以通过一种方法或途径将细胞和/或载体引入受试者，或受试者的某个期望的部位。细胞和/或载体可以在期望的部位(例如损伤或修复的部位)表达本申请的核酸分子(例如，编码gRNA的序列和/或gRNA)，从而产生期望的效果。可以通过任 何合适的途径施用细胞(或它们的分化后代)和/或载体，所述任何合适的途径可以将所述细胞(或它们的分化后代)和/或载体递送至受试者中期望的部位，且至少有一部分植入的细胞(或细胞成分)和/或载体保持活力。在施用于受试者后，细胞的存活期可以短至数小时，例如二十四小时、几天，长达数年，甚至与患者的寿命一致。在某些情形中，所述施用包括注射。例如，可通过全身性给药途径如腹膜内或静脉内途径施用所述载体。例如，所述施用可包括视网膜下腔注射。
本申请所述的gRNA和载体对RHO等位基因突变位点的切割效率可以达约50％以上，例如，约55％以上、约60％以上、约65％以上、约70％以上、约75％以上、约80％以上、约85％以上、约90％以上、约95％以上或约98％以上，如在体外酶切反应中检测的。例如，使用给荧光检测试剂盒，本申请所述的gRNA和载体能够切割靶核酸，具有一定切割效率和特异性。此外，本申请的gRNA和载体不影响宿主细胞存活，具有一定的安全性。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.收集患者的基因诊断的信息
本研究在近年来就诊于北京大学第三医院的千余例RP患者中，收集到多个基因诊断明确的RHO基因突变的RP家系，发现了RHO基因的两个突变热点，见下表2。
表2 RHO的突变热点
家系 外显子 核酸改变 氨基酸改变 是否已报道致病
突变热点1 E1 c.50C>T p.Thr17Met 是
突变热点2 E2 c.403C>T p.Arg135Trp 是
实施例2.SgRNA设计及pX601-SgRNA质粒构建
针对上述2个突变位点，我们使用Benchling网站针对SaCas9体系共设计了5条sgRNA，核苷酸序列见下表3。
表3 SgRNA序列
Figure PCTCN2020105881-appb-000001
Figure PCTCN2020105881-appb-000002
本专利所使用的打靶载体为：pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA；U6::BsaI-sgRNA，图谱如图1所示(载体信息请参见https://www.addgene.org/61591/)。
针对RHO p.Thr17Met设计的sgRNA示意图如图2所示，针对RHO p.Arg135Trp设计的编码gRNA的序列示意图如图3所示。
质粒构建的具体步骤如下：
(1)SgRNA退火
将T4 PNK及10X T4 Ligation Buffer于冰上融化备用。配制如下反应体系：
表4 反应体系
编码gRNA的DNA序列混合物 7μl
10×T4连接缓冲液 1μl
T4 PNK 2μl
总量 10μl
将上述配制的反应体系置于PCR仪上，运行如下反应程序：
表5 反应程序
Figure PCTCN2020105881-appb-000003
(2)载体酶切
使用BSaI酶切释放编码gRNA的DNA序列的结合位点，在1.5ml PCR管中配制如下酶切反应体系：
表6 反应体系
质粒(15ug) Xμl
2×Cutsmart缓冲液 30μl
酶e 12μl
H 2O 258-Xμl
总量 300μl
酶切1-2h/(K过夜酶切)，回收纯化，测定浓度，稀释到50ng/ul。
(3)连接
使用上一步的回收载体和退火的编码gRNA的DNA序列配制如下连接体系(200μl PCR管)：
表7 连接体系
酶切载体 1μl
退火编码gRNA的DNA 1μl
2×T4 Ligation Buffer 5μl
T4 Ligase 1μl
H 2O 3μl
Total 11μl
将上一步的连接反应体系置于37℃连接约1-2h，完成包含编码gRNA的DNA序列载体构建。
(4)质粒转化
1)取出感受态细胞置于冰上解冻
2)取1μl连接至于50μl感受态中，冰上半小时，42度90s，冰上2min
3)无抗培养基500μl摇1h，
4)取100μl铺板
5)第二天挑菌(培养基500μl)摇3-4小时，取200μl送测序
(5)质粒提取(按照Omega去内毒素质粒大提试剂盒进行)
1)将测序正确的px601-SaCas9-RHO-SgRNA进行过夜大摇(50-200mL)，37℃摇床培养12-16h，以扩增质粒，第二天进行提取(摇菌时间摇小于16h)。
2)取50-200mL的菌液，于室温下4000xg离心10min，收集菌体。
3)弃去培养基。往沉淀中加入10mL Solution I/RNaseA混和液，通过移液枪吹打或者漩涡振荡使细胞完全重新悬浮。
4)加入10mL Solution II，盖上盖子，轻轻上下颠倒离心管8-10次以获得澄清裂解物。如有必要，可把裂解液置于室温静置2-3min。
5)加入5mL预冷的N3 Buffer，盖好盖子，并温和地上下颠倒离心管10次，直至形成白色絮狀沉淀，可在室温下静置孵育2min。
6)准备一个针筒过滤器，拉出针筒中的活塞，将针筒竖直放在一个合适的试管架上，在注射器下端出口处放置一个收集管，针筒开口朝上。立即将裂解液倒入过滤器的针筒中。细胞裂解液在针筒中停留5min。此时白色絮状物会漂浮于裂解液表面。细胞裂解液可能已从过 滤注射器口流出。用新的50mL试管收集细胞裂解液。小心轻轻地将注射器活塞插入针筒中，慢慢推动活塞以使裂解液流入到收集试管中。
7)加入0.1倍体积的ETR Solution(蓝色)至已流出的过滤裂解液中，颠倒试管10次，然后于冰浴中静置10min。
8)将上述裂解液于42℃下水浴5min。裂解液又将再次出现浑浊。此时于25℃4,000xg离心5min，ETR Solution将在试管底部形成蓝色分层。
9)将上清液移至另一新的50mL试管中，加入0.5倍体积室温的无水乙醇，轻轻颠倒试管6-7次，室温放置1-2min。
10)将一个
Figure PCTCN2020105881-appb-000004
DNA Maxi结合柱套入50mL收集试管中，加入20mL过滤液至
Figure PCTCN2020105881-appb-000005
DNA Maxi结合柱中。室温下，4,000xg离心3min。弃滤液。
11)将
Figure PCTCN2020105881-appb-000006
DNA Maxi结合柱套入同一个收集管中，重复步骤10直至剩余的过滤液全部结合到
Figure PCTCN2020105881-appb-000007
DNA Maxi结合柱中，按同样条件离心。
12)将
Figure PCTCN2020105881-appb-000008
DNA Maxi结合柱套入同一个收集管中，加入10mL HBC Buffer至
Figure PCTCN2020105881-appb-000009
DNA Maxi结合柱中，室温下4,000xg离心3min，弃滤液。
13)将
Figure PCTCN2020105881-appb-000010
DNA Maxi结合柱套入同一个收集管中，加入15mL DNA Wash Buffer(用无水乙醇稀释的)至
Figure PCTCN2020105881-appb-000011
DNA Maxi结合柱中，室温下4,000xg离心3min，弃去滤液。Note：浓缩的DNA Wash Buffer在使用之前必须按说明书用乙醇稀释。如果DNA洗涤缓冲液在使用之前是置于冰箱中的，须将其拿出置于室温下。
14)将
Figure PCTCN2020105881-appb-000012
DNA Maxi结合柱套入同一个收集管中，加入10mL DNA Wash Buffer(用无水乙醇稀释的)至
