Nucleic acid molecules targeting CYP4V2 gene mutation sites and uses thereof

Abstract

The present application provides a gRNA targeting the CYP4V2 gene and a donor nucleic acid molecule containing a CYP4V2 gene fragment, and discloses the use of the gRNA and the donor nucleic acid molecule in the manufacture of a medicament for treating Bietti crystalline dystrophy.

Description

[Technical field]

[0001] The present invention relates to biopharmaceuticals, and more particularly to gRNA and donor nucleic acid molecules for treating CYP4V2 gene mutation diseases. [Background technology]

[0002] Bietti crystalline dystrophy (BCD), also known as crystalline retinitis pigmentosa, Bietti crystalline corneoretinal dystrophy, crystalline retinopathy, or Bietti's retinal dystrophy, is an autosomal recessively inherited retinal degenerative disease that leads to blindness. The CYP4V2 gene is one of the causative genes for BCD currently discovered (Li et al. Am J Hum Genet. 74:817-826, 200). CYP4V2 (cytochrome P450, family 4, subfamily V, polypeptide 2, also known as CYP4AH1) belongs to the cytochrome P450 superfamily and is a member of the heme-thiolate protein cytochrome P450 subfamily 4 (CYP4).

[0003] Currently, there are several treatments for this disease, such as gene replacement therapy, which uses a viral transfection system or other transfection systems (e.g., AAV, lentivirus, retrovirus) to transfect wild-type CYP4V2 genes into mutant cells, allowing the mutant cells to express wild-type CYP4V2. This method can only partially restore the function of mutant cells, and the effectiveness of the treatment is limited. This is because mutant gene products remain in the cells, and these mutant proteins competitively inhibit normal gene products. Therefore, the development of a safer and more effective treatment method is expected.

[0004] The information disclosed in the Background section is intended only to enhance understanding of the general background of the present application, and is not an admission or any form of suggestion that such information forms prior art publicly known to those skilled in the art. Summary of the Invention [Means for solving the problem]

[0005] The present application provides a gRNA that specifically targets cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2), the gRNA specifically binds to an intron region between exon 6 and exon 7 of the CYP4V2 gene, and the gRNA has a good cleavage effect on the intron region between exon 6 and exon 7 of the CYP4V2, so that the original CYP4V2 gene product is no longer present in the cell. The present application also provides a donor nucleic acid molecule, the donor nucleic acid molecule containing a nucleotide sequence between intron 6 and exon 11 of the CYP4V2 gene, and the donor nucleic acid molecule repairs CYP4V2 exons 7-11 in a gene-mutated cell after endogenous CYP4V2 is cleaved by the gRNA, thereby producing a CYP4V2 protein with normal function, and has an excellent repair effect. The present application provides a vector containing the gRNA and / or the donor nucleic acid molecule, which enables CYP4V2 mutant cells to accurately express cytochrome P450 family 4 subfamily V polypeptide 2 and has good gene editing repair efficiency.

[0006] On the other hand, the present application provides a gRNA that specifically targets the cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene, wherein the gRNA specifically binds to an intron region between exon 6 and exon 7 of the CYP4V2 gene.

[0007] In one embodiment, the gRNA specifically binds to the nucleotide sequence shown in SEQ ID NO:41.

[0008] In one embodiment, the gRNA comprises the nucleotide sequence of any of SEQ ID NOs:48-51.

[0009] In one embodiment, the gRNA comprises 5'-(X)n-SEQ ID NO:48-51-backbone sequence-3', where X is any base selected from A, U, C and G, and n is any integer from 0 to 15.

[0010] In one embodiment, the gRNA is a single-stranded guide RNA (sgRNA).

[0011] On the other hand, the present application provides one or more isolated nucleic acid molecules encoding gRNAs that specifically target the CYP4V2 gene.

[0012] On the other hand, the present application provides a donor nucleic acid molecule comprising a nucleotide sequence between intron 6 and exon 11 of the CYP4V2 gene.

[0013] In one embodiment, the donor nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:39.

[0014] On the other hand, the present application provides a vector containing the isolated nucleic acid molecule and / or the donor nucleic acid molecule.

[0015] In one embodiment, the vector is such that the isolated nucleic acid molecule and the donor nucleic acid molecule are present on the same vector.

[0016] In one embodiment, the vector is a viral vector.

[0017] On the other hand, the present application provides a cell containing the isolated nucleic acid molecule, the donor nucleic acid molecule and / or the vector.

[0018] In one embodiment, the cells comprise HEK293 cells, renal epithelial cells and / or induced pluripotent stem cells.

[0019] In one embodiment, the cells are modified to be differentiation potent.

