WO2022021149A1 - Aav介导的rpgr x连锁视网膜变性的基因编辑治疗 - Google Patents

Aav介导的rpgr x连锁视网膜变性的基因编辑治疗 Download PDF

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WO2022021149A1
WO2022021149A1 PCT/CN2020/105553 CN2020105553W WO2022021149A1 WO 2022021149 A1 WO2022021149 A1 WO 2022021149A1 CN 2020105553 W CN2020105553 W CN 2020105553W WO 2022021149 A1 WO2022021149 A1 WO 2022021149A1
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nucleic acid
grna
rpgr
acid molecule
seq
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PCT/CN2020/105553
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English (en)
French (fr)
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杜娟
杨丽萍
张宏权
乔静
张天赋
张凡
和赛超
曾露颖
裴红杰
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北京中因科技有限公司
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Priority to CN202080001823.XA priority Critical patent/CN114364440B/zh
Priority to PCT/CN2020/105553 priority patent/WO2022021149A1/zh
Publication of WO2022021149A1 publication Critical patent/WO2022021149A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/86—Viral vectors

Definitions

  • the present application relates to the field of biomedicine, in particular to a gRNA and a targeting vector for gene editing to treat RPGR X-linked retinal degeneration.
  • Retinitis pigmentosa is a hereditary blinding eye disease characterized by progressive and selective loss of retinal photoreceptor cells and retinal pigment epithelial cells. It is one of the main causes of irreversible binocular blindness in children and working age people. , there is currently no effective treatment.
  • RP is usually caused by genetic mutations, and gene-based therapies include gene replacement therapy and gene editing therapy.
  • gene-based therapies include gene replacement therapy and gene editing therapy.
  • due to the specificity of the location of the mutated gene causing RP and the difficulty in regulating the expression of exogenous genes safer and more effective treatment methods are urgently needed.
  • the present application provides a gRNA that specifically targets the gene (RPGR gene) of retinitis pigmentosa GTPase regulator, which specifically binds to intron No. 14 of the RPGR gene, and the gRNA is directed to the RPGR gene.
  • the 14th intron has good cleavage efficiency.
  • the application provides nucleic acid molecules comprising a codon-optimized human RPGR ORF15 nucleotide sequence. Compared with the wild-type RPGR ORF15, the codon-optimized human RPGR ORF15 nucleotide sequence is stable in sequence, well sequenced, many clones with correct sequences are obtained, and the cloned sequence is complete and not easily lost.
  • the application provides targeting vectors comprising the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence.
  • the targeting vector described in the present application can improve the accuracy of introducing the nucleic acid molecule into the genome of a subject.
  • the isolated nucleic acid molecule (or the plasmid) encoding the gRNA of the present application and the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence (or the targeting vector) are capable of expressing RPGR mutant cells Correct retinitis pigmentosa GTPase regulator with good gene editing repair efficiency.
  • the present application provides a gRNA that specifically targets the GTPase regulator of retinitis pigmentosa (RPGR) gene, which specifically binds to intron No. 14 of the RPGR gene.
  • RPGR retinitis pigmentosa
  • the gRNA specifically binds to the nucleotide sequence shown in SEQ ID NO: 102, or specifically binds to the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 102 .
  • the gRNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs. 105-126.
  • the gRNA comprises the nucleotide sequence-backbone sequence-3' shown in any one of 5'-(X)n-SEQ ID NO. 105-126, wherein X is selected from A , U, C and G, and n is any integer from 0-15.
  • the gRNA is a single-stranded guide RNA (sgRNA).
  • the application provides one or more isolated nucleic acid molecules encoding the described gRNAs that specifically target the RPGR gene.
  • the application provides plasmids comprising the isolated nucleic acid molecules.
  • the plasmid is a viral vector.
  • the plasmid includes a nucleic acid molecule encoding a DNA endonuclease.
  • the DNA endonuclease comprises a Cas nuclease.
  • the DNA endonucleases include Cas9 nucleases, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.
  • the application provides nucleic acid molecules comprising a codon-optimized human RPGR ORF15 nucleotide sequence.
  • the application provides that the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:44.
  • the application provides targeting vectors comprising the nucleic acid molecules.
  • the targeting vector comprises 1) a 5' upstream target region; 2) the nucleic acid molecule; and 3) a 3' downstream target region; wherein the 5' upstream target region and/or The 3' downstream target region can be recognized and/or cleaved by the gRNA.
  • the 5' upstream target region and/or the 3' downstream target region in the targeting vector comprises a nucleotide mutation that enhances the introduction of the nucleic acid molecule The accuracy of the subject's genome.
  • the 5' upstream target region in the targeting vector comprises the nucleotide sequence shown in any one of SEQ ID NOs: 96-99.
  • the 3' downstream target region in the targeting vector comprises the nucleotide sequence shown in SEQ ID NO: 100.
  • the application provides cells comprising the nucleic acid molecule.
  • the cells comprise HEK cells and/or urinary renal epithelial cells.
  • the cells are modified to have the ability to differentiate.
  • the cells can be differentiated into 3D-retinal organoids.
  • the application provides tissue models comprising 3D-retinal organoids comprising the correct human RPGR cDNA.
  • the present application provides the use of the cells, and/or the tissue models, in evaluating the efficacy and/or safety of gene editing treatments.
  • the present application provides the gRNA, the plasmid, the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence, and/or the targeting vector used in the preparation of the treatment of diseases.
  • the disease includes a disease caused by a mutation in the RPGR gene.
  • the disease comprises retinitis pigmentosa.
  • the disease comprises X-linked inherited retinitis pigmentosa.
  • the present application provides a method for modifying the RPGR gene, the method comprising the steps of: introducing the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence.
  • the application provides a method for treating retinitis pigmentosa, the method comprising the steps of: introducing into a subject in need a nucleic acid molecule comprising a codon-optimized human RPGR ORF15 nucleotide sequence .
  • the retinitis pigmentosa comprises X-linked inherited retinitis pigmentosa.
  • the introduction results in a normal functioning human RPGR protein.
  • the introducing comprises introducing the targeting vector.
  • the introducing comprises introducing the gRNA, and/or the plasmid.
  • the introducing comprises injection.
  • the introduction comprises subretinal injection, or intravitreal injection.
  • Fig. 1 shows the electrophoretic diagram of the shearing result of the gRNA described in this application.
  • Figure 2A shows that gene editing using the four vectors R145, R156, R176 and R193 can amplify a 3kb positive band
  • Figure 2B shows that the four vectors R145, R156, R176 and R193 are used for gene editing Editing can amplify the positive band of the 5'outer
  • Figure 2C shows that gene editing using the four vectors R145, R156, R176 and R193 can amplify the positive band of the 3'outer.
  • Figure 3A shows the analysis of the sequencing results using R145
  • Figure 3B shows the analysis of the sequencing results using R156
  • Figure 3C shows the analysis of the sequencing results using R176
  • Figure 3D shows the analysis of the sequencing results using R193.
  • Figure 4 shows the percentage of GFP-positive cells in HEK293A cells transfected with the four vectors R145, R156, R176 and R193.
  • Figure 5 shows the cytofluorescence of HEK293A cells transfected with the four vectors R145, R156, R176 and R193.
  • Figure 6 shows the fluorescence images after GFP sorting in HEK293A cells transfected with the four vectors R145, R156, R176 and R193.
  • Figure 7 shows the PCR identification results of different transcripts for C primer pair, C1 primer pair, 45F and 45F1 primer pair, 56F and 56F1 primer pair, O15F and O15F1 primer pair.
  • Figure 8 shows the identification of the wild-type ORF15 sequence and the codon-optimized ORF15 sequence by the C primer pair and the C1 primer pair.
  • Figure 9 shows the identification of the wild-type ORF15 sequence and the codon-optimized ORF15 sequence by the 5R primer pair and the 14-ORF15 primer pair.
  • Figure 10 shows the analysis of the sequencing results of three clones HR14WM1 (A), HR14WM2 (B) and HR14WM3 (C) of the vector containing the wild-type RPGR ORF15.
  • Figure 11 shows a fragmented sequencing alignment analysis of vector clone HR15WM2 containing wild-type RPGR ORF15.
  • Figure 12 shows a fragmented sequencing alignment analysis of vector clones HR17WM1 (A) and HR17WM3 (B) containing wild-type RPGR ORF15.
  • Figure 13 shows a fragmented sequencing alignment analysis of vector clone HR19WM2 containing wild-type RPGR ORF15.
  • Figure 14 shows the map of the AAV-saCas9-puro vector.
  • Figure 15 shows a schematic diagram of sequence identification after co-transfection of ZT4-optimized vector and AAV-saCas9-U6-sgRNA vector into HEK293A cell line.
  • the term "specific targeting” generally refers to the interaction between two molecules (eg, molecule A and molecule B) (eg, molecule A specifically recognizes and/or binds molecule B (eg, target )). Molecule A interacts with molecule B to a statistically significant degree compared to interactions with other non-B molecules. The interaction can be covalent or non-covalent.
  • specific targeting can refer to a relationship in which molecule A (or a strand thereof) has a complementary base pairing relationship with molecule B (or a strand thereof).
  • “specific targeting” may refer to the process by which gRNAs recognize and/or bind to target sequences.
  • retinitis pigmentosa generally refers to an inherited blinding eye disease characterized by progressive and selective loss of retinal photoreceptor cells (cones and rods) and retinal pigment epithelial cells (Retinitis Pigmentosa, RP).
  • RP inheritance patterns can include autosomal recessive inheritance (arRP), autosomal dominant inheritance (adRP), and X-linked inheritance (xlRP), with xlRP onset early and with the most severe damage.
  • the clinical manifestations of RP can include night blindness, progressive visual field defect, central vision loss after macular involvement, and eventually blindness.
  • the main fundus change of RP is retinal pigment disorder at the equator, with osteocyte-like pigmentation, which gradually develops towards the posterior pole and the serrated edge.
  • Methods to assess retinal function and morphology may include best corrected visual acuity (BCVA), fundus autofluorescence, perimetry, electroretinography (ERG), fundus color photography, optical coherence tomography, OCT) and fluorescein angiography (fluorescein angiography, FFA).
  • X-linked retinitis pigmentosa generally refers to X-linked retinitis pigmentosa, also known as xlRP.
  • xlRP X-linked retinitis pigmentosa
  • the clinical signs of xlRP include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color perception, decreased visual acuity, decreased photoreceptor cell function, and pigment changes.
  • the English name of the term "human retinitis pigmentosa GTPase regulator” is Retinitis pigmentosa GTPase regulator, which is encoded by the RPGR gene, usually a protein with a series of RCC1-like domains (RLD).
  • the "gene encoding a GTPase regulator of retinitis pigmentosa” may also be referred to herein as the "RPGR gene”.
  • “Retinitis pigmentosa GTPase modulators” may include the full-length gene itself or a functional fragment thereof.
  • the retinitis pigmentosa GTPase modulator can be derived from any mammal that naturally expresses the RPGR gene or a homolog thereof, such as primates (eg, humans), rodents (eg, mice, rats).
  • An "RPGR gene” can encode multiple different isoforms of spliced forms of transcripts, and all spliced forms, transcripts and/or functional variants thereof are included herein.
  • human RPGR subtypes can include subtype A, subtype C, subtype D, subtype E, subtype F, subtype G, subtype I, and subtype J.
  • Subtypes A and C are full-length human RPGR subtypes.
  • an exemplary subtype A nucleotide sequence can be found in NCBI Accession No. NM_000328.3, an amino acid sequence can be found in NCBI Accession No. NP_000319.1, and an exemplary subtype C nucleotide sequence can be found in NCBI Accession No. NM_001034853 .2, the amino acid sequence can be found in NCBI Accession No. NP_001030025.1.
  • Subtypes RPGR ex1-19 (derived from exon 1 to exon 19, corresponding to subtype A above) and RPGR ORF15 (derived from part of exon 1 to intron 15, corresponding to Subtype C) above are two widely expressed subtypes of RPGR.
  • RPGR ORF15 terminates before exon 16 to exon 19, and the termination part of RPGR ORF15 can be referred to as ORF15, and herein, also referred to as "RPGR ORF15".
  • the RPGR ORF15 isoform is required for normal rod and cone function in the retina and is predominantly expressed in photoreceptor cells.
  • normally functioning human retinitis pigmentosa GTPase modulator generally refers to a human retinitis pigmentosa GTPase modulator that does not cause retinitis pigmentosa, ie the protein encoded by the RPGR gene.
  • the RPGR gene which encodes a normal-functioning human retinitis pigmentosa GTPase regulator, usually does not contain pathogenic mutations.
  • RPGR ORF15 generally refers to the terminator portion at the end of the RPGR ORF15 subtype, which may include part of exon 15 and intron 15 of the RPGR gene.
  • RPGR ORF15 contains a long guanine-rich repeat sequence called a highly conserved guanine nucleotide exchange factor, which has poor stability and complex post-transcriptional processing, and is often difficult to clone into cDNA. Unstable in operation.
  • RPGR ORF15 is also a hotspot for RPGR gene mutation.
  • the nucleotide sequence of an exemplary wild-type RPGR ORF15 is set forth in SEQ ID NO:101.