Figure PCTCN2020105881-appb-000013
DNA Maxi结合柱中，室温下4,000xg离心3min，弃去滤液。
15)最高速(不超过6000xg)空甩以干燥
Figure PCTCN2020105881-appb-000014
DNA Maxi结合柱的基质10min。
16)(选做)进一步风干
Figure PCTCN2020105881-appb-000015
DNA Maxi结合柱(可选)选择下面其中一种方法来进一步干燥
Figure PCTCN2020105881-appb-000016
DNA Maxi结合柱，再进行洗脱DNA(有必要的话)：
a)把
Figure PCTCN2020105881-appb-000017
DNA Maxi结合柱放在真空容器中15min来干燥乙醇：在室温下把柱子移到真空室，连接好所有真空室的装置。密封真空室，真空15min。移走
Figure PCTCN2020105881-appb-000018
DNA Maxi结合柱进行下一步操作。b)在真空烘箱烘干柱子或65℃干燥10-15min。移走
Figure PCTCN2020105881-appb-000019
DNA Maxi结合柱，进行下一步骤操作。
17)把
Figure PCTCN2020105881-appb-000020
DNA Maxi结合柱置于一干净的50mL离心管上，直接加入1-3mL Endo-Free Elution Buffer到
Figure PCTCN2020105881-appb-000021
DNA Maxi结合柱基质上(所加的量取决于预期终产物浓度)，室温静置5min。
18)4,000xg离心5min以洗脱出DNA。
19)弃除柱子，把DNA产物保存于-20℃。
构建的pX601-SgRNA质粒载体命名如下：
表8 质粒载体
氨基酸改变 蛋白质改变 SgRNA 质粒名称
c.50C>T p.Thr17Met RHO17-SgRNA1 pX601-R17-sg1
    RHO17-SgRNA2 pX601-R17-sg2
    RHO17-SgRNA3 pX601-R17-sg3
c.403C>T p.Arg135Trp RHO135-SgRNA1 pX601-R135-sg1
    RHO135-SgRNA2 pX601-R135-sg2
实施例3.SaCas9-sgRNA靶点效率检测试剂盒检测RHO各sgRNA体外编辑效率
具体实验步骤如下：
(1)sgRNA的体外转录：
1)SgRNA体外转录的引物设计
表9 SgRNA体外转录的引物序列
Figure PCTCN2020105881-appb-000022
2)sgRNA体外转录模板的构建
PCR反应体系如下：
表10 反应体系
试剂 体积(μL)
2×KOD-FX缓冲液 25
2mM dNTP mix 10
正向引物 1.5
反向引物 1.5
px601质粒 0.5
KOD-FX DNA聚合酶 2
无酶H 2O To 50
PCR反应程序如下：
Figure PCTCN2020105881-appb-000023
3)使用2.0％DNA凝胶跑胶，使用OMEGA胶回收试剂盒回收gRNA体外转录模板胶回收的步骤如下：
a)将PCR反应产物中加入等体积的膜结合液，切胶回收的需要每1mg加入1μL的膜结合液，50-60℃加热7min，直至所有的凝胶溶解完全，涡旋混匀，过柱回收；
b)将上述液体降入到回收柱中，10000x g离心1min，去滤液；
c)加入700μL的Washing Buffer，>13000x g离心1min，去滤液；
d)重复步骤c)；
e)空管>13000x g离心10min；
f)将离心柱转移到新的1.5mL的Ep管中，做好标记，加入20-30μL的Elution Buffer或ddH 2O，室温放置2min；
g)>13000x g离心1min，弃掉吸附柱，将DNA保存于2-8℃，测浓度并记录，长期保存需放置于-20℃。
4)sgRNA的体外转录(20μL体系)：
表11 转录体系
试剂 体积(μL)
10×转录缓冲液 2
rNTP混合液 2
T7 RNA聚合酶混合液 2
gRNA胶回收模板 1μg(≤14μL)
DEPC H 2O 至20
混合后置于37℃恒温孵箱，反应；反应结束，加入2μL Dnase I，37℃反应30min后跑胶。
5)OMEGA胶回收剂盒说明书回收gRNA，步骤同上。
(2)RHO sgRNA切割模板的制备
1)提取RHO-adRP患者来源诱导性多能干细胞iPSCs(p.Thr17Met、p.Arg135Trp)和不携带突变位点的正常iPSCs的基因组DNA，以此为模板制备RHO sgRNA切割模板dsDNA。
基因组DNA提取步骤如下：
a)400x g离心5min收集细胞，弃上清液。加入220μl PBS、10mL RNase Solution和20μL PK工作液至样品中，重悬细胞。室温静置15min以上；
b)加入250μl Buffer GB至细胞重悬液中，涡旋混匀，65℃水浴15-30min，过柱纯化；
c)加入250μl无水乙醇至消化液中，涡旋混匀15-20s；
d)将gDNA Columns吸附柱置于Collection Tubes 2ml收集管中。将上一步所得混合液(包括沉淀)转移至吸附柱中。12,000x g离心1min。若出现堵柱现象，再14,000x g离心3-5min。若混合液超过750μL需分次过柱。
e)弃滤液，将吸附柱置于收集管中。加入500μl Washing Buffer A至吸附柱中。12,000x g离心1min。
f)弃滤液，将吸附柱置于收集管中。加入650μl Washing Buffer B至吸附柱中。12,000x g离心1min。
g)重复步骤4。
h)弃滤液，将吸附柱置于收集管中。12,000×g空管离心2min。
i)将吸附柱置于新的1.5ml离心管。加入30-100μl预热至70℃的Elution Buffer至吸附柱的膜中央，室温放置3min。12,000×g离心1min。
注意：对于DNA含量丰富的组织，可再加入30-100μl Elution Buffer重复洗脱。
j)弃掉吸附柱，将DNA保存于2-8℃，测浓度并记录，长期保存需放置于-20℃。
本研究每个切割位点需要两组dsDNA，一组为包含突变位点的dsDNA，有RHO17-M-dsDNA和RHO135-M-dsDNA，PCR过程以患者iPSCs gDNA为模板；另一组为不包含突变位点的dsDNA，有RHO17-C-dsDNA和RHO135-C-dsDNA，PCR过程以正常人iPSCs gDNA为模板。
使用引物如下表12：
表12 切割模板引物序列
Figure PCTCN2020105881-appb-000024
Figure PCTCN2020105881-appb-000025
2)PCR反应体系如下：
表13 反应体系
试剂 体积(μL)
DNA 0.4
5×PrimeSTAR缓冲液 10
dNTP混合液 4
正向引物 3
反向引物 3
PrimeSTAR HS DNA聚合酶 0.5
DEPC处理水 29.1
PCR反应程序如下:
Figure PCTCN2020105881-appb-000026
3)使用1.5％DNA凝胶跑胶，使用OMEGA胶回收试剂盒回收PCR产物，步骤同前。
(3)SaCas9-SgRNA体外酶切反应：
反应体系如下：
表14 反应体系
试剂 体积(μL)
SaCas9 1
10×SaCas9缓冲液 2
gRNA(体外转录) 50ng
RHO sgRNA切割模板 50ng
DEPC H 2O 至20
充分混匀，37℃反应30min，加入3μL DNA加载缓冲液混合后65℃煮5min，跑2％的琼脂糖凝胶分析酶切结果。