[0020] In one embodiment, the cells are capable of differentiating into 3D-retinal organoids.

[0021] On the other hand, the present application provides a pharmaceutical composition comprising the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, the vector, and a pharma- ceutically acceptable carrier.

[0022] On the other hand, the present application provides a kit containing the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and the vector.

[0023] On the other hand, the present application provides the use of the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector in the manufacture of a medicament for the treatment of a disease, including a disease caused by a mutation in the CYP4V2 gene.

[0024] In one embodiment, the mutation is located after the intron between exon 6 and exon 7 of the CYP4V2 gene.

[0025] In one embodiment, the disease comprises Bietti crystalline dystrophy.

[0026] On the other hand, the present application provides a method for treating Vietti crystalline dystrophy, comprising the step of introducing the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector into a subject in need thereof.

[0027] In one embodiment, the introduction results in a normally functional CYP4V2 protein.

[0028] In one embodiment, the introducing comprises an injection.

[0029] In one embodiment, the introducing comprises a subretinal injection.

[0030] On the other hand, the present application provides a method for regulating expression of the CYP4V2 gene in a cell, comprising introducing the gRNA, the one or more isolated nucleic acid molecules, and / or the vector into a cell.

[0031] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. Those skilled in the art will appreciate that, upon learning the contents of the present application, they may make changes to the specific embodiments disclosed without departing from the spirit and scope of the present invention. Therefore, the drawings and description of the present application are illustrative only and not restrictive.

[0032] Particular features of the present invention are set forth in the appended claims and may be more clearly understood by reference to the following detailed description of exemplary embodiments and drawings, which are briefly described as follows: [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 shows the plasmid map of PX601 described in the present application. [Diagram 2] FIG. 1 shows an electrophoretic image of fragments cleaved by CYP4V2-HITI-sgRNA1-7 described in the present application, and shows that sgRNA1-4 has a good cleavage effect. [Figure 3A] FIG. 1 shows the results of sequencing the fragment containing the cleavage site after CYP4V2 sgRNA1-4 cleavage described in the present application, demonstrating that sgRNA1-4 has good cleavage efficiency. [Figure 3B]FIG. 1 shows the results of sequencing the fragment containing the cleavage site after CYP4V2 sgRNA1-4 cleavage described in the present application, demonstrating that sgRNA1-4 has good cleavage efficiency. [Figure 3C] FIG. 1 shows the results of sequencing the fragment containing the cleavage site after CYP4V2 sgRNA1-4 cleavage described in the present application, demonstrating that sgRNA1-4 has good cleavage efficiency. [Figure 3D] FIG. 1 shows the results of sequencing the fragment containing the cleavage site after CYP4V2 sgRNA1-4 cleavage described in the present application, demonstrating that sgRNA1-4 has good cleavage efficiency. [Figure 4] FIG. 1 shows a map of the minigene fragment described in this application. [Diagram 5] FIG. 1 shows the plasmid map of PMD-19T-MCS described in the present application. [Figure 6A] FIG. 1 shows the effect of sgRNA1-4 described in the present application on splicing, and shows that sgRNA1-4 do not affect mRNA splicing. [Figure 6B] FIG. 1 shows the effect of sgRNA1-4 described in the present application on splicing, and shows that sgRNA1-4 do not affect mRNA splicing. [Figure 6C] FIG. 1 shows the effect of sgRNA1-4 described in the present application on splicing, and shows that sgRNA1-4 do not affect mRNA splicing. [Figure 7] FIG. 1 shows the results of transfected cells in the donor screening experiment described herein, showing that donor-containing vectors successfully transfected cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Hereinafter, embodiments of the present invention will be described with reference to certain specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the contents disclosed herein.

[0035] Definition of Terms In this application, the term "3D-retinal organoid" generally refers to the artificially cultured retina that has a three-dimensional structure, can self-renew, self-organize, and shows the basic functions of the retina (e.g., photosensitivity).3D-retinal organoid can be differentiated from primary tissue or stem cells (e.g., pluripotent stem cells), and has all the cells of the retina that are necessary to respond to light and send signals to the brain.

[0036] As used herein, the term "isolated nucleic acid molecule" refers to a molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. Such an isolated nucleic acid molecule is one that has been removed or separated from its normal or natural environment, or has been produced in a manner that does not cause the molecule to be present in its normal or natural environment, and is separated from polypeptides, peptides, lipids, carbohydrates, other polynucleotides, and other substances in the normal or natural environment. The isolated nucleic acid molecule of the present application can encode an RNA, for example, a gRNA that specifically targets the CYP4V2 gene.