  • fragment or “functional fragment” refers to any fragment that retains the function of a full-length gene, but need not have the same level of expression or activity.
  • intron number 14 generally refers to the 14th intron of the RPGR gene, which is linked to the 5' end of ORF15.
  • codon optimization generally refers to exploiting redundancy in the genetic code to alter the nucleotide sequence while maintaining the same amino acid sequence of the encoded protein.
  • codon optimization can promote the increase or decrease of the expression of the encoded protein, and can promote the accuracy of the expression of the protein.
  • codon-optimized human RPGR ORF15 does not significantly affect the expression level of human RPGR ORF15, and "codon-optimized human RPGR ORF15 nucleotide sequence” has good sequence stability, reducing replication errors and splicing errors .
  • the term "correct human RPGR cDNA" generally refers to the cDNA of the human RPGR gene that can be transcribed and translated into a normally functioning RPGR protein.
  • the correct human RPGR cDNA can be either a human RPGR cDNA that does not contain the pathogenic mutation, or a cDNA derived from codon-optimized nucleotides.
  • the term "targeting vector” generally refers to a vector comprising the nucleic acid molecule described herein comprising the codon-optimized human RPGR ORF15 nucleotide, and the targeting vector can be used to introduce the nucleic acid molecule into cell.
  • the targeting vector may comprise 1) a 5' upstream target region; 2) the nucleic acid molecule; and 3) a 3' downstream target region.
  • the term "5' upstream target region” generally refers to the region in the targeting vector at the 5' end of the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotides, It has a site that can be recognized and/or cleaved by the gRNAs described herein.
  • the 5' upstream target region may comprise intron number 14 or a fragment thereof.
  • the 5' upstream target region may comprise the target region of the gRNA.
  • the 5' upstream target region may contain nucleotide mutations.
  • the term "3' downstream target region” generally refers to the region located at the 3' end of the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotides in the targeting vector, It has a site that can be recognized and/or cleaved by the gRNAs described herein.
  • the 3' downstream target region may comprise a 3' non-coding region (3' UTR) or a fragment thereof.
  • the 3' downstream target region may comprise the target region of the gRNA.
  • the 3' downstream target region may contain nucleotide mutations.
  • the term "gRNA” generally refers to a guide RNA, which recognizes a target sequence and guides a CRISPR-associated protein (Cas protein) to the target sequence.
  • gRNA can form a complex with Cas protein and guide the Cas protein to the target sequence and cleave the target site therein.
  • the degree of complementarity between the gRNA and its corresponding target sequence is at least about 50%.
  • the gRNA can be a double-stranded RNA comprising two RNA strands, the first strand can comprise a crRNA.
  • the second strand may comprise tracrRNA.
  • the gRNA can be a single-stranded RNA comprising a fusion of crRNA and tracrRNA, termed "single-stranded guide RNA (sgRNA)".
  • sgRNA single-stranded guide RNA
  • sgRNA single-stranded guide RNA
  • an sgRNA comprises a sequence that mates with a target sequence (also known as a gRNA mate sequence or sgRNA mate sequence), a backbone sequence (also known as a gRNA backbone sequence), and a transcription terminator.
  • a target sequence also known as a gRNA mate sequence or sgRNA mate sequence
  • backbone sequence also known as a gRNA backbone sequence
  • gRNA and sgRNA are used interchangeably.
  • backbone sequence generally refers to the part of the gRNA, other than the part that recognizes or hybridizes to the target sequence, and may include the sequence between the gRNA pairing sequence and the transcription terminator in the sgRNA.
  • the backbone sequence generally does not change due to changes in the target sequence, nor does it affect the recognition of the target sequence by the gRNA.
  • the backbone sequence can be any sequence available in the art.
  • the structure of the backbone sequence can be found in A and B in Figure 1 ( Figure 1), A, B, C in Figure 3 ( Figure 3), and Figure 4 ( Parts other than the spacer sequence described in A, B, C, D, and E in Fig. 4).
  • target nucleic acid In this application, the terms “target nucleic acid”, “target nucleic acid” and “target region” are used interchangeably, and usually refer to a nucleic acid sequence that can be recognized by a gRNA.
  • the target nucleic acid can refer to a double-stranded nucleic acid or a single-stranded nucleic acid.
  • isolated nucleic acid molecule generally refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs thereof, read from the 5' to 3' end.
  • An isolated nucleic acid molecule can be isolated from the usual or natural environment, or it can be produced synthetically. Such an isolated nucleic acid molecule is removed or isolated from its normal or natural environment, or the molecule is produced in such a way that it is not present in its normal or natural environment, which is different from its normal or natural environment isolated polypeptides, peptides, lipids, carbohydrates, other polynucleotides or other materials.
  • the isolated nucleic acid molecules of the present application can encode RNA, eg, can encode a gRNA that specifically targets the RPGR gene.
  • Plasmid generally refers to any molecule used to transfer encoded information (eg, an isolated nucleic acid molecule or nucleic acid molecule) to a host cell.
  • Plasmids can be linear or circular autonomously replicating sequences, genomic integration sequences, viral, bacteriophage or nucleotide sequences derived from single- or double-stranded DNA or RNA from any source.
  • a number of nucleotide sequences can be ligated or recombined into plasmids to introduce polynucleotide sequences into cells.
  • Plasmids may contain appropriate regulatory sequences, which may include promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, resistance genes, and other sequences as appropriate.
  • DNA endonuclease generally refers to an enzyme capable of recognizing and cleaving a DNA nucleic acid sequence, and usually the site of cleavage is inside the DNA strand.
  • DNA endonucleases may include non-base specific enzymes and enzymes that recognize and cleave specific bases or base sequences.
  • Cas nuclease may also be referred to as “Cas protein” or “CRISPR-associated protein” and generally refers to the ability to use a CRISPR sequence (eg, gRNA) as a guide to recognize and cleave specific DNA strands (eg, target sequences).
  • a CRISPR sequence eg, gRNA
  • Cas nucleases 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, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and/or their homologs, or modified forms thereof.
  • Cas9 nuclease also commonly referred to as Cas9 protein, Csn1 or Csx12, generally refers to a class of proteins involved in both crRNA biosynthesis and destruction of invading DNA in the type II CRISPR/Cas system.
  • Cas9 nucleases typically include a RuvC nuclease domain and an HNH nuclease domain, which cleave two different strands of a double-stranded DNA molecule, respectively. It has been tested in different bacterial species such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.
  • the Cas9 nuclease is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011).
  • the Cas9 protein of Streptococcus pyogenes its amino acid sequence can be found in the SwissProt database accession number Q99ZW2; the Neisseria meningitides Cas9 protein, its amino acid sequence can be found in the UniProt database number A1IQ68; Streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein, its amino acid sequence is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, its amino acid sequence is shown in UniProt database number J7RUA5.
  • the present application also includes the use of variants, derivatives, analogs, homologues, and fragments thereof.
  • a variant of any given sequence refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one Sequence of endogenous functions.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide.
  • the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and /or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie possessing at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics chemical compounds.
  • amino acid substitutions such as at least 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutions, can be made, so long as the modified sequence remains substantially as desired activity or ability.
  • Amino acid substitutions can include the use of non-naturally occurring analogs.
  • proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing uncharged polar headgroups with similar hydrophilicity values include amino acids Paraparagine, Glutamine, Serine, Threonine and Tyrosine.
  • homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • the term “homology” may be equivalent to "identity”.
  • homologous sequences can include sequences that can be at least 70%, 75%, 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical amino acid sequence.
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • recombinant generally refers to a recombinant nucleic acid or recombinant protein formed by linking nucleic acids or proteins from different sources. Recombinants typically possess the functions or properties of each component.
  • modification generally refers to the manipulation of changing a gene encoding an abnormally functional protein (eg, a mutated RPGR gene) to a gene encoding a normal functional protein.
  • the gene encoding a normal functional protein may be a wild-type gene or a codon-optimized gene.
  • modifications may include, but are not limited to, one or more nucleotide changes (including substitutions, insertions or deletions).
  • urine renal epithelial cells generally refers to renal epithelial cells extracted from urine, which can be induced into pluripotent stem cells (iPSCs).
  • iPSCs pluripotent stem cells
  • 3D-retinal organoid generally refers to an artificially grown retina with a three-dimensional structure, capable of self-renewal, self-organization, and display of basic retinal functions (eg, sensing light).
  • 3D-retinal organoids can be differentiated from primary tissue or stem cells (eg, pluripotent stem cells), with all the cells in the retina necessary to receive light and send signals to the brain.
  • introduction generally refers to the transfer of a nucleic acid molecule into a prokaryotic or eukaryotic cell, wherein the nucleic acid molecule can be incorporated into the genome of the cell (eg, chromosome, plasmid, plastid, or mitochondrial DNA). ), into an autonomous replicon, or expression.
  • Introduction can include methods such as “infection”, “transfection”, “transformation” and “transduction”. Suitable methods of introduction may include calcium phosphate transfection, DEAE-Dextran, nucleofection, magnetic transfection, electroporation, liposome-mediated transfection, and transduction using viral vectors, such as vaccinia virus, Or baculovirus for insect cells.
  • injection generally refers to the delivery of a target substance (eg, the gRNA, the plasmid, and/or the target) by puncturing the skin or mucosa of a subject (eg, a human or an animal). carrier) of the liquid process.
  • Injection includes the use of any acceptable form, eg, intraperitoneal injection, intramuscular injection, subcutaneous injection, subcutaneous infusion, intraocular injection, retinal injection, subretinal injection, vitreous injection, and/or epidural injection.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which is capable of transferring an inserted nucleic acid molecule (eg, an exogenous sequence) into and/or between host cells between.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the vectors described herein may include, for example, expression vectors, which may include viral vectors (lentiviral and/or retroviral vectors), phage vectors, phagemids, cosmids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC), and/or plasmids.
  • viral vectors lentiviral and/or retroviral vectors
  • phage vectors phagemids
  • cosmids cosmids
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC), and/or plasmids.
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • 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%, 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 gRNA that specifically targets a gene encoding a GTPase regulator for retinitis pigmentosa (RPGR gene), which specifically binds to intron No. 14 of the RPGR gene.
  • RPGR gene retinitis pigmentosa
  • the gRNA can specifically bind to the nucleotide sequence set forth in SEQ ID NO:102. In certain instances, the gRNA can specifically bind to the nucleotide sequence set forth in SEQ ID NO. 102 with at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleotide sequences.
  • 70% eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity nucleotide sequences.
  • the gRNA can specifically bind to a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 102. In certain instances, the gRNA can specifically bind to the nucleotide sequence set forth in SEQ ID NO. 102 with at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity complementary to nucleotide sequences Nucleotide sequence.
  • 70% eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity complementary to nucleotide sequences Nucleotide sequence
  • the gRNAs described herein can bind to sequences in a target nucleic acid of interest (eg, intron 14 of the RPGR gene).
  • a gRNA can interact with a target nucleic acid in a sequence-specific manner by hybridization (ie, base pairing).
  • the nucleotide sequence of the sgRNA can vary depending on the sequence of the target nucleic acid of interest.
  • the gRNA may include the nucleotide sequence shown in any one of SEQ ID NO. 105-126.
  • the gRNA may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%) of the nucleotide sequence shown in any one of SEQ ID NO. 105-126 , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity .
  • the gRNA may comprise (X)n, the nucleotide sequence and backbone sequence shown in any one of SEQ ID NO. 105-126 from the 5' end to the 3' end, wherein X is selected from A base of any of A, U, C, and G, and n is any integer from 0-15.
  • the gRNA may comprise the nucleotide sequence-backbone sequence-3' shown in any one of 5'-(X)n-SEQ ID NOs: 105-126, wherein X is selected from A, U A base of any one of , C, and G, and n is any integer from 0-15.
  • the backbone sequence used in this application can be derived from any commercially available plasmid as long as it can express Cas nuclease and transcribe gRNA.
  • the backbone sequences described herein can include backbone sequences from AAV-saCas9-puro.
  • the backbone sequence may comprise the nucleotide sequence set forth in SEQ ID NO:103.
  • the gRNA can be a single-stranded guide RNA (sgRNA).
  • sgRNA single-stranded guide RNA
  • the application provides one or more isolated nucleic acid molecules that encode the above-described gRNAs that specifically target the RPGR gene.
  • the present application provides a nucleic acid molecule encoding a DNA endonuclease.
  • the DNA endonucleases can include Endonuclease I, Endonuclease II, Endonuclease IV, Restriction Endonuclease, UvrABC Endonuclease, and/or Engineered Nuclease.
  • engineered nucleases include, but are not limited to, homing endonucleases (also known as meganucleases or meganucleases, Meganucleases), zinc finger nucleases (ZFNs), transcription activators transcription activator-like effector-based nuclease (TALEN), Clustered regularly interspaced short palindromic repeat (CRISPR).
  • homing endonucleases also known as meganucleases or meganucleases, Meganucleases
  • ZFNs zinc finger nucleases
  • TALEN transcription activators transcription activator-like effector-based nuclease
  • CRISPR Clustered regularly interspaced short palindromic repeat
  • the DNA endonuclease may include Cas nuclease.