RHO17-SgRNA体外效率检测结果如图4所示，RHO17-SgRNA1和SgRNA2对实验组的体外模板均有切割，SgRNA1的切割效率达50％以上，SgRNA2的切割效率接近100％；且对对照组的体外模板没有切割，证明这两条SgRNA的切割效率和特异性均好，可应用于后续的细胞实验和体内实验；而RHO17-SgRNA3对实验组和对照组模板均有切割，特异性较差，不可使用。图4中，M：实验组；C：对照组(野生型dsDNA)。
RHO135-SgRNA体外效率检测结果如图5所示，RHO135-SgRNA1对实验组的体外模板有切割，其切割效率近50％，且对对照组的体外模板没有切割，证明该sgRNA的切割效率和特异性均可，可应用于后续的细胞实验和体内实验；而RHO135-SgRNA2对实验组和对照组模板均有切割，特异性差，不可使用。图5中，M：实验组；C：对照组(野生型dsDNA)。
实施例4.使用gRNA活性荧光检测试剂盒体外检测gRNA编辑效率
(1)实验原理
该试剂盒中荧光报告质粒中的荧光报告基因mKate被终止密码子提前终止，这种截短型的mKate没有活性，为检测gRNA检测活性，可将Cas9/gRNA识别的靶位点插入到终止密码子之后，在Cas9和gRNA的作用下，靶点位置的双链DNA被切割形成DSB，细胞通过同源重组效应形成有活性的荧光蛋白，通过荧光显微镜或流式细胞仪来检测荧光蛋白活性是否增加来判断gRNA的活性及敲除效率。
(2)设计构建载体的所需引物
引物序列见表15所示。
表15 引物序列
Figure PCTCN2020105881-appb-000027
Figure PCTCN2020105881-appb-000028
(3)载体构建的实验步骤
1)引物退火
将合成的引物序列按下表中配制体系，退火之后生成含有粘性末端的DNA双链。
表16 反应体系
ddH 2O 12μl
10×缓冲液 2μl
100μM正向引物 2μl
100μM正向引物 2μl
总量 20μl
将上述配制的反应体系置于PCR仪上，运行如下反应程序：
表17 反应程序
Figure PCTCN2020105881-appb-000029
2)载体连接反应
表18 反应体系
退火产物 1μl
BG13701vector 1μl
溶液I 5μl
dd H 2O 3μl
总量 10μl
将上述配制的反应体系置于PCR仪上，程序为：16℃ 30min～1h
3)转化：取5μL连接产物加入到刚解冻的50μL DH5α感受态细胞中，轻弹混匀，冰浴30min后，42℃热激45s，立即放冰上静止2min，加950μL、37℃预热的LB液体培养基，37℃震荡培养45min，取100μL涂于氨苄青霉素抗性的平板。
4)阳性克隆鉴定：用设计的正向sgRNA引物和测序引物TS-SP001配对，进行菌落PCR，产物大小为632bp，挑选2～3个阳性菌落摇菌，提取质粒DNA进行测序，请反向互补测序结果后，再进行序列比对，测序引物为TS-SP001：CTGATAGGCAGCCTGCACCTG(SEQ ID NO:36)。测序序列如下：载体序列I(SEQ IDNO:37)-sgRNA(SEQ ID NO:1-5)-PAM(NNGRR(T)，SEQ ID NO:39)-载体序列II(SEQ IDNO:38)
5)挑选测序正确的菌液，过夜摇菌，提取质粒(步骤同上)。
(4)质粒转染并分析结果
1)复苏293T细胞至于6孔板
2)将构建好的含靶序列的质粒和含有gRNA、Cas9质粒载体，共转染目标细胞，设阴性对照组。
3)荧光显微镜检测荧光信号：转染48h后可以用流式细胞仪观察，与阴性对照组相比：若实验组检测到较强荧光信号，则表明该gRNA活性较高。若实验组检测到荧光信号减弱或检测不到荧光信号，则表明该gRNA活性较弱或者没有活性。
4)流式细胞仪检测gRNA的活性
将构建好的含靶序列的质粒和含有gRNA、Cas9质粒载体，共转染目标细胞，设阴性对照组。每组结果重复三次可进行统计学分析(双尾t检验，p<0.05)。
流式分选的步骤如下：
a.吸去六孔板里的培养基，用DPBS洗2次；
b.加入500μl的0.05％胰酶，37℃孵育消化4min；
c.加入3-5倍体积的DMEM中和胰酶，800r/min，2min离心；
d.吸去上清，加入PBS重悬细胞，800r/min，2min离心，重复一次；
e.吸去上清，加入200ul含2％的FBS的PBS重悬细胞；
f.将上一步所得液体加入过滤管，使其全部通过滤网；
g.上机。
结果如图6-10显示。
图6显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA1编辑效率。其中，A图为实验组，共转染的质粒为RHO17-mkate-mut-sgRNA1和pX601-R17-sg1，pX601-R17-sg1对RHO17-mkate-mut-sgRNA1质粒的靶位点有切割；B图为实验对照组RHO17-mkate- mut-sgRNA1和pX601空质粒，无切割；C图对照组，共转染的质粒为RHO17-mkate-wt-sgRNA1和pX601-R17-sg1，pX601-R17-sg1对RHO17-mkate-wt-sgRNA1质粒的靶位点没有切割。上述结果证明该sgRNA的切割效率和特异性均可，同上述体外切割实验的结果相同，可应用于后续的细胞实验和体内实验。
图7显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA2编辑效率。其中，A图为实验组，共转染的质粒为RHO17-mkate-mut-sgRNA2和pX601-R17-sg2，pX601-R17-sg2对RHO17-mkate-mut-sgRNA2质粒的靶位点有切割；B图为实验对照组RHO17-mkate-mut-sgRNA2和pX601空质粒，无切割；C图对照组，共转染的质粒为RHO17-mkate-wt-sgRNA2和pX601-R17-sg2，pX601-R17-sg2对RHO17-mkate-wt-sgRNA2质粒的靶位点没有切割。上述结果证明该SgRNA的切割效率和特异性均可，同上述体外切割实验的结果相同，可应用于后续的细胞实验和体内实验。
图8显示的是使用gRNA活性荧光检测试剂盒体外检测RHO17-SgRNA3编辑效率。其中，A图为实验组，共转染的质粒为RHO17-mkate-mut-sgRNA3和pX601-R17-sg3，pX601-R17-sg3对RHO17-mkate-mut-sgRNA3质粒的靶位点有切割；B图为实验对照组RHO17-mkate-mut-sgRNA3和pX601空质粒，无切割；C图对照组，共转染的质粒为RHO17-mkate-wt-sgRNA3和pX601-R17-sg3，pX601-R17-sg3对RHO17-mkate-wt-sgRNA3质粒的靶位点有切割。上述结果证明该SgRNA的切割特异性性较差，同上述体外切割实验的结果相同，不可应用于后续的细胞实验和体内实验。
图9显示的是使用gRNA活性荧光检测试剂盒体外检测RHO135-SgRNA1编辑效率。其中，A图为实验组，共转染的质粒为RHO135-mkate-mut-sgRNA1和pX601-R135-sg1，pX601-R135-sg1对RHO135-mkate-mut-sgRNA2质粒的靶位点有切割；B图为实验对照组RHO135-mkate-mut-sgRNA1和pX601空质粒，无切割；C图对照组，共转染的质粒为RHO135-mkate-wt-sgRNA1和pX601-R135-sg1，pX601-R135-sg1对RHO135-mkate-wt-sgRNA1质粒的靶位点没有切割。上述结果证明该SgRNA的切割效率和特异性均可，同上述体外切割实验的结果相同，可应用于后续的细胞实验和体内实验。