[0037] As used herein, the term "donor nucleic acid molecule" generally refers to a nucleic acid molecule that provides a heterologous nucleic acid sequence to a recipient (eg, a recipient nucleic acid molecule).

[0038] In this application, the term "Vietti crystalline dystrophy" generally refers to a group of autosomal recessive genetic eye diseases. The main symptoms include crystals in the cornea (transparent membrane), fine yellow or white crystalline deposits deposited in the light-sensitive tissue of the retina, and progressive atrophy of the retina, choriocapillaris, and choroid. Vietti crystalline dystrophy can include diseases caused by CYP4V2 gene mutations.

[0039] As used herein, the term "kit" generally refers to two or more components packaged in a container, receptacle or other vessel, one of which corresponds to a gRNA, one or more isolated nucleic acid molecules, donor nucleic acid molecules and / or vectors, pharmaceutical compositions or cells described herein. As such, a kit can be described as a set of products and / or equipment sufficient to accomplish a particular goal, which can be sold as a single unit.

[0040] In this application, the term "cell" has its commonly accepted meaning in the art. The term is used in its normal biological sense and does not refer to an intact multicellular organism, such as a human specifically. Cells can be present in avian, plant and mammalian organisms, such as humans, cows, sheep, apes, monkeys, pigs, dogs and cats. Cells can be pronuclear (e.g., bacterial cells) or eukaryotic (e.g., mammalian or plant cells). Cells can be of somatic or germ cell origin, totipotent or pluripotent, dividing or non-dividing. Cells can be derived from or include gametes or embryos, stem cells, or fully differentiated cells.

[0041] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a subject in need thereof. For example, a pharmaceutical composition described herein may contain a gRNA described herein, one or more isolated nucleic acid molecules described herein, a donor nucleic acid molecule described herein, and / or a vector described herein, and a pharmaceutically acceptable carrier. The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to a subject, e.g., a mammalian subject, in need of administration of a pharmaceutical composition of the present application. Animal subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, yellow cattle, dairy cows, etc., e.g., mice. In one embodiment, the pharmaceutical composition includes a composition for subretinal, parenteral, transdermal, intracavitary, intraarterial, intramembrane, and / or intranasal administration or for injection directly into a tissue. For example, the pharmaceutical composition is administered to the subject by subretinal injection.

[0042] In the present application, the term "induced pluripotent stem cells" generally refers to cells that have been converted under certain conditions from somatic cells to a totipotent state. The totipotency refers to the ability to differentiate into any type of cell in the body, to form a complete embryo, or to develop into a new individual. For example, in the present application, the induced pluripotent stem cells include those that have the ability to differentiate into retinal cells, which can be obtained by culturing renal epithelial cells.

[0043] In this application, the term "vector" generally refers to a nucleic acid molecule that can carry another nucleic acid that is linked to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA circle into which other DNA fragments can be ligated. For example, the PMD-19T-MCS plasmid constructed as described herein. Another type of vector is a "viral vector", which refers to a circular double-stranded DNA circle into which other DNA fragments can be ligated into a viral genome. For example, the AAV viral vector constructed as described herein.

[0044] In the present application, the term "CYP4V2" generally refers to a protein that is a member 2 of cytochrome P450 family 4 subfamily V. The term "cytochrome P450", which may also be called cytochrome P450 or CYP450, generally refers to a family of hemoproteins, a type of monooxygenase, involved in the metabolism of endogenous or exogenous substances, including drugs and environmental compounds. According to the homology of amino acid sequences, the members are classified into three groups: family, subfamily, and individual enzyme. The cytochrome P450 enzyme system can be abbreviated as CYP, where family is represented by Arabic numerals, subfamily is represented by capital letters, and individual enzyme is represented by Arabic numerals, for example, CYP4V2 in the present application. The human CYP4V2 gene (HGNC:23198; NCBI ID:285440) is 19.28 kb long, located at 4q35, has 11 exons, and plays an important role in fatty acid metabolism (Kumar S., Bioinformation,2011,7:360-365). The CYP4V2 described in this application may include its functional variants, fragments, homologs, etc. CYP4V2 is expressed in almost all tissues, but is expressed at high levels in the retina and retinal pigment epithelium and at somewhat lower levels in corneal tissue. Mutations in the CYP4V2 gene may be associated with Vietti crystalline dystrophy and / or retinitis pigmentosa.