  • the DNA nuclease can include Cas9 nuclease, homologues thereof, recombinants of naturally occurring molecules thereof, codon-optimized versions thereof, and/or modified versions thereof.
  • the gRNA sequence can be designed to hybridize to a target nucleic acid adjacent to a PAM sequence recognizable by the Cas nuclease.
  • the gRNA may or may not be fully complementary to the target sequence.
  • the degree of complementarity between the gRNA and its corresponding target sequence is at least about 50% (eg, at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or more).
  • Cas proteins generally have a specific PAM sequence that can be recognized in the target DNA (eg, target sequence).
  • the PAM may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 23-41.
  • the DNA nuclease may or may not be modified.
  • the gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.
  • modifications known in the art that can be used. For example, deletion, insertion, translocation, inactivation and/or activation of nucleotides. Such modifications may include introducing one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, cross-linking, breaking specific stretches of nucleotides, and other modifications. Modifications can include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof.
  • the nucleotides may be modified at ribose, phosphate and/or base moieties.
  • the gRNAs and/or isolated nucleic acid molecules described herein can be delivered using vectors.
  • DNA endonucleases may be delivered individually as one or more polypeptides.
  • the nucleic acid molecule encoding the DNA endonuclease is delivered separately, or pre-complexed together, with one or more guide RNAs, or one or more crRNAs and tracrRNA.
  • the nucleic acid molecule of the present application eg, the isolated nucleic acid molecule encoding the sgRNA that specifically targets the RPGR gene
  • the nucleic acid molecule encoding the Cas9 nuclease can be located in the same vector (eg, a plasmid).
  • the vector may include viral or non-viral vectors known in the art.
  • Non-viral delivery vehicles can include, but are not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes.
  • the isolated nucleic acid molecule and/or the nucleic acid molecule encoding the DNA endonuclease may be delivered by a plasmid.
  • the plasmid can be a viral vector, eg, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus.
  • viral vectors comprising nucleic acid molecules (eg, isolated nucleic acid molecules described herein) as part of the vector genome are well known in the art and can be performed by those of skill in the art without undue experimentation.
  • the associated vector may be a recombinant AAV virion that packages the nucleic acid molecules described herein.
  • Methods of producing recombinant AAV can include introducing the nucleic acid molecules described herein into a packaging cell line, producing AAV infection, helper functions of the AAV cap and rep genes, 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 adeno-associated vector (AAV).
  • AAV adeno-associated vector
  • AAV adenovirus-associated vector
  • the AAV may comprise different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, as well as any AAV variant or mixture.
  • the AAV genome is usually flanked by inverted terminal repeats (ITRs).
  • ITR inverted terminal repeat
  • AAV vectors are standard in the art and include providing to cells the polynucleotide to be delivered, the rep and cap genes, and the AAV genome to be packaged for helper virus function.
  • the production of AAV vectors generally requires the presence of the following components within a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (eg, not in) the rAAV genome, and a helper virus.
  • the AAV rep and cap genes can be from any AAV serotype, or from an AAV serotype different from the AAV genomic ITR, including but not limited to the AAV serotypes described herein.
  • the application provides a plasmid that can include the isolated nucleic acid molecule.
  • the plasmid may include a nucleic acid molecule encoding a DNA endonuclease.
  • the isolated nucleic acid molecule eg, the isolated nucleic acid molecule encoding the sgRNA that specifically targets the RPGR gene
  • the nucleic acid encoding the Cas9 nuclease can be on the same plasmid.
  • the isolated nucleic acid molecule (eg, the isolated nucleic acid molecule encoding the sgRNA that specifically targets the RPGR gene) and the nucleic acid molecule encoding the Cas9 nuclease may be located on different plasmids.
  • the application also provides a nucleic acid molecule that can comprise a human RPGR ORF15 nucleotide sequence.
  • a "nucleic acid molecule” described herein that may comprise a human RPGR ORF15 nucleotide sequence is different from an "isolated nucleic acid molecule” described herein that specifically targets an RPGR gene sgRNA.
  • the human RPGR ORF15 nucleotide sequence described herein may be a wild-type human RPGR ORF15 nucleotide sequence.
  • the wild-type human RPGR ORF15 nucleotide sequence may comprise the nucleotide sequence set forth in SEQ ID NO:101.
  • the human RPGR ORF15 nucleotide sequence described herein may be a codon-optimized human RPGR ORF15 nucleotide sequence.
  • the codon-optimized human RPGR ORF15 nucleotide sequence may comprise the nucleotide sequence set forth in SEQ ID NO:44.
  • the codon-optimized human RPGR ORF15 nucleotide sequence may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 85%) of the nucleotide sequence set forth in SEQ ID NO:44 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleosides acid sequence.
  • the codon optimization can improve the sequence stability of the human RPGR ORF15.
  • the degree of sequence integrity, the degree of sequence correctness and/or the amplification in the genome is improved by at least 10% (eg, at least 10%). 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or higher).
  • the application provides a targeting vector that can include the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence.
  • the targeting vector may include a 5' upstream target region.
  • the 5' upstream target region described in the present application may include the 14th intron of the human RPGR gene.
  • the 5' upstream target region described in the present application may also include the target region of the gRNA.
  • the 5' upstream target region may include the target region of the gRNA and the 14th intron of the human RPGR gene from the 5' end to the 3' end.
  • the 5' upstream target region may comprise nucleotide mutations.
  • intron 14 of the human RPGR gene in the 5' upstream target region may include a nucleotide mutation.
  • the nucleotide mutation can occur at the target region of the gRNA.
  • the nucleotide mutation may not occur in the 5' upstream target region and the nucleic acid molecule contained in the targeting vector (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence) junction.
  • the nucleotide mutation can occur at least about 50 nucleotides away from the nucleic acid molecule contained in the targeting vector (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence) (eg, at least about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 110 nucleotides, about 120 nucleotides nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides or more amino acids).
  • the mutation may not occur at the junction of intron 14 of the human RPGR gene and the nucleic acid molecule (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence).
  • the targeting vector may include a 3' downstream target region.
  • the 3' downstream target region may include the 3' non-coding region and/or the target region of the gRNA.
  • the 3' downstream target region may include the 3' non-coding region and the target region of the gRNA from the 5' end to the 3' end.
  • the 3' downstream target region may comprise nucleotide mutations.
  • the nucleotide mutation can occur at the target region of the gRNA.
  • the nucleotide mutation may not occur in the 3' downstream target region and the nucleic acid molecule contained in the targeting vector (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence) junction.
  • the nucleotide mutation in the 3' downstream target region can occur in the nucleic acid molecule comprised by the targeting vector (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence) separated by at least about 50 nucleotides (eg, at least about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 110 nucleotides) Nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides or more amino acids ) at.
  • the targeting vector eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence
  • at least about 50 nucleotides eg, at least about 60 nucleotides, about 70 nucleotides, about 80 nucle
  • the nucleotide mutation may increase the accuracy with which the nucleic acid molecule (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence) is introduced into the genome of a subject.
  • the accuracy rate may include the accuracy rate of the location of the nucleic acid molecule in the subject's genome and/or the sequence integrity of the nucleic acid molecule in the subject's genome.
  • the 5' upstream target region may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 96-99.
  • the 5' upstream target region may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 85%) of the nucleotide sequence set forth in any of SEQ ID NOs: 96-99 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleosides acid sequence.
  • the 3' downstream target region may comprise the nucleotide sequence set forth in SEQ ID NO: 100.
  • the 3' downstream target region may comprise at least 70% (eg, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%) of the nucleotide sequence set forth in SEQ ID NO: 100 , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.
  • the targeting vector may include the 5' upstream target region, the nucleic acid molecule (eg, the nucleic acid molecule comprising the human RPGR ORF15 nucleotide sequence), and the 3' downstream target area.
  • the targeting vector may sequentially include the 5' upstream target region from the 5' end to the 3' end, the nucleic acid molecule (eg, the nucleic acid comprising the human RPGR ORF15 nucleotide sequence) molecule), and the 3' downstream target region.
  • the application provides cells that can comprise the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence.
  • the cells described in this application can express a normally functional RPGR protein.
  • the cells may include mammalian cells, eg, cells derived from humans.
  • the cells can include COS cells, COS-1 cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, NSO cells or myeloma cells, stem cells (eg, pluripotent stem cells and/or totipotent stem cells), and/or epithelial cells (eg, renal epithelial cells and/or retinal epithelial cells).
  • stem cells eg, pluripotent stem cells and/or totipotent stem cells
  • epithelial cells eg, renal epithelial cells and/or retinal epithelial cells.
  • the cells may include HEK cells and/or urinary renal epithelial cells.
  • the cells can be modified to have differentiation ability.
  • the ability to differentiate may include the ability to differentiate into any cell type of the body: neurons, astrocytes, oligodendrocytes, retinal epithelial cells, epidermis, hair and keratinocytes, hepatocytes, pancreatic beta cells, intestinal Epithelial cells, alveolar cells, hematopoietic cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, kidney cells, adipocytes, chondrocytes and/or osteocytes.
  • the cells can be reprogrammed into induced pluripotent stem cells (iPSCs) with overexpression of key reprogramming genes (eg, OCT4, KLF4, SOX2, cMYC, NANOG, and/or LIN28).
  • iPSCs induced pluripotent stem cells
  • key reprogramming genes eg, OCT4, KLF4, SOX2, cMYC, NANOG, and/or LIN28.
  • the cells described herein can be used to evaluate the efficacy and safety of substances required for gene editing therapy (eg, sgRNA and CRISPR systems).
  • substances required for gene editing therapy eg, sgRNA and CRISPR systems.
  • tissue models can include 3D-retinal organoids containing the correct human RPGR cDNA.
  • the tissue model can be used to evaluate the efficacy and safety of substances required for gene editing therapy (eg, sgRNA and CRISPR systems).
  • the present application provides the use of the cells, and/or the tissue models, in evaluating the efficacy and/or safety of gene editing treatments. For example, after adding a polynucleotide encoding the gRNA, a plasmid, a nucleic acid molecule comprising a codon-optimized ORF15, and/or a targeting vector to the cell and/or the tissue model, the detectable gRNA, codon Expression of suboptimized ORF15, e.g., using PCR sequencing or gel electrophoresis; alternatively, the cell and/or tissue model does not produce immune rejection, toxicity, and/or the introduced substance does not affect the cells and/or all Other features of the organizational model are described.
  • a repair efficiency assay can be used as an indicator for evaluating the effectiveness of gene editing, and an exemplary method is shown in Example 3.
  • off-target efficiency can be detected as an indicator for evaluating the safety of gene editing, for example, off-target efficiency can be detected using whole genome sequencing.
  • the application provides the gRNA, the plasmid, the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence, and/or the application of the targeting vector in the preparation of a medicine for treating diseases , wherein the diseases include diseases caused by mutations in the RPGR gene.
  • the disease may include retinitis pigmentosa.
  • the disease may include X-linked inherited retinitis pigmentosa.
  • the present application provides a method for modifying the RPGR gene, and the method may comprise the steps of: introducing the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence.
  • the present application provides a method of treating retinitis pigmentosa, the method may comprise the steps of: introducing the nucleic acid molecule comprising the codon-optimized human RPGR ORF15 nucleotide sequence into a subject in need thereof.
  • the retinitis pigmentosa can include X-linked inherited retinitis pigmentosa.
  • compositions may also contain pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other substances known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • pharmaceutically acceptable carriers e.g, subretinal injection, direct retinal injection, or intravitreal injection.
  • the formulations described herein can be introduced by a variety of methods, for example, including, but not limited to, intravitreal injection (eg, anterior, intermediate, or posterior vitreous injection), subconjunctival injection, intracameral injection, via temporal Injections into the anterior chamber, intrastromal injections, injections into the subchoroidal space, intracorneal injections, subretinal injections, and intraocular injections are administered locally to the eye.
  • the introduction may include subretinal injection, which is injection into the subretinal space, ie, beneath the sensorineural retina.
  • the injected material eg, the targeting vector, the gRNA, and/or the plasmid
  • the injected material is introduced directly between the photoreceptor cells and the retinal pigment epithelium (RPE) layer and is Create space in between.
  • RPE retinal pigment epithelium
  • the methods described herein can include ex vivo methods.
  • subject-specific induced pluripotent stem cells iPSCs
  • the genomic DNA of these iPSC cells can then be edited using the methods described herein.
  • the method can include editing in or near the mutated site of the RPGR gene of the iPSC so that it does not encode a mutated RPGR ORF15.
  • the gene-edited iPSCs can be differentiated into other cells, such as photoreceptor cells or retinal progenitor cells.
  • the differentiated cells eg, photoreceptor cells or retinal progenitor cells
  • the differentiated cells can be implanted into the subject.
  • photoreceptor cells or retinal progenitor cells can be isolated from the subject.
  • the genomic DNA of these photoreceptor cells or retinal progenitor cells can be edited using the methods described herein.
  • the method can include editing in or near the mutated site of the RPGR gene of the photoreceptor or retinal progenitor cell so that it does not have the mutated RPGR ORF15.
  • the gene-edited photoreceptor cells or retinal progenitor cells can be implanted into the subject.
  • mesenchymal stem cells can be isolated in vivo, in other instances, from bone marrow or peripheral blood in other instances.