图10显示的是使用gRNA活性荧光检测试剂盒体外检测RHO135-SgRNA2编辑效率。其中，A图为实验组，共转染的质粒为RHO135-mkate-mut-sgRNA2和pX601-R135-sg2，pX601-R135-sg2对RHO135-mkate-mut-sgRNA2质粒的靶位点有切割；B图为实验对照组RHO135-mkate-mut-sgRNA2和pX601空质粒，无切割；C图对照组，共转染的质粒为RHO135-mkate-wt-sgRNA2和pX601-R135-sg2，pX601-R135-sg2对RHO135-mkate-wt-sgRNA2质粒的靶位点 有切割。上述结果证明该SgRNA的切割特异性性较差，同上述体外切割实验的结果相同，不可应用于后续的细胞实验和体内实验。
图11显示的是使用gRNA活性荧光检测试剂盒体外检测sgRNA编辑效率的流式分选结果统计。由图A可以看出，实验组(RHO17-m1)和对照组(RHO17-W1)的切割效率有统计学差异；由图B可以看出，实验组(RHO17-m2)和对照组(RHO17-W2)的切割效率有统计学差异；由图C可以看出，实验组(RHO135-m1)和对照组(RHO135-W1)的切割效率有统计学差异，可用于后续细胞和体内实验。实验组和对照组各设置三个重复组用于流式分选。
实施例5.使用293T细胞体外验证gRNA安全性和特异性
(1)293T细胞培养
293T细胞使用的培养基为添加10％胎牛血清和100U/ml青/链霉素双抗的高糖DMEM，5％CO 2于37℃培养。
1)冻存细胞的复苏
a)将恒温水浴锅温度调至37℃，将冻存细胞从液氮中取出，用镊子夹住盖子，在水中快速晃动。
b)将冻存液转移到15ml刻度离心管中，缓慢地加入10ml的细胞培养液，并轻轻的晃动混匀液体。拧紧盖子，过火，1000rpm/min，离心3分钟。
c)过火，加入适量培养液，轻轻吹打底部的细胞沉淀，然后将细胞转移至培养瓶中放到培养箱中培养。
2)细胞传代
a)倒置显微镜下观察细胞的形态和密度，当细胞在培养瓶中的汇合度到达80％-90％时，开始对细胞进行传代。
b)将细胞培养瓶中的旧培养液洗出来，用PBS清洗3次。向培养瓶中加入500μl的含EDTA的胰蛋白酶，放入培养箱中孵育1分钟左右，带细胞间隙变大，细胞变圆时，立即向培养瓶中加入1ml的培养液终止消化，并用吸管轻轻吹打细胞，待细胞全部从瓶底飘起后，将培养瓶中的液体转移到离心管中，1000rpm/min离心2分钟。
c)弃掉上清，再向离心管中加入2ml培养基使沉淀的细胞重新悬浮。将细胞悬浮液分装到4个新培养瓶中，每个加入4ml的培养液，轻摇培养瓶，使细胞混合均匀铺满培养瓶，放入细胞培养箱中进行培养。
d)转染之前1-2d，将293T细胞接种到6孔板，待细胞长到80-90％汇合度时进行质粒转染。
(2)293T细胞转染
1)我们将实施例2构建的pX601-SaCas9质粒进行编号，见下表：
表19 质粒载体
氨基酸改变 蛋白质改变 质粒名称
c.50C>T p.Thr17Met pX601-R17-sg1
    pX601-R17-sg2
    pX601-R17-sg3
c.403C>T p.Arg135Trp pX601-R135-sg1
    pX601-R135-sg2
2)质粒转染过程及步骤如下：
取1.5mL的EP管，按以上顺序进行编号，每管中加入250μL的DMEM培养基(无血清)，依次按上述表格加入1.5μg pX601-RHO-SgRNA质粒和1μg的pLenti-GFP质粒，充分涡旋混匀后，每管加入7.5μL的PEI转染试剂，手指轻弹混匀(不要涡旋混匀)，室温放置20min后进行转染。
将培养有293T的6孔板取出，镜下观察细胞汇合度，对细胞进行换液，去除废液，加入新的完全培养基1.75mL，然后对各空细胞按上述进行编号，将配好的转染体系按编号加入各孔中，过夜培养。
次日，荧光显微镜下观察GFP的表达情况，以评估转染效率，转染效率良好的情况下继续培养转染后的质粒。
转染后两日，在每个孔中(包括阴性对照组)加入适量的嘌呤霉素(注意：抗生素浓度可从0.1μg/mL逐渐增加至0.5μg/mL)，开始筛选转染阳性的细胞，以后每日观察细胞的存活情况，每2日换液，换液时加上相应量的嘌呤霉素。
待到阴性对照的孔的细胞完全死亡，而实验组和对照组的细胞有存活的(说明转染成功)，停止抗生素筛选，改用正常培养基，待6孔板细胞长到80-90％汇合度后，传代到6cm培养皿培养，待细胞长到80-90％汇合度后，收细胞，准备提取基因组DNA，整个过程在7-10d左右。
3)293T细胞基因组DNA的提取
步骤同前。
(3)gRNA效率验证
1)T7E1酶切实验
按上述的体系进行PCR，跑胶，并回收PCR产物，回收步骤同前。
将上述获得的PCR回收或切胶回收产物进行T7E1酶切反应
a)T7E1酶切退火体系(19.5μL)：
表20 反应体系
试剂 体积(μL)
NEB Buffer 2 2
PCR或切胶回收产物 X(500ng或1000ng)
去离子H 2O 至19.5
b)T7E1酶切退火程序：
95℃ 2min
95℃至85℃ 温度-2℃/s
85℃至25℃ 温度-0.1℃/s
16℃ ∞。
b)T7E1酶切反应体系
表21 反应体系
试剂 体积(μL)
退火产物 9.75或9.5
T7E1酶 0.25或0.5
37℃ 20min
d)酶切产物跑胶
配胶：2.5％凝胶，加双倍染料
跑胶程序：140V，20min到30min
e)查看跑胶结果。结果如图12所示，如图12中的A所示，使用pX601-R17-Sg1转染293T细胞后，该质粒对靶基因组没有切割，证明RHO17-SgRNA1的特异性良好；如图12中的B所示，使用pX601-R17-Sg2转染293T细胞后，该质粒对靶基因组没有切割，证明RHO17-SgRNA2的特异性良好；如图12中的C所示，使用pX601-R135-Sg1转染293T细胞后，该质粒对靶基因组没有切割，证明RHO135-SgRNA1的特异性良好。图12中，M：pX601-R17-Sg1、pX601-R17-Sg2或pX601-R135-Sg1，C：pX601。
实施例6.在患者来源的iPSCs验证gRNA的编辑效率
(1)患者肾上皮细胞的提取和培养
本申请中患者A携带RHO c.50C>T突变，患者B携带RHO c.403C>T，正常人C不患病也不携带任何基因的突变位点。
使用北京赛贝公司提供的肾上皮细胞分离和培养试剂盒进行，实验步骤如下：
1)将UrinEasy分离完全培养基、添加剂、Gelatin、洗涤液带到细胞间。冰箱解冻添加剂， 其余置于外面。
2)照上紫外灯及12孔板、50ml离心管、15ml离心管、电动移液器、移液管、吸管、5ml枪及枪头、1ml枪及枪头；打开37度水浴锅
3)取尿：戴手套、消毒、最好中段尿、并用PARAFILM封口。
4)Gelatin750μL/孔，包被皿底(3个孔)不少于半小时，置于37度。
5)75％酒精消毒尿瓶外表面，分装至50mL锥形底离心管中，封口后400xg 10min。
6)取出UrinEasy分离完全培养基+添加剂并配置(每0.5mL添加剂与5mL基础培养基混合)。
7)最小速度移液管，沿上液面，缓慢吸取上清至1ml。
8)重悬至15ml离心管中，加入10ml洗涤液，混匀，200xg 10min。
9)取出12孔板，吸去Gelatin，用洗涤液清洗一次(500μL)，每孔加入750μL UrinEasy分离完全培养基，置于37度。
10)取出15ml离心管，剩余0.2mL细胞团。
11)UrinEasy分离完全培养基重悬细胞团：男性一孔，女性两孔，记为D0。
12)观察：
D1：观察有无污染；
D2：补充分离培养基——女性：500ul/孔；男性：250ul/孔；