[0045] In the present application, the term "gRNA" generally refers to a guide RNA, which is an RNA molecule. In nature, crRNA and tracrRNA usually exist as two separate RNA molecules, constituting gRNA. The term "crRNA", which may be referred to as CRISPR RNA, generally refers to a stretch of nucleotide sequence complementary to the targeted target DNA, and the term "tracrRNA" generally refers to a scaffold-type RNA that can bind to Cas nuclease. The crRNA and tracRNA may also be fused into a single strand, in which case the gRNA is also referred to as a single guide RNA (sgRNA). Since sgRNA has become the most common form of gRNA used by those skilled in the art in CRISPR technology, the terms "sgRNA" and "gRNA" may have the same meaning in the present application. sgRNA can be artificially synthesized or produced in vitro or in vivo from a DNA template. The sgRNA can bind to a Cas nuclease and target a target DNA, directing the Cas nuclease to cleave the DNA site complementary to the gRNA.

[0046] In this application, the term "HEK293 cells" generally refers to "human embryonic kidney cells 293," which are cells derived from human embryonic kidney cells. They are characterized by being easy to culture and having high transfection efficiency, and are a cell line that is commonly used in the technical field for research on exogenous genes.

[0047] In this application, the term "renal epithelial cells" generally refers to kidney epithelial cells collected from human urine. In this application, it is the source of induced pluripotent stem cells. In the art, the induction of pluripotent stem cells using renal epithelial cells in urine is cost-effective, versatile, and can be adapted to various ages, genders, and races. With this technology, it is much easier and more economical to obtain a large number of patient samples than other existing methods.

[0048] In the present application, the term "subretinal injection" generally refers to the introduction of a substance between photoreceptor cells and the retinal pigment epithelium (RPE) layer. During subretinal injection, a substance (e.g., a gRNA described herein, one or more isolated nucleic acid molecules described herein, a donor nucleic acid molecule described herein, a vector described herein, and a pharma- ceutically acceptable carrier) is introduced between the photoreceptor cells and the retinal pigment epithelium (RPE) layer, forming a space therebetween.

[0049] In addition to the specific proteins and nucleic acid molecules described herein, the present application includes functional variants, derivatives, analogs, homologs and fragments thereof.

[0050] The term "functional variant" refers to a polypeptide having essentially the same amino acid sequence as a naturally occurring sequence or encoded by essentially the same nucleotide sequence and having one or more activities of the naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (either amino acid residues or nucleotide residues) has been modified such that said polypeptide or polynucleotide essentially retains at least one endogenous function. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide, as long as the original functional activity is retained.

[0051] As used herein, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that includes any substitution, mutation, modification, substitution, deletion and / or addition of one (or more) amino acid residues from / to the sequence, so long as the resulting polypeptide or polynucleotide essentially retains at least one endogenous function thereof.

[0052] As used herein, the term "analog", generally with respect to a polypeptide or polynucleotide, includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that has at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0053] Typically, the modified sequence can have, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence essentially retains the desired activity or ability. The amino acid substitutions can be non-naturally occurring analogs.

[0054] Proteins or polypeptides used herein may also have deletions, insertions or substitutions of amino acid residues that result in non-phenotypic changes to said amino acid residues and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, so long as the intrinsic function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with similar hydrophilicity values ​​that do not have an electrically polar head group include asparagine, glutamine, serine, threonine and tyrosine.

[0055] In the present application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has a certain homology with the amino acid sequence to which it is compared and with the nucleotide sequence to which it is compared. The term "homology" may be understood equivalently to sequence "identity". A homologous sequence may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% homologous to the subject amino acid sequence. Typically, a homolog contains an active site, etc. that is homologous to the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions) or may be expressed in terms of sequence identity. In this application, a sequence having a percent identity to any one of the SEQ ID NOs of a referenced amino acid or nucleotide sequence refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NO.

[0056] To determine sequence identity, sequence alignment can be performed. This can be done in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment over the entire length of the sequences being compared.

[0057] In this application, the term "and / or" should be understood to mean either one or both of the alternatives.

[0058] In this application, the terms "comprise" or "comprise" generally refer to the inclusion of the explicitly specified features, but are not intended to exclude other elements.

[0059] In this application, the term "about" generally refers to a variation within 0.5%-10% above or below the specified numerical value, for example, a variation within 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% above or below the specified numerical value.

[0060] Detailed Description of the Invention gRNA On the other hand, the present application provides a gRNA that specifically targets the cytochrome P450 family 4 subfamily V polypeptide 2 gene (CYP4V2 gene), and the gRNA specifically binds to the intron region between the 6th and 7th exons of the CYP4V2 gene.

[0061] In certain embodiments, the gRNA can specifically bind to a nucleotide sequence as set forth in SEQ ID NO: 41. In certain embodiments, the gRNA can specifically bind to a nucleotide sequence having at least 70% (e.g., 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 to the nucleotide sequence as set forth in SEQ ID NO: 41. In the present application, the term "identity" refers to different nucleotide sequences that have identical base sequences.