  • the genomic DNA of these mesenchymal stem cells can be edited using the methods described in this application.
  • the method can include editing in or near the mutated site of the RPGR gene of the mesenchymal stem cell so that it does not have the mutated RPGR ORF15.
  • the gene-edited mesenchymal stem cells can be differentiated into any type of cell, 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 can include a comprehensive 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 at genomic locations associated with minimal risk to the subject.
  • specific cell populations including clonal cell populations, can be isolated prior to implantation.
  • the methods described herein may include methods of using a site-directed nuclease to cleave DNA at a precise target location in the genome, thereby producing single- or double-stranded DNA breaks at specific locations within the genome. Such breaks can be periodically repaired by endogenous cellular processes such as Homology directed repair (HDR), Non-Homologous End Joining (NHEJ), and microhomology-mediated end joining (Microhomology-Mediated End Joining, MMEJ).
  • HDR Homology directed repair
  • NHEJ Non-Homologous End Joining
  • MMEJ microhomology-mediated end joining
  • the methods described herein can include creating one or two DNA breaks, which can be double-stranded breaks or two single-stranded breaks, in a locus of interest proximate the target sequence.
  • the cleavage can be achieved by site-directed polypeptides.
  • Site-directed polypeptides eg, DNA endonucleases
  • nucleic acids eg, genomic DNA
  • Double-strand breaks can stimulate a cell's endogenous DNA repair pathway, eg, HDR, NHEJ, or MMEJ.
  • the exogenous polynucleotide sequence can be inserted into the target nucleic acid cleavage site using HDR.
  • the exogenous polynucleotide sequence may be referred to as a donor polynucleotide (or donor, or donor sequence, or polynucleotide donor template).
  • a donor polynucleotide, a portion of a donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site.
  • the donor polynucleotide may be an exogenous polynucleotide sequence, ie, a sequence that is not naturally present at the target nucleic acid cleavage site.
  • HDR uses homologous sequences or donor sequences (e.g., the targeting vector) as templates to insert specific DNA sequences at breakpoints.
  • homologous sequences can be in the endogenous genome, eg, sister chromatid.
  • the donor may be an exogenous nucleic acid, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, double-stranded oligonucleotide, or virus.
  • the donor may comprise the targeting vector described herein.
  • exogenous nucleic acids may contain regions of high homology to the DNA nuclease-cleavable locus, and may also contain additional sequences or sequence changes (including deletions that can incorporate cleavable target loci).
  • exogenous donor template additional nucleic acid sequences (eg, the targeting vector) or modifications (eg, single- or polybasic changes or deletions) can be introduced between the homologous flanking regions, so that the Additional or altered nucleic acid sequences are incorporated into the locus of interest, and exogenous donors can be delivered by plasmid vectors, eg, AAV vectors and/or TA cloning vectors (eg, ZT4 vectors).
  • plasmid vectors eg, AAV vectors and/or TA cloning vectors (eg, ZT4 vectors).
  • NHEJ directly joins the ends of DNA resulting from double-strand breaks, sometimes missing or adding nucleotide sequences, which can disrupt or enhance gene expression.
  • MMEJ also known as "alternative NHEJ (ANHEJ)"
  • ANHEJ alternative NHEJ
  • MMEJ can utilize homologous sequences of several base pairs flanking the DNA break site to drive more favorable DNA end-joining repair outcomes. In some cases, it may be possible to predict possible repair outcomes based on analysis of potential microscopic homology at DNA break sites.
  • Example 1 In vitro screening of gRNAs with editing efficiency
  • gRNAs were designed for the 14th intron of human RPGR, which are gRNA-1 to gRNA-22, and the corresponding targets are R1 to R22.
  • the sequences are shown in Table 1 below, where 1 and -1 represent the 14th intron The forward and reverse strands of the duplex in the target region.
  • the above 22 gRNAs (the target sites recognized by the gRNAs are numbered R1 to R22) were constructed into the AAV-saCas9-puro vector (the vector is pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA, from addgene , numbered #61592, the vector was transformed to obtain puromycin puro resistance), transfected into HEK293A cells, and the genomic DNA was extracted after screening with puromycin, and primers were designed near the target for PCR sequencing, the primer is hRPGR -exin14-F and hRPGR-exin14-R, the sequences are shown in Table 2 below.
  • the AAV-saCas9-puro vector map is shown in Figure 14
  • the PCR products were treated with T7E1 enzyme to recover the products, and agarose gel electrophoresis was performed to quantitatively analyze the cleavage mutation efficiency.
  • the mutation rates are shown in Figure 1 and Table 3, indicating that all the gRNAs of the present application can cleave the 14th intron of RPGR.
  • the nucleotide sequence of the codon-optimized sequence of human RPGR ORF15 is shown in SEQ ID NO: 44, and PCR amplification primers are designed for this sequence.
  • the upstream primer HR-ORF15co-F is upstream of ORF15
  • the downstream primer HR-ORF15co-R is in ORF15. downstream of the area.
  • Design primers for the 14th intron (HR-int14-F and HR-int14-R) and primers for the 3' untranslated region (3'UTR) (HR-3'UTR-F and HR-3' UTR-R) the sequence is shown in 4. PCR amplification was performed.
  • Multi-fragment recombination of the amplified product was carried out with Novozyme's rapid cloning kit C115 and the matching linear vector of the kit, then transformed, plated, cloned and sequenced, and the vector with the correct sequencing was selected for amplification.
  • the vector was named ZRC03, and the sequence was 14 intron-ORF15-3'UTR.
  • the R14, R15, R17 and R19 targets in intron 14 were mutated in ZRC03 vector using Novozan's Mut Express II Fast Mutagenesis Kit V2 mutation kit.
  • the primers for the mutation target R14 are Zt4-HRa14-mut-F (SEQ ID NO: 51) and Zt4-HRa14-mut-R (SEQ ID NO: 52), the primers for the mutation target R15
  • the primers are Zt4-HRa15-mut-F (SEQ ID NO: 53) and Zt4-HRa15-mut-R (SEQ ID NO: 54)
  • the primer for the mutation target R17 is Zt4-HRa17-mut-F (SEQ ID NO: 54).
  • Each targeting vector was amplified by PCR, digested with Dpn1 enzyme, and the methylated template plasmid was removed for recombination, transformation, plating and clone identification.
  • the vector sequence after R14 target mutation is shown in SEQ ID NO: 59
  • the vector sequence after R15 target mutation is shown in SEQ ID NO: 60
  • the vector sequence after R17 target mutation is shown as SEQ ID NO: 61
  • the vector sequence after R19 target mutation is shown in SEQ ID NO: 62.
  • the ZT4 vector was constructed using the zero-background ZT4-Blunt rapid cloning kit (Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., product number ZC205).
  • the ZT4 vector sequence is shown in SEQ ID NO: 104, and the vector insertion site is after 371bp. .
  • the clones were picked and sequenced, and the optimized vectors numbered R145, R156, R176 and R193 were obtained accordingly.
  • the primer sequences of the optimized vector for the R14 mutation are hR-saHITI-F14 (SEQ ID NO: 63) and hR-saHITI-R14 (SEQ ID NO: 64), and the primer sequences for the optimized vector for the R15 mutation are hR-saHITI- F15 (SEQ ID NO:65) and hR-saHITI-R15 (SEQ ID NO:66), the primer sequences for the R17 mutated optimized vector are hR-saHITI-F17 (SEQ ID NO:67) and hR-saHITI-R17 (SEQ ID NO:68), and the primer sequences for the R19 mutated optimized vector are hR-saHITI-F19 (SEQ ID NO:69) and hR-saHITI-R19 (SEQ ID NO:70).
  • ZT4 optimized vector and AAV-saCas9-U6-sgRNA vector were co-transformed into HEK293A cell line, and the genomic DNA was extracted after flow sorting for identification.
  • the identification primer sequences are shown in Table 5, and the identification schematic diagram is shown in Figure 15, wherein the 5'outer is the upstream and downstream sequences near the 5' end of the inserted codon-optimized RPGR ORF15.
  • Figure 3A is the sequencing result of R145
  • Figure 3B is the sequencing result of R156
  • Figure 3C is the sequencing result of R176
  • Figure 3D is the sequencing result of R193
  • the first sequence in Figure 3 is the correct sequence, indicating that the target region is correctly inserted into the genome of the host cell and expressed.
  • the sequence of the sequence), P2A-GFP and 3'UTR sequence, the primers of part of the 14th intron+ORF15 optimized sequence are ZT4-HR-int14co-F (SEQ ID NO: 76) and ZT4-HR-int14co-R ( SEQ ID NO: 77), primers for P2A-GFP are ZT4-HR-P2AEG-F (SEQ ID NO: 78) and ZT4-HR-P2AEG-R (SEQ ID NO: 79), and primers for 3'UTR are ZT4-HR-3'UTR-F (SEQ ID NO:80) and ZT4-HR-3'UTR-R (SEQ ID NO:81).
  • the HITI vector was obtained, the vector number corresponding to R14 was 145G, the vector number corresponding to R15 was 156G, the vector number corresponding to R17 was 176G, and the vector number corresponding to R19 was 193G.
  • AAV-saCas9-sagRNA, HITI-GFP targeting vector and pMcherry-N1 vector (Clontech, USA, product number 632523) were co-transformed into HEK293A cell line, and the efficiency was evaluated by GFP flow sorting. After the first round of sorting Mcherry, the culture was continued, and the second round of sorting was for GFP.
  • the pMcherry-N1 vector can express Mcherry protein, and red fluorescence can be observed under a fluorescence microscope. Comparing the percentages of GFP-positive cells among the four groups, the results showed that the percentages of positive cells in the four groups were not statistically significant (Figure 4).
  • the cell fluorescence after GFP sorting is shown in Figure 5, wherein the respective fluorescence of the four groups of cells after GFP sorting is shown in Figure 6. It shows that the four groups of cells can express GFP protein, and the sorting efficiency is good.
  • the ZT4 optimized vector of Example 2 and the AAV-saCas9-U6-sgRNA vector were co-transformed into HEK293A cell line, and after flow sorting, the RNA was cultured and extracted, and the cDNA was reverse transcribed to identify the repair effect.
  • the primer sequences for identifying different transcripts of RPGR are shown in Table 6.
  • the C, C1 and 5R primer pairs were used to identify optimized repair primers; the 45F and 45F1 primer pairs were used to identify the common exons of RPGR ex1-19 transcripts and RPGR ORF15 transcripts; the 56F and 56F1 primer pairs were used to identify RPGRex 1-19 transcripts; O15F and O15F1 primer pairs were used to identify the RPGR ORF15 transcript.
  • Figure 7 shows that the 45F and 45F1 primer pairs, the 56F and 56F1 primer pairs, and the O15F and O15F1 primer pairs all have good specificity, and it also shows that the wild-type HEK293A cell line has the expression of RPGR ex1-19 transcripts and RPGR ORF15 transcripts. While the C primer pair was able to identify editing effects in the wild-type HEK293A cell line, indicating poor specificity.
  • Embodiment 6 detects the repair efficiency of non-codon-optimized RPGR ORF15
  • the four sgRNAs for the R14, R15, R17, R19 target sites were designed to contain non-optimized codons, namely wild-type RPGR ORF15 (SEQ ID NO: 101) and codon-optimized RPGR ORF15 (SEQ ID NO: 44) donor vectors , PCR cloned and transformed into HEK293 cells with GT115 competent cells.
  • non-optimized codons namely wild-type RPGR ORF15 (SEQ ID NO: 101) and codon-optimized RPGR ORF15 (SEQ ID NO: 44) donor vectors , PCR cloned and transformed into HEK293 cells with GT115 competent cells.
  • the codon-optimized sequence was well sequenced, and many clones obtained the correct sequence, while the non-optimized sequence had poor sequencing signal. Sequencing alignments are shown in Figures 10-13.
  • the three clones (HR14WM1, HR14WM2 and HR14WM3) of the vector containing the wild-type RPGR ORF15 corresponding to the gRNA of the R14 target site were sequenced, and it was found that part of the sequence was lost in the sequencing results of the three clones ( Figure 10A-10C).
  • the three clones (HR15WM1, HR15WM2 and HR15WM3) of the vector containing the wild-type RPGR ORF15 corresponding to the gRNA of the R15 target were sequenced, and the preliminary segmental sequencing of HR15WM1 and HR15WM3 was poor.
  • Figure 11 The three clones (HR15WM1, HR15WM2 and HR15WM3) of the vector containing the wild-type RPGR ORF15 corresponding to the gRNA of the R15 target were sequenced, and the preliminary segmental sequencing of HR15WM1 and HR15WM3 was poor.
  • the three clones (HR17WM1, HR17WM2 and HR17WM3) of the vector containing the wild-type RPGR ORF15 corresponding to the gRNA of the R17 target were sequenced.
  • the preliminary segmental sequencing of HR17WM2 was poorly aligned.
  • Three clones (HR19WM1, HR19WM2 and HR19WM3) of the vector containing the wild-type RPGR ORF15 corresponding to the gRNA of the R19 target site were sequenced. When the two clones were subjected to preliminary segmental sequencing, the alignment of HR19WM1 and HR19WM3 was poor.