D4：若无贴壁：半量换液，每两天换液，缓慢沿壁加入1mL分离完全培养基。
13)直到出现贴壁：细胞出现贴壁(3～7天或9～10天)后，UrinEasy扩增完全培养基
培养两天，500μL，全量换液。贴壁后大概9～12D(不超过14D)80～90％汇合度时传代，依次传代至6孔板，6cm培养皿和10cm培养皿后冻存，备用。
(2)iPSCs的诱导
将患者来源的(p.Arg135Trp和p.Thr17Met)肾上皮细胞诱导成iPSCs，步骤如下：
1)体细胞汇合度达到70-90％即可进行消化传代，将细胞接种于96孔板中；接种密度控制在5000—15000个/孔，可根据细胞情况设置3个密度梯度，每个梯度设置3个复孔。细胞接种当天记为第-1天。
2)第0天：镜下观察细胞的汇合度以及状态，选择不同梯度的复孔进行消化计数，选择细胞量达到10000-20000个的孔进行重编程。请按下表配置重编程培养基A：
表22 培养基配方
重编程培养基A 体积
体细胞培养基 10mL
重编程添加剂Ⅰ 10μL
3)先将重编程添加剂Ⅱ离心，再将97μL重编程培养基A到加入到重编程添加剂Ⅱ管中，混匀配成重编程培养基B，将100μL重编程培养基B加入选定的符合条件的一个96孔中，将培养板放回培养箱。
4)第1-2天：镜下观察，并拍照记录细胞的形态变化。若细胞形态变化明显即可撤去重编程培养基B，换为重编程培养基A继续培养；若形态变化不明显，可不换液。
5)第3天：若细胞形态在前两天就已经发生了明显的形变，且细胞生长速度较快，可以进行胰酶消化传代。根据细胞状态和细胞量将细胞传至六孔板的2-6个孔，加入重编程培养基C，尽量形成单细胞贴壁。请按下表配置重编程培养基C：
表23 培养基配方
重编程培养基C 体积
重编程培养基A 9.8mL(上述剩余)
重编程添加剂Ⅲ 5μL
6)第4天：观察细胞的贴壁情况，若大部分细胞贴壁良好，则更换新鲜的体细胞培养基继续培养。
7)第5天：镜下观察，若有小簇克隆(4个细胞以上的克隆团块)形成，可将体细胞培养基换为Reproeasy人体细胞重编程培养基。若暂无小簇克隆形成，可继续观察一到两天，再更换Reproeasy人体细胞重编程培养基。
8)第6-8天：镜下观察，若小簇克隆变大，一个克隆团块有10个以上的细胞，可直接将Reproeasy人体细胞重编程培养基换为PSCeasy人多潜能干细胞培养基(或PGM1人多潜能干细胞培养基)。若换液前观察到死细胞较多，可用室温平衡后的PBS清洗后，再进行换液。
9)第9-20天：镜下观察，并拍照记录细胞形态变化。每天更换经过室温平衡的新鲜PSCeasy人多潜能干细胞培养基。
10)第21天：镜下观察，若单个细胞克隆能填满整个10倍镜视野，可用1mL注射器针头(或其他器具如玻璃针)切割克隆，并将其挑取至提前包被Matrigel的48孔板中(若克隆状态良好，细胞厚实且生长较快，可直接挑取到24孔板中)。
11)克隆挑出后用PSCeasy人多潜能干细胞复苏培养基接种，细胞贴壁后可以更换为PSCeasy人多潜能干细胞培养基继续培养至所需的代数。
(3)iPSCs电转
1)将pX601质粒进行改造，使之带有嘌呤霉素抗性，以备电转后筛选使用，改造后的质 粒命名为pX601-R17-puro-sg1、pX601-R17-puro-sg2及pX601-R135-puro-sg1,空白对照质粒为pX601-GFP-puro质粒。
2)iPSCs电转步骤如下：
a)复苏冻存的iPSCs至6孔板，待细胞长至60-80％汇合度时进行电转。
b)弃去培养基，加1mLDPBS清洗细胞，弃去废液，重复两次。
c)加入1mL 0.05％的EDTA消化液至iPSCs，37℃静置消化3min；
d)取出6孔板，光镜下确认细胞消化情况，加入完全培养基中和消化液，轻轻敲打培养皿壁，并使用枪头轻轻吹打细胞，收集细胞悬液至15mL离心管中，计数，室温，200xg离心5min。
e)尽可能弃去上清，用配置好的100μL电转液(82μL电转基础液+18μL电转添加剂+5μg质粒)重悬细胞，所有电转细胞数为1*10 6个。轻轻吹打混匀后用吸管吸至电极杯杯底部，确保杯底被完全覆盖且无气泡产生，将电极杯置于Lonza电转仪内进行电转，电转程序为CA-137。
f)电转完成后，将iPSCs重新接种至6孔板中培养。
g)转染后24h后观察细胞存活情况，转染后的第二天开始使用嘌呤霉素进行筛选。
h)后期的细胞培养、筛选以及见实施例5。
(4)gRNA编辑效率的验证
1)提取iPSCs的gDNA
方法同前。
2)PCR反应
所用引物如下表24，方法同前。
表24 引物序列
Figure PCTCN2020105881-appb-000030
3)T7E1酶切实验
步骤同前。
患者A的实验结果如图13所示，左图的Sanger测序结果显示患者A携带RHO c.50C>T的杂合突变，而正常人不携带；右图显示RHO17-SgRNA1和RHO-SgRNA2在患者A和正常 人C的iPSCs中的基因编辑效果，A-GFP表示患者A的iPSCs仅转染了pX601-GFP-puro空白对照质粒；A-17sgRNA1或A-17sgRNA2表示患者A的iPSCs转染了pX601-R17-puro-sg1或pX601-R17-puro-sg2质粒；C-17sgRNA1或C-17sgRNA2表示正常人的iPSCs转染了pX601-R17-puro-sg1或pX601-R17-puro-sg2质粒，T7E1的结果显示，RHO17-SgRNA1和RHO17-SgRNA2仅对患者A的iPSCs的RHO基因突变位点有编辑效果，而对正常人的相应基因位置无编辑效果。和以上体外实验的结果一致。
患者B的实验结果如图14所示，左图的Sanger测序结果显示患者B携带RHO c.403C>T的杂合突变，而正常人不携带；右图显示RHO135-SgRNA1在患者B和正常人C的iPSCs中基因编辑效果，B-GFP表示患者B的iPSCs仅转染了pX601-GFP-puro空白对照质粒；B-135sgRNA1表示患者B的iPSCs转染了pX601-R135-puro-sg1质粒；C-135sgRNA1表示正常人的iPSCs转染了pX601-R135-puro-sg1质粒，T7E1的结果显示，RHO135-SgRNA1仅对患者B的iPSCs的RHO基因突变位点有编辑效果，而对正常人的相应基因位置无编辑效果。和以上体外实验的结果一致。
实施例7.使用3D视网膜组织体外验证gRNA的基因编辑效率
(1)将患者A、B及正常人C来源的iPSCs诱导成为3D Retina组织。
具体步骤见下表25：
表25 实验步骤
Figure PCTCN2020105881-appb-000031
Figure PCTCN2020105881-appb-000032
(2)AAV8病毒的构建和包被
RHO17-SgRNA1和RHO17-SgRNA2编辑同一位点，鉴于体外实验和干细胞实验的结果，后者的切割效果强于前者，3D视网膜实验和小鼠实验均采用RHO17-SgRNA2进行实验。
将上述pX601质粒进行改造，使之带有GFP荧光蛋白，以备后续筛选GFP+细胞，改造后的质粒命名为pX601-R17-GFP-sg2及pX601-R135-GFP-sg1,空白对照质粒为pX601-GFP质粒。
1)质粒扩增。构建好的AAV载体、包装质粒和辅助质粒需经过大量去内毒素抽提，使用Qiagen大抽试剂盒进行质粒的大量抽提，步骤同前。
2)AAV8-293T细胞转染。转染当天观察细胞密度，80-90％满即可将载体质粒、包装质粒和辅助质粒进行转染。
3)AAV8病毒收毒：病毒颗粒同时存在于包装细胞和培养上清中。可以将细胞和培养上清都收集下来以获得最好的收率。
4)AAV的纯化、-80℃长期保存。
(3)3D视网膜组织体外验证gRNA基因编辑效率