[0062] In certain embodiments, the gRNA is capable of specifically binding to a nucleotide sequence complementary to the nucleotide sequence depicted in SEQ ID NO: 41. In certain embodiments, the gRNA is capable of specifically binding to a nucleotide sequence complementary to a nucleotide sequence having at least 70% (e.g., 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 to the nucleotide sequence depicted in SEQ ID NO: 41.

[0063] The gRNA described herein can bind to the sequence of the target nucleic acid (e.g., the intron region between the 6th and 7th exons of the CYP4V2 gene). The gRNA can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the sgRNA can vary depending on the sequence of the target nucleic acid.

[0064] In the present application, the gRNA can comprise any of the nucleotide sequences of SEQ ID NOs: 48-51. In the present application, the gRNA can comprise a nucleotide sequence having at least 70% (e.g., 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 to any of the nucleotide sequences of SEQ ID NOs: 48-51.

[0065] In the present application, the gRNA may contain, from the 5' to 3' end, (X)n, any nucleotide sequence of SEQ ID NO: 48-51 and a backbone sequence, where X is any base selected from A, U, C, and G, and n is any integer from 0 to 15. In the present application, the gRNA may contain 5'-(X)n-any nucleotide sequence of SEQ ID NO: 48-51-backbone sequence-3', where X is any base selected from A, U, C, and G, and n is any integer from 0 to 15.

[0066] For example, the backbone sequence described in the present application generally refers to a portion of the gRNA other than the portion that recognizes or hybridizes to the target sequence, and may include a sequence between the gRNA pairing sequence and the transcription terminator in the sgRNA. The backbone sequence generally does not change with changes in the target sequence, and does not affect the recognition of the target sequence by the gRNA. Therefore, the backbone sequence may be any sequence available in the prior art. The structure of the backbone sequence may refer to portions other than the spacer sequences described in A and B in Figure 1 (Figure 1), A, B, and C in Figure 3 (Figure 3), and A, B, C, D, and E in Figure 4 (Figure 4) in Nowak et al. Nucleic Acids Research 2016.44:9555-9564.

[0067] In certain embodiments, the gRNA may be a single-stranded or double-stranded guide RNA. For example, the gRNA may be a single-stranded guide RNA (e.g., sgRNA).

[0068] The present application provides one or more isolated nucleic acid molecules, which can encode a gRNA that specifically targets the CYP4V2 gene described above. For example, the isolated nucleic acid molecule can contain the nucleotide sequence of any of SEQ ID NOs:1-7.

[0069] The present application provides one or more isolated nucleic acid molecules, which can encode a gRNA that specifically targets the CYP4V2 gene described above. For example, the isolated nucleic acid molecule can contain the nucleotide sequence of any of SEQ ID NOs:1-4.

[0070] In the present application, the gRNA sequence can be designed to hybridize to a target nucleic acid near a PAM sequence recognizable by a Cas nuclease. The gRNA can be fully or incompletely complementary to the target sequence. The degree of complementarity between the gRNA and its corresponding target sequence is at least 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or more). The "Cas nuclease" generally refers to an enzyme that has the ability to recognize and cleave a specific DNA strand using a CRISPR sequence (e.g., gRNA) as a guide. Examples include Cas9 nuclease, Csn1, or Csx12. Cas9 nuclease usually contains a RuvC nuclease domain and an HNH nuclease domain, each of which cleaves two different strands of a double-stranded DNA molecule. Cas9 nuclease has been identified in different bacterial species, such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. pyogenes (Deltcheva, Chylinski et al. 2011). For example, the amino acid sequence of the Cas9 protein from Streptococcus pyogenes is shown in SwissProt database accession number Q99ZW2; the amino acid sequence of the Cas9 protein from Neisseria meningitides is shown in UniProt database accession number A1IQ68; the amino acid sequence of the Cas9 protein from Streptococcus thermophilus is shown in UniProt database accession number Q03LF7; and the amino acid sequence of the Cas9 protein from Staphylococcus aureus (e.g., SaCas in the vectors described herein) is shown in UniProt database accession number J7RUA5.Cas nucleases are typically capable of recognizing a specific PAM sequence from DNA, for example, the PAM can contain a nucleotide sequence set forth in any of SEQ ID NOs:8-14.