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Abstract

提供一种特异性靶向编码视网膜色素变性GTP酶调节剂(RPGR)基因的gRNA，所述gRNA特异性结合所述RPGR基因的第14号内含子，还提供包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子，及所述gRNA和所述核酸分子在治疗RPGR X连锁视网膜变性中的应用。

Description

AAV介导的RPGR X连锁视网膜变性的基因编辑治疗 技术领域
本申请涉及生物医药领域，具体的涉及一种用于基因编辑治疗RPGR X连锁视网膜变性的gRNA和靶向载体。
背景技术
视网膜色素变性(retinitis pigmentosa，RP)是由于视网膜光感受器细胞及视网膜色素上皮细胞进行性、选择性丧失为主要特征的遗传性致盲眼病，是儿童和工作年龄人群不可逆双眼盲的主要病因之一，目前无有效疗法。
RP通常是由基因突变引起的，基于基因的治疗有基因替代治疗和基因编辑治疗。但由于引起RP的突变基因位置的特殊性和外源基因的表达难以调控，亟需更加安全有效的治疗方法。
发明内容
本申请提供了一种特异性靶向视网膜色素变性GTP酶调节剂的基因(RPGR基因)的gRNA，其特异性结合所述RPGR基因的第14号内含子，所述gRNA对所述RPGR基因的第14号内含子具有好的切割效率。本申请提供了核酸分子，其包含密码子优化的人RPGR ORF15核苷酸序列。与野生型RPGR ORF15相比，所述密码子优化的人RPGR ORF15核苷酸序列的序列稳定，测序良好，得到正确序列的克隆多，克隆序列完整不易丢失。本申请提供了靶向载体，其包含所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。本申请所述的靶向载体能够提高所述核酸分子导入受试者基因组的准确率。本申请的编码所述gRNA的分离的核酸分子(或所述质粒)和包含密码子优化的人RPGR ORF15核苷酸序列(或所述靶向载体)的核酸分子，能够使RPGR突变的细胞表达正确的视网膜色素变性GTP酶调节剂，具有好的基因编辑修复效率。
一方面，本申请提供了一种特异性靶向视网膜色素变性GTP酶调节剂(RPGR)基因的gRNA，其特异性结合所述RPGR基因的第14号内含子。
在某些实施方式中，所述的gRNA特异性结合SEQ ID NO:102所示的核苷酸序列，或特异性结合与SEQ ID NO:102所示的核苷酸序列互补的核苷酸序列。
在某些实施方式中，所述gRNA包含SEQ ID NO.105-126中任一项所示的核苷酸序列。
在某些实施方式中，所述gRNA包含5’-(X)n-SEQ ID NO.105-126中任一项所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
在某些实施方式中，所述gRNA为单链向导RNA(sgRNA)。
另一方面，本申请提供了一种或多种分离的核酸分子，其编码所述的特异性靶向RPGR基因的gRNA。
另一方面，本申请提供了质粒，其包含所述的分离的核酸分子。
在某些实施方式中，所述的质粒为病毒载体。
在某些实施方式中，所述的质粒包括编码DNA核酸内切酶的核酸分子。
在某些实施方式中，所述DNA核酸内切酶包括Cas核酸酶。
在某些实施方式中，所述DNA核酸内切酶包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
另一方面，本申请提供了核酸分子，其包含密码子优化的人RPGR ORF15核苷酸序列。
另一方面，本申请提供了所述的核酸分子包含SEQ ID NO:44所示的核苷酸序列。
另一方面，本申请提供了靶向载体，其包含所述的核酸分子。
在某些实施方式中，所述的靶向载体包含1)5’上游靶区域；2)所述的核酸分子；以及3)3’下游靶区域；其中所述5’上游靶区域和/或所述3’下游靶区域能够被所述gRNA所识别和/或切割。
在某些实施方式中，所述的靶向载体中的所述5’上游靶区域和/或所述3’下游靶区域包含核苷酸突变，所述突变提高了所述的核酸分子被导入受试者的基因组的准确率。
在某些实施方式中，所述的靶向载体中的所述5’上游靶区域包含SEQ ID NO:96-99中任一项所示的核苷酸序列。
在某些实施方式中，所述的靶向载体中的所述3’下游靶区域包含SEQ ID NO:100所示的核苷酸序列。
另一方面，本申请提供了细胞，其包含所述的核酸分子。
在某些实施方式中，所述的细胞包括HEK细胞和/或尿液肾上皮细胞。
在某些实施方式中，所述的细胞经修饰后具备分化能力。
在某些实施方式中，所述的细胞可分化为3D-视网膜类器官。
另一方面，本申请提供了组织模型，其包括包含正确的人RPGR cDNA的3D-视网膜类器官。
另一方面，本申请提供了所述的细胞，和/或所述的组织模型在评价基因编辑治疗有效性和/或安全性中的用途。
另一方面，本申请提供了所述的gRNA，所述的质粒，所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子，和/或所述的靶向载体在制备治疗疾病的药物中的应用，其中所述疾病包括RPGR基因中的突变所导致的疾病。
在某些实施方式中，所述疾病包括视网膜色素变性。
在某些实施方式中，所述疾病包括X连锁遗传的视网膜色素变性。
另一方面，本申请提供了一种修饰RPGR基因的方法，所述方法包括以下的步骤：导入所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。
另一方面，本申请提供了一种治疗视网膜色素变性的方法，所述方法包括以下的步骤：向有需要的受试者导入所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。
在某些实施方式中，所述视网膜色素变性包括X连锁遗传的视网膜色素变性。
在某些实施方式中，所述导入获得了正常功能的人RPGR蛋白。
在某些实施方式中，所述导入包括导入所述的靶向载体。
在某些实施方式中，所述导入包括导入所述的gRNA，和/或所述的质粒。
在某些实施方式中，所述导入包括注射。
在某些实施方式中，所述导入包括视网膜下腔注射，或玻璃体注射。
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gRNA的剪切结果电泳图。
图2A显示的是使用R145，R156，R176和R193这四个载体进行基因编辑均能扩增出3kb的阳性条带，图2B显示的是使用R145，R156，R176和R193这四个载体进行基因编辑均能 扩增出5’outer的阳性条带，图2C显示的是使用R145，R156，R176和R193这四个载体进行基因编辑均能扩增出3’outer的阳性条带。
图3A显示的是使用R145的测序结果分析，图3B显示的是使用R156的测序结果分析，图3C显示的是使用R176的测序结果分析，图3D显示的是使用R193的测序结果分析。
图4显示的是使用R145，R156，R176和R193这四个载体转染HEK293A细胞后GFP阳性细胞百分率。
图5显示的是使用R145，R156，R176和R193这四个载体转染HEK293A细胞后的细胞荧光图。
图6显示的是使用R145，R156，R176和R193这四个载体转染HEK293A细胞后，GFP分选后的荧光图。
图7显示的是C引物对、C1引物对、45F和45F1引物对、56F和56F1引物对、O15F和O15F1引物对对不同转录本的PCR鉴定结果。
图8显示的是C引物对和C1引物对对野生型ORF15序列和密码子优化ORF15序列的鉴定结果。
图9显示的是5R引物对和14-ORF15引物对对野生型ORF15序列和密码子优化ORF15序列的鉴定结果。
图10显示的是包含野生型RPGR ORF15的载体三个克隆HR14WM1(A)，HR14WM2(B)和HR14WM3(C)的测序结果分析。
图11显示的是包含野生型RPGR ORF15的载体克隆HR15WM2的分段测序比对分析。
图12显示的是包含野生型RPGR ORF15的载体克隆HR17WM1(A)和HR17WM3(B)的分段测序比对分析。
图13显示的是包含野生型RPGR ORF15的载体克隆HR19WM2的分段测序比对分析。
图14显示的是AAV-saCas9-puro载体图谱。
图15显示的是将ZT4优化载体和AAV-saCas9-U6-sgRNA载体共转到HEK293A细胞系后的序列鉴定示意图。
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“特异性靶向”通常是指两种分子(例如，分子A和分子B)之间的相互作用(例如，分子A特异性识别和/或结合分子B(如，靶标))。同与其他非B分子的相互作用相比，分子A以统计学显著的程度与分子B相互作用。所述相互作用可以是共价的或非共价的。当涉及多核苷酸时，特异性靶向可以指分子A(或其一条链)与分子B(或其一条链)具有碱基互补配对的关系。在本申请中，“特异性靶向”可以指gRNA识别和/或结合至靶序列的过程。
在本申请中，术语“视网膜色素变性”通常是指一种由于视网膜光感受器细胞(视锥细胞和视杆细胞)及视网膜色素上皮细胞进行性、选择性丧失为主要特征的遗传性致盲眼病(Retinitis Pigmentosa，RP)。RP遗传方式可包括常染色体隐性遗传(arRP)、常染色体显性遗传(adRP)，以及X连锁遗传(xlRP)，其中xlRP发病早、而且损害最为严重。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。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，视网膜色素上皮细胞(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。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、视网膜电图(electroretinography，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。
在本申请中，术语“X连锁遗传的视网膜色素变性”通常是指X连锁遗传的视网膜色素变性，也称为xlRP。目前大约70％～75％的xlRP由RPGR基因突变所致，其中超过75％的RPGR突变位于RPGR ORF15亚型最后一个外显子ORF15区。所述xlRP的临床体征包括但不限于周边视力减弱、中央(阅读)视力减弱、夜晚视力减弱、颜色感觉丧失、视敏度下降、光感受器细胞功能下降、色素变化。
在本申请中，术语“人视网膜色素变性GTP酶调节剂”英文名称为Retinitis pigmentosa GTPase regulator，由RPGR基因编码，通常为一种具有一系列RCC1样结构域(RLD)的蛋白质。“编码视网膜色素变性GTP酶调节剂的基因”在本文中也可以称为“RPGR基因”。“视网膜色素变性GTP酶调节剂”可以包括全长基因本身或其功能片段。视网膜色素变性GTP酶调节剂可来源于天然表达RPGR基因或其同源物的任何哺乳动物，如灵长类动物(例如，人)、啮齿类动物(如小鼠、大鼠)。“RPGR基因”可编码多种不同的亚型(isoform)的剪接形式的转录本，本文可包括其所有剪接形式、转录本和/或功能性变体。例如，人RPGR 亚型可包括亚型A、亚型C、亚型D、亚型E、亚型F、亚型G、亚型I和亚型J。亚型A和亚型C为全长的人RPGR亚型。例如，示例性的亚型A的核苷酸序列可参见NCBI登录号NM_000328.3，氨基酸序列可参见NCBI登录号NP_000319.1，示例性的亚型C的核苷酸序列可参见NCBI登录号NM_001034853.2，氨基酸序列可参见NCBI登录号NP_001030025.1。
亚型RPGR ex1-19(源自第1外显子至第19外显子，对应上文的亚型A)和RPGR ORF15(源自第1外显子至第15内含子的一部分，对应上文的亚型C)为RPGR的两种广泛表达的亚型。其中，RPGR ORF15的在第16外显子至第19外显子之前终止，RPGR ORF15的终止部分可称为ORF15，在本文中，也称为“RPGR ORF15”。RPGR ORF15亚型是视网膜中正常视杆和视锥功能所必需的，并且主要在感光细胞中表达。
在本申请中，术语“正常功能的人视网膜色素变性GTP酶调节剂”通常是指不会引起视网膜色素变性的人视网膜色素变性GTP酶调节剂，即由RPGR基因编码的蛋白。编码正常功能的人视网膜色素变性GTP酶调节剂的RPGR基因通常不包含致病突变。
在本申请中，术语“RPGR ORF15”或“ORF15”通常是指RPGR ORF15亚型末端的终止子部分，其可包括RPGR基因的第15外显子和第15内含子的一部分。RPGR ORF15包含一段较长的富含鸟嘌呤的重复序列，称为高度保守的鸟嘌呤核苷酸交换因子，该序列稳定性差，而且具有复杂的转录后处理，通常难以克隆为cDNA，在重组DNA操作中不稳定。RPGR ORF15也是RPGR基因突变的热点。示例性的野生型RPGR ORF15的核苷酸序列如SEQ ID NO:101所示。
术语“片段”或“功能片段”指保留全长基因功能的任何片段，但无需具有相同水平的表达或活性。
在本申请中，术语“第14号内含子”通常是指RPGR基因的第14个内含子，其与ORF15的5’端连接。
在本申请中，术语“密码子优化”通常是指利用遗传密码中的冗余来改变核苷酸序列而保持编码的蛋白质的相同的氨基酸序列。通常，进行密码子优化可以促进编码的蛋白质的表达增加或降低，可以促进蛋白质的表达的准确性。本申请中，“密码子优化的人RPGR ORF15”并不显著影响人RPGR ORF15的表达水平，“密码子优化的人RPGR ORF15核苷酸序列”具有好的序列稳定性，减少复制错误和剪接错误。
在本申请中，术语“正确的人RPGR cDNA”通常是指能够转录翻译为正常功能的RPGR蛋白的人RPGR基因的cDNA。正确的人RPGR cDNA可以是不包含致病突变的人RPGR cDNA，也可以是源自密码子优化的核苷酸的cDNA。
在本申请中，术语“靶向载体”通常是指包含本申请所述包含了所述密码子优化的人RPGR ORF15核苷酸的核酸分子的载体，靶向载体可用于将所述核酸分子导入细胞。在某些情形中，所述靶向载体可以包含1)5’上游靶区域；2)所述的核酸分子；以及3)3’下游靶区域。
在本申请中，术语“5’上游靶区域”通常是指在所述靶向载体中，位于所述包含了所述密码子优化的人RPGR ORF15核苷酸的核酸分子的5’端的区域，其具有能被本申请所述gRNA识别和/或切割的位点。在某些情形中，所述5’上游靶区域可包含第14号内含子或其片段。在某些情形中，所述5’上游靶区域可包含所述gRNA的靶区域。在某些情形中，所述5’上游靶区域可以包含核苷酸突变。