1)使用AAV8感染诱导成功的3D Retina组织。感染后3天荧光显微镜下观察GFP的表达情况，收集GFP+的3D视网膜组织，使用木瓜蛋白酶体系消化3D组织，制成单细胞悬液，通过流式细胞仪器筛选并GFP+阳性的视网膜细胞，提取gDNA，方法同前。
2)PCR反应
所用引物如下表26，方法同前。
表26 引物序列
Figure PCTCN2020105881-appb-000033
3)T7E1酶切实验
方法同前。
结果如图15所示。图15A显示的是RHO-SgRNA2在患者A和正常人C的3D视网膜组织的基因编辑效果，A-GFP表示患者A的3D视网膜仅感染了pX601-GFP空白对照病毒；A-17sgRNA2表示患者A的3D视网膜感染了pX601-R17-GFP-sg2病毒；C-17sgRNA2表示正常人的3D视网膜转染了pX601-R17-GFP-sg2病毒，T7E1的结果显示，RHO-17SgRNA2仅对患者A的3D视网膜的RHO基因突变位点有编辑效果，而对正常人的相应基因位置无编辑效果；图15B显示RHO135-SgRNA1在患者B和正常人C的3D视网膜组织的基因编辑效果，B-GFP表示患者B的3D视网膜仅感染了pX601-GFP空白对照质粒；B-135sgRNA1表示患者B的iPSCs感染了pX601-R135-GFP-sg1病毒；C-135sgRNA1表示正常人的3D视网膜感染了pX601-R135-GFP-sg1病毒，T7E1的结果显示，RHO135-SgRNA1仅对患者B的3D视网膜的RHO基因突变位点有编辑效果，而对正常人的相应基因位置无编辑效果。和以上体外实验的结果一致。
实施例8.在人源化小鼠模型上验证基因编辑效率
(1)人源化小鼠的构建
人源化小鼠的构建工作由北京百奥塞图公司完成。
1)人源化小鼠的制作方案
针对人的突变位点，我们委托北京百奥赛图基因生物技术有限公司构建了RHO人源化的小鼠模型。该人源化小鼠模型有两种，一种为携带RHO基因突变位点(p.Arg135Trp或p.Thr17Met)的人源化小鼠模型，一种为只敲入人源化片段不带突变位点的小鼠模型。
2)步骤如下：
a)开发RHO基因人源化携带突变(p.Thr17Met或p.Arg135Trp或)点突变小鼠：
①设计构建识别靶序列的gRNA；按照双方共同决定的方案1进行试验；即Targeting strategy-1-EGE-System；
②构建至靶基因切割的CRISPR/Cas9载体；
③sgRNA/Cas9的活性检测；
④设计构建基因敲进的打靶载体；按照双方共同商定的方案1的内容来执行，coding region在基因组水平上的替换(4.9kb替换4.7kb)，同时引入突变位点，即c.50C>T,p.Thr17Met或c.403C>T,p.Arg135Trp；
⑤体外转录sgRNA/Cas9 mRNA；
⑥小鼠受精卵注射sgRNA/Cas9 mRNA和打靶载体；
⑦RHO基因敲进F0代小鼠检测及扩繁；
⑧RHO基因敲进F1代杂合子小鼠的获得及基因型鉴定
b)开发RHO基因人源化敲进小鼠：
①设计构建识别靶序列的sgRNA；按照双方共同商议的方案1进行，即实验方案中的Targeting strategy-1-EGE-System；
②构建致靶基因切割的CRISPR/Cas9载体；
③sgRNA/Cas9的活性检测；
④设计构建基因敲进的打靶载体；按照方案1内容执行，coding region在基因组水平上的替换(4.9kb替换4.7kb)；
⑤体外转录sgRNA/Cas9 mRNA；
⑥小鼠受精卵注射sgRNA/Cas9 mRNA和打靶载体；
⑦RHO基因敲进F0代小鼠检测及扩繁；
⑧RHO基因敲进F1代杂合子小鼠的获得及基因型鉴定。
(2)饲养和繁育
1)获得两种人源化小鼠之后，让F1代杂合的小鼠进行内交，尽快获得足够数量的F2代或F3代人源化纯合的小鼠，用作AAV病毒注射，以评估上述获得的AAV8-pX601-RHO-SgRNA在体内的编辑效率。
(3)人源化小鼠基因型鉴定
引物对WT-F/WT-R设计在野生基因序列中，这对引物进行PCR时，不能扩增出突变型等位基因的产物，只能扩增出野生型等位基因的产物，而引物/Mut-R设计在小鼠人源化的RHO基因序列中，使用引物对WT-F/Mut-R进行PCR时，不能扩增出野生型等位基因的产物，只能扩增出突变型等位基因的产物。
引物序列如下表27：
表27 引物序列
Figure PCTCN2020105881-appb-000034
WT-F/WT-R这对引物主要用于鉴定野生型等位基因的存在，并结合WT-F/Mut-R这对引物的PCR结果判断动物的具体基因型：纯合/杂合/野生型。
基因型判定的标准如下表28：
表28 基因型判定标准
Figure PCTCN2020105881-appb-000035
说明：Y：凝胶电泳检测到预期长度的PCR产物；N：凝胶电泳未检测到预期长度的PCR产物；H/H：纯合子基因型；H/+：杂合子基因型；+/+：野生型。
PCR反应体系及程序见“实施例3”，PCR产物送测序，检测人源化小鼠RHO基因是否包括期望敲入的突变位点。
(4)小鼠视网膜下腔注射AAV8病毒
小鼠注射的AAV病毒即为3D视网膜组织感染所用病毒。
1)注射前30分钟用1％阿托品散瞳；麻醉前再次散瞳。
2)按80mg/kg氯胺酮+8mg/kg甲苯噻嗪安腹腔内注射麻醉后将小鼠放置在眼外科手术显微镜的动物实验平台前方，在小鼠眼睛上滴一滴0.5％的丙美卡因局麻。以100：1的浓度在AAV病毒里加入荧光素钠原液，低速离心混匀。
3)用胰岛素针在小鼠眼睛睫状体平坦部预扎一个小孔，用微量注射器的针头穿过该小孔后进入小鼠眼睛玻璃体腔，这时在小鼠眼睛上滴加适量2％羟甲基纤维素使在镜下能清晰见到小鼠眼底，再继续将针头避开玻璃体插入对侧周边的视网膜下，缓慢推入带有荧光素钠的AAV病毒，每只眼睛注射量为1ul,以荧光素钠为指示剂判断是否注射入视网膜下腔。
4)术后观察小鼠有无异常，给予新霉素眼膏预防感染。
(5)评估AAV8-pX601-RHO-SgRNA在人源化小鼠中的编辑效率
1)手术后3个月，使用小鼠活体成像系统观察小鼠眼球中GFP的表达，取GFP+小鼠眼球，使用木瓜蛋白酶体系消化3D组织，制成单细胞悬液，通过流式细胞仪器筛选并GFP+阳性的视网膜细胞，提取gDNA，方法同前。
2)PCR反应
所用引物如下表29，方法同前。
表29 引物序列
Figure PCTCN2020105881-appb-000036
3)T7E1酶切实验
方法同前。
图16A显示的是RHO人源化小鼠基因型鉴定的PCR跑胶结果，编号20的小鼠为携带杂合突变的人源化小鼠，编号21和22的小鼠为携带WT的人源化小鼠，编号23的小鼠为携带纯合突变的人源化小鼠；图16B显示携带RHO c.50C>T纯合突变的人源化小鼠，图16C显示携带RHO c.403C>T纯合突变的人源化小鼠。
图17A显示的是RHO17-SgRNA2对人源化小鼠的基因编辑效率，M-17sgRNA2表示，携带c.50C>T纯合突变的人源化小鼠注射AAV-R17-sg2-SaCas9病毒，而C-17sgRNA2表示携带WT的人源化小鼠注射AAV-R17-sg2-SaCas9病毒，T7E1结果显示RHO-17SgRNA2仅对携带突变的人源化小鼠有编辑效果，而对携带WT的人源化小鼠的相应位点无编辑效果；图17B显示的是RHO135-SgRNA1对人源化小鼠的基因编辑效率，M-135sgRNA1表示，携带c.403C>T纯合突变的人源化小鼠注射AAV-R135-sg1-SaCas9病毒，而C-135sgRNA1表示携带WT的人源化小鼠注射AAV-R135-sg1-SaCas9病毒，T7E1结果显示RHO-135SgRNA1仅对携带突变的人源化小鼠有编辑效果，而对携带WT的人源化小鼠的相应位点无编辑效果。体内实验的结果同上述体外实验的结果一致。