[0071] The gRNA and / or isolated nucleic acid molecule described herein can be delivered using a vector. In the present application, the vector (e.g., pX601) may or may not contain a nucleic acid encoding a Cas nuclease. In the present application, the Cas nuclease can be delivered separately as one or more polypeptides. Alternatively, the nucleic acid molecule encoding the Cas nuclease can be delivered separately or in a complex with one or more guide RNAs, or one or more crRNAs and tracrRNAs. For example, the nucleic acid molecule of the present application (e.g., an isolated nucleic acid molecule encoding an sgRNA that specifically targets the CYP4V2 gene) and the nucleic acid molecule encoding the Cas9 nuclease can be present in the same vector (e.g., a plasmid). The vector can include viral or non-viral vectors known in the art.

[0072] Non-viral delivery vectors include, but are not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras and RNA fusion protein complexes.

[0073] In the present application, the isolated nucleic acid molecule and / or the nucleic acid molecule encoding the DNA endonuclease can be delivered via a plasmid.

[0074] In certain embodiments, the vector may be a viral vector, including, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus. Methods for producing viral vectors containing a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are known in the art and can be performed by one of ordinary skill in the art without undue experimentation. In other embodiments, the vector may be a recombinant AAV viral particle that packages a nucleic acid molecule described herein. The method for producing the recombinant AAV may include introducing a nucleic acid molecule described herein into a packaging cell line, introducing a packaging plasmid of rep and cap genes expressing AAV into the cell line, and collecting the recombinant AAV from the packaging cell line supernatant. The packaging cell line may be various types of cells. For example, packaging cell lines that can be used include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

[0075] Donor nucleic acid molecules and vectors On the other hand, the present application provides a donor nucleic acid molecule. In the present application, the term "donor nucleic acid molecule" generally refers to a nucleic acid molecule that provides a heterologous nucleic acid sequence to a recipient (e.g., a recipient nucleic acid molecule). In certain embodiments, the donor nucleic acid molecule can be introduced into a recipient cell to repair a DNA fragment (e.g., a fragmented double-stranded DNA) cleaved by the isolated nucleic acid molecule. In other specific embodiments, the fragmented DNA can be repaired through the donor nucleic acid molecule. Repair methods include, but are not limited to, repair by homologous recombination (HR) that depends on DNA homology and repair by non-homologous end joining (NHEJ). HR is a method in which a specific DNA sequence is inserted into the cleavage site using a homologous sequence or a donor sequence (e.g., a vector that targets the above) as a template. The homologous sequence may be present in an endogenous genome, such as a sister chromatid. Alternatively, the donor may be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus. For example, the donor may contain a donor nucleic acid molecule as described herein. These exogenous nucleic acids may contain regions of high homology to the locus cleaved by the donor Cas nuclease, and may further contain additional sequences or sequence changes, including deletions that can be integrated into the cleaved target locus. NHEJ directly joins the DNA ends resulting from the double-strand break, but may also result in the deletion or addition of nucleotide sequences, which may disrupt or enhance gene expression. Among them are NHEJ-based Microhomology-mediated end joining (MMEJ), Homology-independent targeted integration (HITI), and HR-mediated end joining (HMEJ).For example, in the present application, a HITI repair method can be used to join a donor nucleic acid molecule to a DNA fragment (e.g., a CYP4V2 gene fragment) cleaved by the isolated nucleic acid molecule (e.g., an isolated nucleic acid molecule encoding an sgRNA that specifically targets the CYP4V2 gene).

[0076] With respect to the donor nucleic acid molecule described herein, the donor nucleic acid molecule may be a wild-type human nucleotide sequence or a gene fragment containing different numbers of introns and exons. In certain embodiments, the donor nucleic acid molecule may contain or not contain introns (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11). In certain embodiments, it may contain the nucleotide sequence between intron 6 and exon 11 of the CYP4V2 gene. For example, it may contain the nucleotide sequence of one or more (e.g., 2, 3, 4, 5 or 6) exons from intron 6 to exon 11, such as one or more nucleotide sequences in exon 7, exon 8, exon 9, exon 10 and / or exon 11. For example, in the present application, the donor nucleic acid molecule may contain CYP4V2 no. 7 to exon 11. For example, the donor nucleic acid molecule contains a nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, the donor nucleic acid can contain a nucleotide sequence having at least 70% (e.g., 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 to the nucleotide sequence set forth in SEQ ID NO: 39. The "nucleic acid molecule" that can contain a CYP4V2 nucleotide sequence described herein is distinct from the gRNA or "isolated nucleic acid molecule" that specifically targets CYP4V2 described herein.

[0077] On the other hand, the present application provides a vector containing the isolated nucleic acid molecule (e.g., an isolated nucleic acid molecule encoding an sgRNA that specifically targets the CYP4V2 gene) and / or the donor nucleic acid molecule (e.g., a nucleotide molecule encoding the human CYP4V2 gene).