在本申请中，术语“3’下游靶区域”通常是指在所述靶向载体中，位于所述包含了所述密码子优化的人RPGR ORF15核苷酸的核酸分子的3’端的区域，其具有能被本申请所述gRNA识别和/或切割的位点。在某些情形中，所述3’下游靶区域可包含3’非编码区(3’UTR)或其片段。在某些情形中，所述3’下游靶区域可包含所述gRNA的靶区域。在某些情形中，所述3’下游靶区域可以包含核苷酸突变。
在本申请中，术语“gRNA”通常是指向导RNA，其识别靶序列，并引导CRISPR关联蛋白(Cas蛋白)至靶序列。gRNA crRNA上与靶序列互补的核苷酸和由crRNA与tracrRNA碱基配对形成的RNA骨架。gRNA可以与Cas蛋白形成复合体并将Cas蛋白引导至靶序列并切割其中的靶位点。gRNA与其相应的靶序列之间的互补程度至少为约50％。在某些情形中，gRNA可以为包含两条RNA链的双链RNA，第一链可包含crRNA。第二链可包含tracrRNA。在某些情形中，gRNA可以为包含crRNA和tracrRNA融合而成的单链RNA，称为“单链向导RNA(sgRNA)”。
在本申请中，术语“单链向导RNA(sgRNA)”通常是指包含crRNA和tracrRNA融合而成的单链RNA。通常，如本领域技术人员所熟知的，sgRNA包含与靶序列配对的序列(也被称为gRNA配对序列或sgRNA配对序列)、骨架序列(也被称为gRNA骨架序列)和转录终止子。在本申请中，除非特别指出，gRNA和sgRNA可以互换使用。
在本申请中，术语“骨架序列”通常是指gRNA中，除识别或杂交靶序列的部分的其他部分，可包括sgRNA中gRNA配对序列与转录终止子之间的序列。骨架序列一般不会因为靶序列的变化而变化，也不影响gRNA对靶序列的识别。因此，骨架序列可以是现有技术中任何可行的序列。骨架序列的结构可参见如文献Nowak et al.Nucleic Acids Research 2016.44:9555-9564中的Figure 1(图1)中A和B，Figure 3(图3)中A、B、C，以及Figure 4(图 4)中A、B、C、D、E中所记载的除spacer序列之外的部分。
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。
在本申请中，术语“分离的核酸分子”通常是指从5’至3’末端阅读的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双链聚合物或其类似物。分离的核酸分子可以是从通常的或天然的环境中分离出的，也可以是人工合成的方式生产而成的。这种分离的核酸分子从其通常的或天然的环境中移出的或分离的，或者生产所述分子的方式使其不存在于其通常的或天然的环境中，其与通常的或天然的环境中的多肽、肽、脂质、糖类、其他的多核苷酸或其它材料分离。本申请中的分离的核酸分子可编码RNA，例如，可编码特异性靶向RPGR基因的gRNA。
在本申请中，术语“质粒”通常是指用于将编码信息(例如，分离的核酸分子或核酸分子)转移至宿主细胞的任何分子。质粒可以是来源于任何来源的单链或双链DNA或RNA的线性或环状的自主复制序列、基因组整合序列、病毒、噬菌体或核苷酸序列。可以将许多核苷酸序列连接或重组到质粒中，从而将多核苷酸序列引入到细胞中。质粒可以包含适当调节序列，所述调节序列可包括启动子序列、终止子序列、多聚腺苷酸化序列、增强子序列、标记基因、抗性基因以及视情况而定的其他序列。
在本申请中，术语“DNA核酸内切酶”通常是指能够识别并切割DNA核酸序列的酶，并且通常切割的位点在DNA链的内部。DNA核酸内切酶可包含非碱基特异性的酶和识别并切断特定的碱基或碱基序列的酶。
在本申请中，术语“Cas核酸酶”也可称为“Cas蛋白”或“CRISPR相关蛋白”，通常是指能够使用CRISPR序列(例如，gRNA)作为指导(guide)，从而识别和切割特定的DNA链(例如，靶序列)。Cas核酸酶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。
在本申请中，术语“Cas9核酸酶”通常也称为Cas9蛋白，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核酸酶通常包括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核酸酶。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。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括变体、衍生物、类似物、同源物及其片段的用途。
在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于“同一性”。同源序列可以包括可以与主题序列是至少70％、75％、80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基 酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“重组体”通常是指不同来源的核酸或蛋白连接形成的重组核酸或重组蛋白。重组体通常具备各组成部分的功能或性质。
在本申请中，术语“修饰”通常是指将编码异常功能蛋白的基因(例如，突变的RPGR基因)改变为编码正常功能蛋白的基因的操作。所述编码正常功能蛋白的基因可以为野生型的基因，也可以为经过密码子优化的基因。所述修饰可以包括但不限于一个或多个核苷酸变化(包括取代、插入或缺失)。
在本申请中，术语“尿液肾上皮细胞”通常是指提取自尿液的肾上皮细胞，其可以被诱导为多能干细胞(iPSC)。
在本申请中，术语“3D-视网膜类器官”通常是指一种具有三维结构、能够自我更新、自我组织并显示视网膜基本功能(例如，感受光)的人工培育的视网膜。3D-视网膜类器官可以由原代组织或干细胞(例如，多功能干细胞)分化而成，具有视网膜中所有接收光线并向大脑发出信号所必需的细胞。
在本申请中，术语“导入”通常是指将核酸分子转移到原核细胞或真核细胞中，其中所述核酸分子可以被并入到细胞的基因组(例如，染色体、质粒、质体或者线粒体DNA),转变为自主复制子，或者表达。导入可包括方法如“感染”、“转染”、“转化”和“转导”。合适的导入的方法可包括磷酸钙转染、DEAE-Dextran、核转染(nucleofection)、磁转染、电穿孔、脂质体介导的转染以及使用病毒载体的转导，例如痘苗病毒，或者对于昆虫细胞使用的杆状病毒。
在本申请中，术语“注射”通常是指通过穿刺受试者(例如，人或动物)的皮肤或粘膜而传送含有目标物质(例如，所述gRNA、所述质粒和/或所述靶向载体)的液体的过程。注射包括使用任何可接受的形式，例如，腹膜内注射、肌内注射、皮下注射、皮下输注、眼内注射、视网膜注射、视网膜下注射、玻璃体注射和/或硬膜外注射。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其能够将插入的核酸分子(例如，外源性序列)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。本申请所述的载体可以包括，例如，表达载体，可包括病毒载体(慢病毒载体和/或逆转录病毒载体)，噬菌体载体，噬菌粒，粘粒，cosmid，人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)，和/或质粒。
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
gRNA
一方面，本申请提供一种特异性靶向编码视网膜色素变性GTP酶调节剂的基因(RPGR基因)的gRNA，其特异性结合所述RPGR基因的第14号内含子。
在某些情形中，所述gRNA可特异性结合SEQ ID NO:102所示的核苷酸序列。在某些情形中，所述gRNA可特异性结合与SEQ ID NO.102所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在某些情形中，所述gRNA可特异性结合与SEQ ID NO:102所示的核苷酸序列互补的核苷酸序列。在某些情形中，所述gRNA可特异性结合与SEQ ID NO.102所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列互补的核苷酸序列。
本申请所述的gRNA可以与目标靶核酸(例如，所述RPGR基因的第14号内含子)中的序列结合。gRNA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。
在本申请中，所述gRNA可包括SEQ ID NO.105-126中任一项所示的核苷酸序列。本申请中，所述gRNA可包括与SEQ ID NO.105-126中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在本申请中，所述gRNA自5’端至3’端可包含(X)n、SEQ ID NO.105-126中任一项所示的核苷酸序列和骨架序列，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。在本申请中，所述gRNA可包含5’-(X)n-SEQ ID NO:105-126任一项所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
本申请使用的骨架序列可以来自任何商购可得的质粒，只要其可表达Cas核酸酶和转录gRNA即可。例如，本申请所述的骨架序列可以包括来自AAV-saCas9-puro的骨架序列。例如，所述骨架序列可包含SEQ ID NO:103所示的核苷酸序列。
在某些情形中，所述gRNA可以为单链向导RNA(sgRNA)。
本申请提供了一种或多种分离的核酸分子，所述分离的核酸分子可编码上文所述的特异性靶向RPGR基因的gRNA。
本申请提供了一种编码DNA核酸内切酶的核酸分子。所述DNA核酸内切酶可以包括脱氧核糖核酸内切酶I，脱氧核糖核酸内切酶II，脱氧核糖核酸内切酶IV，限制性内切酶，UvrABC核酸内切酶，和/或工程化核酸酶。工程化核酸酶的例子有，包括但不限于，归巢核酸内切酶(也称为兆核酸酶或大范围核酸酶，Meganuclease)，锌指核酸酶(zinc finger nuclease，ZFN)，转录激活子样效应因子核酸酶(transcription activator-like effector-based nuclease，TALEN)，规律性间隔的短回文序列重复簇(Clustered regularly interspaced short palindromic repeat，CRISPR)。
在本申请中，所述DNA核酸内切酶可包括Cas核酸酶。例如，所述DNA核酸酶可包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
在本申请中，所述gRNA序列可以设计成与所述Cas核酸酶可识别的PAM序列临近处的靶核酸杂交。所述gRNA可以与靶序列完全互补或不完全互补。gRNA与其相应的靶序列之间的互补程度至少为约50％(例如，至少为约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约98％、或更多)。Cas蛋白通常都有一个可以在目标 DNA(例如，靶序列)中识别的特定的PAM序列。例如，所述PAM可包含SEQ ID NO:23-41中任一项所示的核苷酸序列。
所述DNA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。
本申请所述的gRNA和/或分离的核酸分子可以使用载体递送。在本申请中，DNA核酸内切酶可以作为一种或多种多肽单独地递送。或者，编码所述DNA核酸内切酶的核酸分子，与一种或多种引导RNA，或一种或多种crRNA以及tracrRNA，单独地递送，或者一起预复合地递送。例如，所述本申请的核酸分子(例如，编码所述特异性靶向RPGR基因的sgRNA的分离的核酸分子)和编码Cas9核酸酶的核酸分子可以位于同一载体(例如，质粒)中。所述载体可包括本领域已知的病毒或非病毒载体。
非病毒递送载体可以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。
在本申请中，所述分离的核酸分子和/或所述编码DNA核酸内切酶的核酸分子可以通过质粒递送。
在某些情形中，所述质粒可以为病毒载体，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。
另一方面，本申请提供了一种质粒，所述质粒可包括所述分离的核酸分子。在某些情形中，所述质粒可包括编码DNA核酸内切酶的核酸分子。所述分离的核酸分子(例如，编码所述特异性靶向RPGR基因的sgRNA的分离的核酸分子)和编码Cas9核酸酶的核酸可以位于同一质粒。所述分离的核酸分子(例如，编码所述特异性靶向RPGR基因的sgRNA的分离的核酸分子)和编码Cas9核酸酶的核酸分子可以位于不同的质粒。
ORF15和靶向载体
另一方面，本申请还提供了一种核酸分子，其可包含人RPGR ORF15核苷酸序列。本申请所述可包含人RPGR ORF15核苷酸序列的“核酸分子”与本申请所述特异性靶向RPGR基因的sgRNA的“分离的核酸分子”不同。
本申请所述人RPGR ORF15核苷酸序列可以是野生型的人RPGR ORF15核苷酸序列。例如，所述野生型的人RPGR ORF15核苷酸序列可以包含SEQ ID NO:101所示的核苷酸序列。
本申请所述人RPGR ORF15核苷酸序列可以是经过密码子优化的人RPGR ORF15核苷酸序列。例如，所述密码子优化的人RPGR ORF15核苷酸序列可以包含SEQ ID NO:44所示的核苷酸序列。例如，所述密码子优化的人RPGR ORF15核苷酸序列可以包含与SEQ ID NO:44所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。所述密码子优化可提高所述人RPGR ORF15的序列稳定性。例如，经过所述密码子优化后，所述人RPGR ORF15导入受试者基因组中后，在基因组中的序列完整程度提高、序列正确程度提高和/或扩增情况提高至少10％(例如，至少15％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或更高)。
另一方面，本申请提供了一种靶向载体，其可包括包含所述人RPGR ORF15核苷酸序列的所述核酸分子。