Claims (53)

  1. 治疗视网膜色素变性的方法，其包括以下步骤：使有需要的受试者具有功能性的RHO基因，其中所述功能性的RHO基因不包含选自下组的突变位点：c.C50T和c.C403T。
  2. 根据权利要求1所述的方法，其包括以下步骤：去除有需要的受试者中RHO基因的所述突变位点。
  3. 根据权利要求2所述的方法，其中所述去除包括敲除所述突变位点和/或降低包含所述突变位点的RHO基因的表达水平。
  4. 根据权利要求2-3中任一项所述的方法，其中所述去除包括不影响所述受试者中野生型RHO基因的表达水平和/或功能。
  5. 根据权利要求2-4中任一项所述的方法，其中所述去除包括使包含所述突变的RHO等位基因发生双链断裂。
  6. 根据权利要求2-5中任一项所述的方法，其中所述去除包括向有需要的受试者施用至少一个能够去除所述突变位点的载体。
  7. 根据权利要求6所述的方法，其中所述载体包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
  8. 根据权利要求7所述的方法，其中所述gRNA与包含所述突变位点的RHO等位基因中的至少部分核酸特异性结合。
  9. 根据权利要求7-8中任一项所述的方法，其中所述gRNA与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补。
  10. 根据权利要求7-9中任一项所述的方法，其中所述gRNA包含SEQ ID NO.44-45中任一项所示的核苷酸序列。
  11. 根据权利要求7-10中任一项所述的方法，其中所述编码gRNA的序列包含SEQ ID NO.1-2中任一项所示的核苷酸序列。
  12. 根据权利要求7-8中任一项所述的方法，其中所述gRNA与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补。
  13. 根据权利要求12所述的方法，其中所述gRNA包含SEQ ID NO.47所示的核苷酸序列。
  14. 根据权利要求12-13中任一项所述的方法，其中所述编码gRNA的序列包含SEQ ID NO.4所示的核苷酸序列。
  15. 根据权利要求6-14中任一项所述的方法，其中所述载体包括编码Cas蛋白的核酸。
  16. 根据权利要求15所述的方法，其中所述Cas蛋白包括Cas9蛋白。
  17. 根据权利要求15-16中任一项所述的方法，其中所述编码gRNA的序列与所述编码Cas蛋白的核酸位于同一个所述载体中。
  18. 根据权利要求6-17中任一项所述的方法，其中所述载体包括病毒载体。
  19. 根据权利要求6-18中任一项所述的方法，其中所述载体为腺相关病毒载体(AAV)。
  20. 根据权利要求6-19中任一项所述的方法，其中所述载体为AAV8。
  21. 根据权利要求1-20中任一项所述的方法，其中所述受试者包括东亚人。
  22. 根据权利要求1-21中任一项所述的方法，其中所述方法在包括体外、体内或离体的条件下进行。
  23. 根据权利要求6-22中任一项所述的方法，其中所述施用包括注射。
  24. 根据权利要求6-23中任一项所述的方法，其中所述施用包括视网膜下腔注射。
  25. 编辑RHO基因的方法，其包括以下步骤：特异性去除所述RHO基因中选自下组的突变位点：c.C50T和c.C403T。
  26. 根据权利要求25所述的方法，其中所述去除包括敲除所述突变位点和/或降低包含所述突变位点的RHO基因的表达水平。
  27. 根据权利要求25-26中任一项所述的方法，其中所述去除包括不影响所述受试者中野生型RHO基因的表达水平和/或功能。
  28. 根据权利要求25-27中任一项所述的方法，其中所述去除包括使包含所述突变的RHO等位基因发生双链断裂。
  29. 根据权利要求25-28中任一项所述的方法，其中所述去除包括施用至少一个能够去除所述突变位点的载体。
  30. 根据权利要求29所述的方法，其中所述载体包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
  31. 根据权利要求30所述的方法，其中所述gRNA与包含所述突变位点的RHO等位基因中的至少部分核酸特异性结合。
  32. 根据权利要求30-31中任一项所述的方法，其中所述gRNA与包含c.C50T突变的RHO等位基因的外显子1的至少部分核酸序列特异性互补。
  33. 根据权利要求30-32中任一项所述的方法，其中所述gRNA包含SEQ ID NO.44-45中任一项所示的核苷酸序列。
  34. 根据权利要求30-33中任一项所述的方法，其中所述编码gRNA的序列包含 SEQ ID NO.1-2中任一项所示的核苷酸序列。
  35. 根据权利要求30-31中任一项所述的方法，其中所述gRNA与包含c.C403T突变的RHO等位基因的外显子2的至少部分核酸序列特异性互补。
  36. 根据权利要求35所述的方法，其中所述gRNA包含SEQ ID NO.47所示的核苷酸序列
  37. 根据权利要求35-36中任一项所述的方法，其中所述编码gRNA的序列包含SEQ ID NO.4所示的核苷酸序列。
  38. 根据权利要求29-37中任一项所述的方法，其中所述载体包括编码Cas蛋白的核酸。
  39. 根据权利要求38所述的方法，其中所述Cas蛋白包括Cas9蛋白。
  40. 根据权利要求38-39中任一项所述的方法，其中所述编码gRNA的序列与所述编码Cas蛋白的核酸位于同一个所述载体中。
  41. 根据权利要求29-40中任一项所述的方法，其中所述载体包括病毒载体。
  42. 根据权利要求29-41中任一项所述的方法，其中所述载体为腺相关病毒载体(AAV)。
  43. 根据权利要求29-42中任一项所述的方法，其中所述载体为AAV8。
  44. 用于治疗受试者视网膜色素变性的组合物，其包括去除RHO基因的突变位点的活性成分和药学上的可接受载体，其中所述突变位点选自下组：c.C50T和c.C403T。
  45. 根据权利要求44所述的组合物，其中所述活性成分包括编码gRNA的序列，所述gRNA与所述突变位点特异性结合。
  46. 根据权利要求45所述的组合物，其中所述gRNA包含SEQ ID NO.44、45和47中任一项所示的核苷酸序列。
  47. 根据权利要求44-45中任一项所述的组合物，其中所述编码gRNA的序列包含SEQ ID NO.1、2和4中任一项所示的核苷酸序列。
  48. 根据权利要求46-47中任一项所述的组合物，其中所述活性成分包括Cas蛋白。
  49. 根据权利要求48所述的组合物，其中所述Cas蛋白包括Cas9蛋白。
  50. 根据权利要求48-49中任一项所述的组合物，其中所述编码gRNA的序列与编码所述Cas蛋白的核酸位于同一个载体中。
  51. 根据权利要求44-50中任一项所述的组合物，其中所述载体包括病毒载体。
  52. 根据权利要求44-51中任一项所述的组合物，其中所述载体为腺相关病毒载体(AAV)。
  53. 根据权利要求44-52中任一项所述的组合物，其中所述载体为AAV8。
PCT/CN2020/105881 2020-04-21 2020-07-30 RHO-adRP基于基因编辑的方法和组合物 WO2021212686A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202080001820.6A CN113038971B (zh) 2020-04-21 2020-07-30 RHO-adRP基于基因编辑的方法和组合物
US17/996,786 US20230149439A1 (en) 2020-04-21 2020-07-30 Rho-adrp gene editing-based methods and compositions