[0078] In certain embodiments, the isolated nucleic acid molecule and the donor nucleic acid molecule can be present on different vectors.In other specific embodiments, the isolated nucleic acid molecule and the donor nucleic acid molecule can be present on the same vector.In the present application, when the vector containing the isolated nucleic acid molecule and the vector containing the donor nucleic acid molecule are simultaneously introduced into a cell, Cas nuclease simultaneously cuts the donor nucleic acid molecule and the cell genome DNA, and inserts the donor nucleic acid molecule into the cell genome at a precise position (for example, CYP4V2 gene fragment), and in certain embodiments, the insertion efficiency of this method is very high.

[0079] In one embodiment, the vector is a viral vector, such as AAV, lentivirus, retrovirus, adenovirus, herpesvirus and hepatitis virus. Methods for producing viral vectors that contain a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are known in the art and can be performed by one of ordinary skill in the art without undue experimentation.

[0080] Cells, pharmaceutical compositions, uses and methods The present application provides a cell, which may contain the isolated nucleic acid molecule and / or the donor nucleic acid molecule. The cell described herein may express sgRNA and Cas nuclease and have excellent DNA cleavage effect. The cell described herein may express CYP4V2 protein with normal function. The cell may include a mammalian cell, for example, a cell derived from a human. For example, the cell may include a COS cell, a COS-1 cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a NS0 cell, or a myeloma cell, a stem cell (for example, a pluripotent stem cell and / or a totipotent stem cell), and / or an epithelial cell (for example, a renal epithelial cell and / or a retinal epithelial cell). In the present application, the cell may include a HEK293 cell and / or a urinary renal epithelial cell. In the present application, the cell may have differentiation potential by modification. The differentiation ability can include the ability to differentiate into any type of cell in the body (neurons, astrocytes, oligodendrocytes, retinal epithelial cells, epidermis, hair and keratinocytes, hepatocytes, beta cells, intestinal epithelial cells, lung foam cells, hematopoietic cells, endothelial cells, cardiac myocytes, smooth muscle cells, skeletal muscle cells, kidney cells, adipocytes, chondrocytes and / or bone cells). For example, the cells can be reprogrammed into induced pluripotent stem cells (iPSCs) that overexpress important reprogramming genes (e.g., OCT4, KLF4, SOX2, cMYC, NANOG and / or LIN28).

[0081] The cells described herein can be used to evaluate the efficacy and safety of materials required for gene editing therapy (e.g., sgRNA and donor nucleic acid molecules).

[0082] The present application provides a pharmaceutical composition, comprising the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, the vector, and a pharma- ceutically acceptable carrier, which should be non-toxic and should not interfere with the efficacy of the active ingredient.

[0083] The present application provides a kit, which typically includes two or more components packaged in a container, receptacle, or other vessel, such as the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and the vector.

[0084] The pharmaceutical compositions described herein can be administered locally to the eye by various methods, including, but not limited to, intravitreal injection (e.g., anterior, medial or posterior intravitreal injection), subconjunctival injection, intracameral injection, injection into the anterior chamber via the temporal limbus, intrastromal injection, injection into the subchoroidal space, intracorneal injection, subretinal injection and intraocular injection. The administration can include subretinal injection, which is an injection into the subretinal space, i.e., under the neurosensory retina. During subretinal injection, the injected (e.g., the targeting vector, the gRNA, and / or the plasmid) is introduced directly between the photoreceptor cells and the retinal pigment epithelium (RPE) layer, forming a space between them.

[0085] The present application provides a use of any one of the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector in the manufacture of a medicament for the treatment of a disease, wherein the disease can include a disease caused by a CYP4V2 gene mutation, wherein the mutation is located behind the intron between exon 6 and exon 7 of the CYP4V2 gene. For example, the disease can include Vietti crystalline dystrophy.

[0086] The present application provides a method of treating Vietti crystalline dystrophy, comprising introducing into a subject in need thereof the gRNA (e.g., an sgRNA specifically targeting the CYP4V2 gene), the one or more isolated nucleic acid molecules (an isolated nucleic acid molecule encoding an sgRNA specifically targeting the CYP4V2 gene), the donor nucleic acid molecule (a nucleotide molecule encoding the human CYP4V2 gene), and / or the vector, wherein said introduction results in the subject obtaining a normally functioning CYP4V2 protein.