本申请中，所述靶向载体可包括5’上游靶区域。本申请所述5’上游靶区域可包括人RPGR基因的第14内含子。本申请所述5’上游靶区域还可包括所述gRNA的靶区域。例如，所述5’上游靶区域自5’端至3’端可包括所述gRNA的靶区域和所述人RPGR基因的第14内含子。
在本申请中，所述5’上游靶区域可包含核苷酸突变。例如，所述5’上游靶区域的人RPGR基因的第14内含子可包括核苷酸突变。所述核苷酸突变可以发生在所述gRNA的靶区域处。例如，所述核苷酸突变可以不发生在所述5’上游靶区域与所述靶向载体包含的所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)的交界处。例如，所述核苷酸突变可发生在与所述靶向载体包含的所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)相隔至少约50个核苷酸(例如，至少约60个核苷酸、约70个核苷酸、约80个核苷酸、约90个核苷酸、约100个核苷酸、约110个核苷酸、约120个核苷酸、约130个核苷酸、约140个核苷酸、约150个核苷酸、约160个核苷酸、约170个核苷酸或更多个氨基酸)处。例如，所述突变可不发生在所述人RPGR基因的第14内含子和所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)的交界处。
本申请中，所述靶向载体可包括3’下游靶区域。所述3’下游靶区域可包括3’非编码区和/或所述gRNA的靶区域。例如，所述3’下游靶区域自5’端至3’端可包括所述3’非编码区和所述gRNA的靶区域。
在本申请中，所述3’下游靶区域可包含核苷酸突变。所述核苷酸突变可以发生在所述gRNA的靶区域处。例如，所述核苷酸突变可以不发生在所述3’下游靶区域与所述靶向载体包含的所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)的交界处。例如，所述3’下游靶区域的所述核苷酸突变可发生在与所述靶向载体包含的所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)相隔至少约50个核苷酸(例如，至少约60个核苷酸、约70个核苷酸、约80个核苷酸、约90个核苷酸、约100个核苷酸、约110个核苷酸、约120个核苷酸、约130个核苷酸、约140个核苷酸、约150个核苷酸、约160个核苷酸、约170个核苷酸或更多个氨基酸)处。
在本申请中，所述核苷酸突变可以提高所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)被导入受试者的基因组的准确率。例如，所述准确率可包括所述核酸分子在所述受试者基因组的位置的准确率和/或所述核酸分子在所述受试者基因组的序列完整性。
例如，所述5’上游靶区域可包含SEQ ID NO:96-99中任一项所示的核苷酸序列。例如， 所述5’上游靶区域可包含与SEQ ID NO:96-99中任一项所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
例如，所述3’下游靶区域可包含SEQ ID NO:100所示的核苷酸序列。例如，所述3’下游靶区域可包含与SEQ ID NO:100所示的核苷酸序列具有至少70％(例如，至少75％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的核苷酸序列。
在本申请中，所述靶向载体可包括所述5’上游靶区域，所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)，以及所述3’下游靶区域。在本申请中，所述靶向载体自5’端至3’端可依次包括所述5’上游靶区域，所述核酸分子(例如，所述包含所述人RPGR ORF15核苷酸序列的核酸分子)，以及所述3’下游靶区域。
细胞、组织模型、用途和方法
本申请提供了细胞，所述细胞可包含所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。本申请所述的细胞可表达具备正常功能的RPGR蛋白。所述细胞可以包括哺乳动物细胞，例如，来自人的细胞。例如，所述细胞可包括COS细胞、COS-1细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、HEK293细胞、NS0细胞或骨髓瘤细胞、干细胞(例如，多能干细胞和/或全能干细胞)、和/或上皮细胞(例如，肾上皮细胞和/或视网膜上皮细胞)。在本申请中，所述的细胞可包括HEK细胞和/或尿液肾上皮细胞。在本申请中，所述的细胞可以经修饰后具备分化能力。所述分化能力可包括分化成身体任何细胞类型的能力：神经元、星形胶质细胞、少突胶质细胞、视网膜上皮细胞、表皮、毛发和角质形成细胞、肝细胞、胰岛β细胞、肠上皮细胞、肺泡细胞、造血细胞、内皮细胞、心肌细胞、平滑肌细胞、骨骼肌细胞、肾细胞、脂肪细胞、软骨细胞和/或骨细胞。例如，所述细胞可被重编程为具有关键重编程基因(例如，OCT4、KLF4、SOX2、cMYC、NANOG和/或LIN28)过表达的诱导多能干细胞(iPSC)。
本申请所述细胞可用于评价基因编辑治疗所需物质(例如，sgRNA和CRISPR系统)的有效性和安全性。
本申请提供了组织模型，所述组织模型可包括包含正确的人RPGR cDNA的3D-视网膜类器官。所述组织模型可用于评价基因编辑治疗所需物质(例如，sgRNA和CRISPR系统)的有效性和安全性。
本申请提供了所述的细胞，和/或所述的组织模型在评价基因编辑治疗有效性和/或安全性中的用途。例如，将编码所述gRNA的多核苷酸、质粒、包含密码子优化的ORF15的核酸分子和/或靶向载体到所述细胞和/或所述组织模型后，所述能检测到gRNA、密码子优化的ORF15的表达，例如，使用PCR测序或凝胶电泳；或者，该细胞和/或组织模型不产生免疫排斥反应、毒性，和/或，导入的物质不影响所述细胞和/或所述组织模型的其他功能。例如，可使用修复效率检测作为评价基因编辑有效性的指标，示例性的方法如实施例3所示。例如，可检测脱靶效率作为评价基因编辑安全性的指标，例如，可使用全基因组测序法检测脱靶效率。本申请提供了所述的gRNA，所述的质粒，所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子，和/或所述的靶向载体在制备治疗疾病的药物中的应用，其中所述疾病包括RPGR基因突变所导致的疾病。例如，所述疾病可包括视网膜色素变性。例如，所述疾病可包括X连锁遗传的视网膜色素变性。
本申请提供了一种修饰RPGR基因的方法，所述方法可包括以下的步骤：导入所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。
本申请提供了一种治疗视网膜色素变性的方法，所述方法可包括以下的步骤：向有需要的受试者导入所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子。例如，所述视网膜色素变性可包括X连锁遗传的视网膜色素变性。
本申请所述方法可包括将所述的gRNA，所述的质粒，所述包含密码子优化的人RPGR ORF15核苷酸序列的核酸分子，和/或所述的靶向载体配制成组合物。这些组合物还可包含药学上可接受的载体、稀释剂、赋形剂、缓冲剂、稳定剂或本领域公知的其它物质。此类材料应当是无毒的，并且不应干扰活性成分的功效。本领域技术人员根据施用途径(例如，视网膜下注射、直接视网膜注射或玻璃体内注射)可以确定载体或其它物质的确切性质。
本申请所述的调配物可通过各种方法导入，例如，包括但不限于，玻璃体内注射(例如，前部、中间或后部玻璃体注射)、结膜下注射、前房内注射、经由颞侧注射到前房中、基质内注射、注射到脉络膜下间隙中、角膜内注射、视网膜下注射和眼内注射局部地投予眼睛。所述导入可包括视网膜下注射，视网膜下注射为注射到视网膜下空间，即感觉神经性视网膜下面。在视网膜下注射期间，将注射的材料(例如，所述的靶向载体，所述的gRNA，和/或所述的质粒)直接导入感光细胞和视网膜色素上皮(RPE)层之间，并在其间创建空间。
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RPGR基因的突变位点内或附近进行编辑，使得其不编码具 有突变的RPGR ORF15。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RPGR基因的突变位点内或附近进行编辑，使得其不具有突变的RPGR ORF15。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RPGR基因的突变位点内或附近进行编辑，使得其不具有突变的RPGR ORF15。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。
本申请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，例如同源重组(Homology directed repair，HDR)，非同源末端连接(Non-Homologous End Joining，NHEJ)，和微同源介导的末端连接(Microhomology-Mediated End Joining，MMEJ)。
本申请所述的方法可包括在目标基因座中靠近靶序列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。所述断裂可通过定点(site-directed)多肽来实现。定点多肽(例如DNA核酸内切酶)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。
在某些情况下，可以使用HDR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。HDR利用同源序列或供体序列(例如，所述靶向载体)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、双链寡核苷酸或病毒。例如，所述供体可以包含本申请所述靶向载体。这些外源核酸可以包含与DNA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)。
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，所述靶向载体)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，外源供体可以由质粒载体递送，例如，AAV载体和/或TA克隆载体(例如，ZT4载体)。
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。还可以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。
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体外筛选有编辑效率的gRNA
针对人RPGR第14内含子设计22条gRNA，分别为gRNA-1至gRNA-22，对应的靶点分别为R1至R22，序列见下表1，其中1和-1代表第14内含子靶区域双链中的正反两条链。
表1 gRNA结合的靶点
Figure PCTCN2020105553-appb-000001
Figure PCTCN2020105553-appb-000002
将上述22个gRNA(gRNA识别的靶位点分别编号为R1至R22)分别构建到AAV-saCas9-puro载体(载体为pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA，来自addgene，编号为#61592，对载体进行改造使其获得嘌呤霉素puro抗性)上，转染至HEK293A细胞，用嘌呤霉素筛选后提取基因组DNA，在靶点附近设计引物PCR测序，引物为hRPGR-exin14-F和hRPGR-exin14-R，序列如下表2所示。AAV-saCas9-puro载体图谱如图14所示
表2 PCR鉴定引物序列
引物名称 引物序列
hRPGR-exin14-F GAACTGACGCAGGATACA(SEQ ID NO:42)
hRPGR-exin14-R CTGACAAGCGATCACATT(SEQ ID NO:43)
将PCR测定产物用T7E1酶处理后回收产物，进行琼脂糖凝胶电泳定量分析切割突变效率。以靶向R13-R19的gRNA为例，突变率如图1和表3所示，说明本申请的gRNA均能切割RPGR第14内含子。以下选择结合R14、R15、R17和R19靶点的gRNA进行下一步的载体设计及筛选。
表3示例性位点的突变率
靶点 R13 R14 R15 R16 R17 R18 R19
突变率 0.32 0.67 0.54 0.37 0.58 0.27 0.53
实施例2构建人RPGR基因编辑优化载体
(1)构建打靶载体
人RPGR ORF15密码子优化序列的核苷酸序列如SEQ ID NO:44所示，针对该序列设计PCR扩增引物，上游引物HR-ORF15co-F在ORF15上游，下游引物HR-ORF15co-R在ORF15区下游。设计针对第14内含子的引物(HR-int14-F和HR-int14-R)和针对3’端非翻译区(3’UTR) 的引物(HR-3'UTR-F和HR-3'UTR-R)，序列如4所示。进行PCR扩增。
表4用于鉴定的引物序列
引物名称 引物序列
HR-int14-F GTTTTTCAGCAAGATGTCCTTTCTTTGGCAACTT(SEQ ID NO:45)
HR-int14-R TCTCCTCGGGGATCTCTGACAAGCGATCACATT(SEQ ID NO:46)
HR-ORF15co-F GTGATCGCTTGTCAGAGATCCCCGAGGAGAAGG(SEQ ID NO:47)
HR-ORF15co-R CACATTTAAGGTTTGTCACTTCAGCTCCAGGTAGTG(SEQ ID NO:48)
HR-3'UTR-F CTGGAGCTGAAGTGACAAACCTTAAATGTGACCCGAT(SEQ ID NO:49)
HR-3'UTR-R ATCTTCTAGAAAGATTATAGCCCTTAAGCATCTG(SEQ ID NO:50)
将扩增产物用诺唯赞公司的快速克隆试剂盒C115与试剂盒配套的线性载体进行多片段重组，转化铺板挑克隆测序，选测序正确的载体进行扩增，载体命名为ZRC03，序列为第14内含子-ORF15-3’UTR。
(2)打靶载体突变