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010318429 2020-04-21
CN202010318429.0 2020-04-21

Publications (1)

Publication Number Publication Date
WO2021212686A1 true WO2021212686A1 (zh) 2021-10-28

Family

ID=78271083

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/105881 WO2021212686A1 (zh) 2020-04-21 2020-07-30 RHO-adRP基于基因编辑的方法和组合物

Country Status (1)

Country Link
WO (1) WO2021212686A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023173120A1 (en) * 2022-03-11 2023-09-14 Epicrispr Biotechnologies, Inc. Systems and methods for genetic modulation to treat ocular diseases

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044649A1 (en) * 2015-09-08 2017-03-16 Precision Biosciences, Inc. Treatment of retinitis pigmentosa using engineered meganucleases
CN109890424A (zh) * 2016-07-05 2019-06-14 约翰霍普金斯大学 用于治疗视网膜变性的基于crispr/cas9的组合物和方法
CN110241202A (zh) * 2019-06-25 2019-09-17 复旦大学附属眼耳鼻喉科医院 视网膜色素变性突变位点及其应用
WO2019183630A2 (en) * 2018-03-23 2019-09-26 The Trustees Of Columbia University In The City Of New York Gene editing for autosomal dominant diseases
WO2019232517A1 (en) * 2018-06-01 2019-12-05 University Of Florida Research Foundation, Incorporated Compositions and methods for treatment of dominant retinitis pigmentosa

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044649A1 (en) * 2015-09-08 2017-03-16 Precision Biosciences, Inc. Treatment of retinitis pigmentosa using engineered meganucleases
CN109890424A (zh) * 2016-07-05 2019-06-14 约翰霍普金斯大学 用于治疗视网膜变性的基于crispr/cas9的组合物和方法
WO2019183630A2 (en) * 2018-03-23 2019-09-26 The Trustees Of Columbia University In The City Of New York Gene editing for autosomal dominant diseases
WO2019232517A1 (en) * 2018-06-01 2019-12-05 University Of Florida Research Foundation, Incorporated Compositions and methods for treatment of dominant retinitis pigmentosa
CN110241202A (zh) * 2019-06-25 2019-09-17 复旦大学附属眼耳鼻喉科医院 视网膜色素变性突变位点及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI SHUXIAN, LIU TIECHENG CHEN XIAOFEI ACTING FOR AI AI GAO XUHUI LI RUNPU: "Advances in Gene Therapy for Retinitis Pigmentosa", JIEFANGJUN YIXUEYUAN XUEBAO - JOURNAL OF CHINESE PLA POSTGRADUATE MEDICAL SCHOOL, JIEFANGJUN ZONGYIYUAN, CN, vol. 38, no. 1, 31 January 2017 (2017-01-31), CN , XP055860605, ISSN: 2095-5227, DOI: 10.3969/j.issn.2095-5227.2017.01.022 *
SULLIVAN LORI S., BOWNE SARA J., BIRCH DAVID G., HUGHBANKS-WHEATON DIANNA, HECKENLIVELY JOHN R., LEWIS RICHARD ALAN, GARCIA CHARLE: "Prevalence of Disease-Causing Mutations in Families with Autosomal Dominant Retinitis Pigmentosa: A Screen of Known Genes in 200 Families", INVESTIGATIVE OPTHALMOLOGY & VISUAL SCIENCE, ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY, US, vol. 47, no. 7, 1 July 2006 (2006-07-01), US , pages 3052, XP055860603, ISSN: 1552-5783, DOI: 10.1167/iovs.05-1443 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023173120A1 (en) * 2022-03-11 2023-09-14 Epicrispr Biotechnologies, Inc. Systems and methods for genetic modulation to treat ocular diseases

Similar Documents

Publication Publication Date Title
JP7197363B2 (ja) ヌクレアーゼを使用するヒト神経幹細胞のゲノム編集
JP7418957B2 (ja) タイチン系ミオパチー及び他のタイチノパチーの治療のための材料及び方法
Burnight et al. CRISPR-Cas9 genome engineering: treating inherited retinal degeneration
JP2022008560A (ja) 無カプシドａａｖベクター、組成物ならびにベクター製造および遺伝子運搬のための方法
JP2021503945A (ja) 常染色体優性網膜色素変性の処置のための材料および方法
EP3516058A1 (en) Compositions and methods for gene editing
US20230067480A1 (en) Method for treating usher syndrome and composition thereof
AU2017290614C1 (en) Materials and methods for treatment of friedreich ataxia and other related disorders
WO2021212686A1 (zh) RHO-adRP基于基因编辑的方法和组合物
US20230149439A1 (en) Rho-adrp gene editing-based methods and compositions
WO2020000641A1 (zh) 编码人nadh脱氢酶亚单位蛋白的核酸及其应用
WO2022021149A1 (zh) Aav介导的rpgr x连锁视网膜变性的基因编辑治疗
CN111926044B (zh) 结合突变rho基因的核酸分子和试剂盒
CN113201499A (zh) Hbb基因cd17突变细胞及其制备方法与应用
CN113015804B (zh) 用于治疗结晶样视网膜变性的核酸分子及其用途
WO2024088175A1 (zh) 基因编辑系统及其应用
CN113166763B (zh) 靶向cyp4v2基因突变位点的核酸分子及其用途
Fang et al. Gene editing in regenerative medicine
WO2024098383A1 (zh) 蛋白突变体及其治疗与hbb基因突变相关疾病的应用
WO2020187272A1 (zh) 一种用于基因治疗的融合蛋白及其应用
CN116334141A (zh) 基于基因编辑的RHO-R135W-adRP基因编辑药物
Jaskula-Ranga 23 Genome Editing for Retinal Diseases
Naessens Recurrent coding and rare non-coding targets for treatment in inherited retinal diseases
KR20240029030A (ko) 미오신 중쇄 염기 편집을 위한 조성물 및 방법
CN115427568A (zh) Rp1相关视网膜变性的基于单倍型的治疗

Legal Events

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

Ref document number: 20932735

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20932735

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20932735

Country of ref document: EP

Kind code of ref document: A1