用诺唯赞公司的Mut Express II Fast Mutagenesis Kit V2突变试剂盒，在ZRC03载体中，分别对第14内含子中的R14、R15、R17和R19靶点进行突变。
设计靶点突变引物，针对突变靶点R14的引物为Zt4-HRa14-mut-F(SEQ ID NO:51)和Zt4-HRa14-mut-R(SEQ ID NO:52)，针对突变靶点R15的引物为Zt4-HRa15-mut-F(SEQ ID NO:53)和Zt4-HRa15-mut-R(SEQ ID NO:54)，针对突变靶点R17的引物为Zt4-HRa17-mut-F(SEQ ID NO:55)和Zt4-HRa17-mut-R(SEQ ID NO:56)，以及，针对突变靶点R19的引物为Zt4-HRa19-mut-F(SEQ ID NO:57)和Zt4-HRa19-mut-R(SEQ ID NO:58)。
PCR扩增各打靶载体，使用Dpn1酶消化，去除甲基化模板质粒后进行重组、转化、涂板和克隆鉴定。
得到突变后的载体序列，R14靶点突变后的载体序列如SEQ ID NO:59所示，R15靶点突变后的载体序列如SEQ ID NO:60所示，R17靶点突变后的载体序列如SEQ ID NO:61所示，以及，R19靶点突变后的载体序列如SEQ ID NO:62所示。
(3)优化载体
在上述构建成功的相应靶点突变载体的基础上，在打靶区域的上下游分别加上R14、R15、R17和R19靶点序列，gRNA靶点序列插入14内含子的5’端和3’UTR的3’端。使用零背景ZT4-Blunt快速克隆试剂盒(北京庄盟国际生物基因科技有限公司，产品编号为ZC205)构建到ZT4载体，ZT4载体序列如SEQ ID NO:104所示，371bp后为载体插入位点。挑克隆 测序，分别相应得到编号为R145、R156、R176和R193的优化载体。针对R14突变的优化载体的引物序列为hR-saHITI-F14(SEQ ID NO:63)和hR-saHITI-R14(SEQ ID NO:64)，针对R15突变的优化载体的引物序列为hR-saHITI-F15(SEQ ID NO:65)和hR-saHITI-R15(SEQ ID NO:66)，针对R17突变的优化载体的引物序列为hR-saHITI-F17(SEQ ID NO:67)和hR-saHITI-R17(SEQ ID NO:68)，以及，针对R19突变的优化载体的引物序列为hR-saHITI-F19(SEQ ID NO:69)和hR-saHITI-R19(SEQ ID NO:70)。
实施例3检测体外基因编辑修复效率
将ZT4优化载体和AAV-saCas9-U6-sgRNA载体共转到HEK293A细胞系中，流式分选后培养提取基因组DNA进行鉴定。鉴定引物序列如表5所示，鉴定示意图如图15所示，其中，5’outer是插入的密码子优化的RPGR ORF15的5’端附近上下游序列。
表5用于鉴定的引物序列
引物名称 引物序列
HRco-KJJD-F(P4-F) GGCTGACACTGATGGAGA(SEQ ID NO:71)
HRco-KJJD-R(P4-R) GGATCTAAGCTCTGAACACA(SEQ ID NO:72)
HR-5’outer-F(P4-F) GGCTGACACTGATGGAGA(SEQ ID NO:71)
HR-5’outer-R(P5-R) CAGATCGTCGGACAGGAT(SEQ ID NO:73)
HR-3’outer-F(P6-F) CAATGGCAAGGAGCAGAG(SEQ ID NO:74)
HR-3’outer-R(P6-R) TGTCTGTAAGGTCATCTGATAG(SEQ ID NO:75)
(1)全长跨界引物(HRco-KJJD-F/R引物对)鉴定：R145，R156，R176和R193这四个候选载体均能扩增出3kb的阳性条带(图2A)。
(2)5’outer(HRco-KJJD-F与HR-5’outer-R引物对)和3’outer(HR-3’outer-F与HR-3’outer-R引物对)编辑PCR鉴定：结果如图2B和图2C所示，阳性条带由星号(*)标出。
(3)将片段鉴定产物TA克隆后测序分析克隆序列检测编辑效果，结果如图3所示，其中，图3A为R145的测序结果，图3B为R156的测序结果，图3C为R176的测序结果，图3D为R193的测序结果，图3各种中第一条序列为正确的序列，说明打靶区域正确插入宿主细胞的基因组中并表达。
实施例4检测修复细胞的分选效率
(1)构建带有GFP表达盒的HITI载体，即在实施例2得到的优化载体上插入P2A-GFP。 具体来说，用无缝克隆试剂盒在ZT4载体(北京庄盟国际生物基因科技有限公司，产品编号为ZC205)中插入优化序列(该处的优化序列至包含部分第14内含子+ORF15优化序列的序列)、P2A-GFP和3’UTR序列，部分第14内含子+ORF15优化序列的引物为ZT4-HR-int14co-F(SEQ ID NO:76)和ZT4-HR-int14co-R(SEQ ID NO:77)，P2A-GFP的引物为ZT4-HR-P2AEG-F(SEQ ID NO:78)和ZT4-HR-P2AEG-R(SEQ ID NO:79)，以及，3’UTR的引物为ZT4-HR-3'UTR-F(SEQ ID NO:80)和ZT4-HR-3'UTR-R(SEQ ID NO:81)。得到HITI载体，对应R14的载体编号为145G、对应R15的载体编号为156G、对应R17的载体编号为176G和对应R19的载体编号为193G。
(2)将AAV-saCas9-sagRNA、HITI-GFP打靶载体和pMcherry-N1载体(美国Clontech公司，产品编号632523)共转到HEK293A细胞系中，通过GFP流式分选评价效率。第一轮分选Mcherry后继续培养，第二轮分选GFP。pMcherry-N1载体可以表达Mcherry蛋白，在荧光显微镜下可以观察到红色荧光。比较四组GFP阳性细胞百分比，结果显示，四组阳性细胞数百分率无统计学意义(图4)。GFP分选后的细胞荧光如图5所示，其中，四组细胞GFP分选后各自的荧光如图6所示。说明四组细胞均能表达GFP蛋白，分选效率好。
实施例5不同引物对的修复效率检测
将实施例2的ZT4优化载体和AAV-saCas9-U6-sgRNA载体共转到HEK293A细胞系中，流式分选后培养提取RNA并反转录cDNA进行修复效果鉴定。鉴定RPGR不同转录本的引物序列如表6所示。其中，C、C1和5R引物对用来鉴定优化修复引物；45F和45F1引物对用来鉴定RPGR ex1-19转录本和RPGR ORF15转录本的公共外显子；56F和56F1引物对用来鉴定RPGRex 1-19转录本；O15F和O15F1引物对用来鉴定RPGR ORF15转录本。
表6用于鉴定的引物序列
Figure PCTCN2020105553-appb-000003
Figure PCTCN2020105553-appb-000004
PCR鉴定结果如图7所示。图7显示45F和45F1引物对、56F和56F1引物对、O15F和O15F1引物对均有良好的特异性，也表明野生型HEK293A细胞系中存在RPGR ex1～19转录本和RPGR ORF15转录本的表达。而C引物对在野生型HEK293A细胞系中能够鉴定编辑效果，表明特异性差。
(2)鉴定修复序列(C引物对，C1引物对和5R引物对)和野生型ORF15(O15F引物对)序列。PCR结果表明C引物对特异性差，C1引物对和5R引物对均能在基因编辑组中检测出密码子优化的ORF15阳性条带，在野生型HEK293A细胞系中和对照组细胞中不能检测出密码子优化的ORF15的阳性条带。结果如图8和图9所示。
(3)测序结果比对，将5R引物对扩增的G145，G156，G176和G193组的PCR产物正向测序，176条带弱没有信号，其余比对正确，表明剪切正常。
实施例6检测非密码子优化的RPGR ORF15的修复效率
对R14、R15、R17、R19靶位点的四个sgRNA设计包含非优化密码子即野生型RPGR ORF15(SEQ ID NO:101)和密码子优化RPGR ORF15(SEQ ID NO:44)的供体载体，PCR克隆并用GT115感受态细胞转化至HEK293细胞，在分析克隆测序结果中，我们发现，在同等条件下，密码子优化序列测序良好，得到正确序列的克隆多，而非优化序列测序信号差，测序比对如图10-13所示。
R14靶位点的gRNA对应的包含野生型RPGR ORF15的载体三个克隆(HR14WM1，HR14WM2和HR14WM3)测序，可以发现三个克隆测序结果中部分序列丢失(图10A-10C)。R15靶点的gRNA对应的包含野生型RPGR ORF15的载体三个克隆(HR15WM1，HR15WM2和HR15WM3)测序，HR15WM1和HR15WM3初步分段测序差，将HR15WM2进行分段测序比对，发现有部分序列丢失(图11)。R17靶点的gRNA对应的包含野生型RPGR ORF15的载体三个克隆(HR17WM1，HR17WM2和HR17WM3)测序，HR17WM2初步分段测序比对差，我们对HR17WM1和HR17WM3进行分段测序发现部分序列丢失(图12)。R19靶位点的gRNA对应的包含野生型RPGR ORF15的载体三个克隆(HR19WM1，HR19WM2和HR19WM3)测序，两个克隆进行初步分段测序时，HR19WM1和HR19WM3比对差，我们对HR19WM2进行分段测序发现部分序列丢失(图13)。结果显示，包含野生型RPGR ORF15载体的测序结果较差。通过具体分析包含野生型RPGR ORF15载体的扩增测序情况以及转染细胞后对细胞基因组测序，发现基因组中野生型RPGR ORF15序列的稳定性也差。而包含所述密码子优化的RPGR ORF15的载体测序结果好，序列完整，序列稳定性高。

Claims (35)

  1. 特异性靶向编码视网膜色素变性GTP酶调节剂的基因(RPGR基因)的gRNA，其特异性结合所述RPGR基因的第14号内含子。
  2. 根据权利要求1所述的gRNA，其特异性结合SEQ ID NO:102所示的核苷酸序列，或特异性结合与SEQ ID NO:102所示的核苷酸序列互补的核苷酸序列。
  3. 根据权利要求1-2中任一项所述gRNA，其中所述gRNA包含SEQ ID NO.105-126中任一项所示的核苷酸序列。
  4. 根据权利要求1-3中任一项所述gRNA，其中所述gRNA包含5’-(X)n-SEQ ID NO:105-126中任一项所示的核苷酸序列-骨架序列-3’，其中X为选自A、U、C和G中任一个的碱基，且n为0-15中的任一整数。
  5. 根据权利要求1-4中任一项所述gRNA，其中所述gRNA为单链向导RNA(sgRNA)。
  6. 一种或多种分离的核酸分子，其编码权利要求1-5中任一项所述的特异性靶向RPGR基因的gRNA。
  7. 质粒，其包含权利要求6所述的分离的核酸分子。
  8. 根据权利要求7所述的质粒，其为病毒载体。
  9. 根据权利要求7-8中任一项所述的质粒，其包括编码DNA核酸内切酶的核酸分子。
  10. 根据权利要求9所述的质粒，其中所述DNA核酸内切酶包括Cas核酸酶。
  11. 根据权利要求9-10中任一项所述的质粒，其中所述DNA核酸内切酶包括Cas9核酸酶、其同源物、其天然存在分子的重组体、其密码子优化版本，和/或其经修饰版本。
  12. 核酸分子，其包含密码子优化的人RPGR ORF15核苷酸序列。
  13. 根据权利要求12所述的核酸分子，其包含SEQ ID NO:44所示的核苷酸序列。
  14. 靶向载体，其包含权利要求12-13中任一项所述的核酸分子。
  15. 根据权利要求14所述的靶向载体，其包含1)5’上游靶区域；2)权利要求12-13中任一项所述的核酸分子；以及3)3’下游靶区域；其中所述5’上游靶区域和/或所述3’下游靶区域能够被权利要求1-4中任一项所述gRNA所识别和/或切割。
  16. 根据权利要求15所述的靶向载体，其中所述5’上游靶区域和/或所述3’下游靶区域包含核苷酸突变，所述突变提高了权利要求12-13中任一项所述的核酸分子被导入受试者的基因组的准确率。
  17. 根据权利要求15-16中任一项所述的靶向载体，其中所述5’上游靶区域包含SEQ ID NO:96-99中任一项所示的核苷酸序列。
  18. 根据权利要求15-17中任一项所述的靶向载体，其中所述3’下游靶区域包含SEQ ID NO:100所示的核苷酸序列。
  19. 细胞，其包含权利要求12-13中任一项所述的核酸分子。
  20. 根据权利要求19所述的细胞，其包括HEK293细胞和/或尿液肾上皮细胞。
  21. 根据权利要求19-20中任一项所述的细胞，其经修饰后具备分化能力。
  22. 根据权利要求19-21中任一项所述的细胞，其可分化为3D-视网膜类器官。
  23. 组织模型，其包括包含正确的人RPGR cDNA的3D-视网膜类器官。
  24. 权利要求19-22中任一项所述的细胞，和/或权利要求23所述的组织模型在评价基因编辑治疗有效性和/或安全性中的用途。
  25. 权利要求1-5中任一项所述的gRNA，权利要求7-11中任一项所述的质粒，权利要求12-13中任一项所述的核酸分子，和/或权利要求14-18所述的靶向载体在制备治疗疾病的药物中的应用，其中所述疾病包括RPGR基因中的突变所导致的疾病。
  26. 根据权利要求25所述的应用，其中所述疾病包括视网膜色素变性。
  27. 根据权利要求25-26中任一项所述的应用，其中所述疾病包括X连锁遗传的视网膜色素变性。
  28. 一种修饰RPGR基因的方法，所述方法包括以下的步骤：导入权利要求12-13中任一项所述的核酸分子。
  29. 一种治疗视网膜色素变性的方法，所述方法包括以下的步骤：向有需要的受试者导入权利要求12-13中任一项所述的核酸分子。
  30. 根据权利要求29所述的方法，其中所述视网膜色素变性包括X连锁遗传的视网膜色素变性。
  31. 根据权利要求29-30中任一项所述的方法，其中所述导入获得了正常功能的人视网膜色素变性GTP酶调节剂。
  32. 根据权利要求29-31中任一项所述的方法，其中所述导入包括导入权利要求14-18所述的靶向载体。
  33. 根据权利要求29-32中任一项所述的方法，其中所述导入包括导入权利要求1-5中任一项所述的gRNA，和/或权利要求7-11中任一项所述的质粒。
  34. 根据权利要求29-33中任一项所述的方法，其中所述导入包括注射。
  35. 根据权利要求29-34中任一项所述的方法，其中所述导入包括视网膜下腔注射，或玻璃体注射。
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