[0087] The method described herein can include ex vivo methods. In certain embodiments, subject-specific induced pluripotent stem cells (iPSCs) can be obtained. In addition, induced pluripotent stem cells can be differentiated into any type of cell, such as photoreceptor cells or retinal progenitor cells. In the present application, it can be 3D retinal organoids. Then, the genomic DNA of these 3D retinal organoid cells can be edited using the method described herein. For example, the method can include editing the 3D retinal organoid cells in or near the mutation site of the CYP4V2 gene so that they do not encode the CYP4V2 protein having the mutation. Finally, the 3D retinal organoid cells can be transplanted into the subject's body.

[0088] In other specific embodiments, photoreceptor cells or retinal progenitor cells can be isolated from a subject. The methods described herein can then be used to edit the genomic DNA of these photoreceptor cells or retinal progenitor cells. For example, the methods can include editing the CYP4V2 gene of photoreceptor cells or retinal progenitor cells at or near the mutation site so that they do not encode CYP4V2 with the mutation. Finally, the gene-edited photoreceptor cells or retinal progenitor cells can be transplanted into the subject.

[0089] The method can include performing a full analysis of the therapeutic agent prior to administration. For example, whole genome sequencing can be performed on the calibration cells to ensure that there are no off-target effects (if any) at genomic locations associated with minimal risk to the subject. Additionally, specific populations of cells, including clonal cell populations, can be isolated prior to transplantation.

[0090] Methods described herein include using site-specific nucleases to cleave DNA at precise target locations in the genome, thereby generating single- or double-stranded DNA breaks at specific locations in the genome, which can be periodically repaired by endogenous cellular processes, such as homologous recombination and non-homologous end joining.

[0091] The method described herein can include generating one or two DNA breaks at a position adjacent to a target sequence in a target locus, and the two DNA breaks can be double-stranded breaks or two single-stranded breaks. The breaks can be achieved by a site-directed polypeptide. The site-directed polypeptide (e.g., a DNA endonuclease) can introduce a double-stranded break or a single-stranded break into a nucleic acid (e.g., genomic DNA). The double-stranded break can stimulate the cell's endogenous DNA repair pathway, e.g., HR, NHEJ.

[0092] An exogenous donor template can be used to introduce additional nucleic acid sequences (e.g., the targeting vector) or modifications (e.g., single or multiple base modifications or deletions) between the homologous flanking regions to integrate additional or modified nucleic acid sequences into the target locus. The exogenous donor can be carried by a plasmid vector, e.g., an AAV vector and / or a TA cloning vector (e.g., a ZT4 vector).

[0093] The present application provides a method for regulating expression of the CYP4V2 gene in a cell, comprising introducing into the cell the gRNA, the one or more isolated nucleic acid molecules, and / or the vector.

[0094] In the present application, the method may be to introduce the gRNA into the cell.For example, the gRNA targets the CYP4V2 gene fragment in the genome of the recipient cell and cleaves it with nuclease, which reduces the chance that the CYP4V2 gene is translated into protein, and the translated protein cannot exert its normal function.

[0095] In the present application, the method may be to introduce the one or more isolated nucleic acid molecules into the cell. For example, the isolated nucleic acid molecule encoding sgRNA specifically targeting the CYP4V2 gene disrupts the CYP4V2 gene fragment, thereby reducing the chance of the CYP4V2 gene being translated into protein, and the translated protein cannot function normally.

[0096] In the present application, the method may be to introduce the vector into the cell. The vector contains the isolated nucleic acid molecule and / or the donor nucleic acid molecule. In a specific embodiment, the vector contains the isolated nucleic acid molecule and the donor nucleic acid molecule, and the CYP4V2 gene in the cell is replaced by the donor nucleic acid molecule, resulting in a change in the chance that the CYP4V2 gene is translated into a protein (e.g., the expression of the CYP4V2 protein changes from no expression to a normal amount of expression), and the function of the translated protein changes from abnormal to normal. In a specific embodiment, the vector contains the isolated nucleic acid molecule, and the CYP4V2 gene fragment is destroyed, resulting in a decrease in the chance that the CYP4V2 gene is translated into a protein, and the translated protein cannot perform its normal function. In another specific embodiment, the vector contains the donor nucleic acid molecule, and thus the cell contains more CYP4V2 gene fragments, and can be transcribed and translated into more CYP4V2 proteins.

[0097] Without wishing to be bound by any particular theory, the following examples are intended only to illustrate the nucleic acid molecules, production methods, uses, and the like of the present invention, and are not intended to limit the scope of the present invention.

[0098] Working Example Example 1 Design of sgRNA 1. sgRNA screening Based on the DNA sequence of the CYP4V2 gene, we designed a 21-bp-long sgRNA with PAM sequences NNGRRT and NNGRR (Staphylococcus aureus, SA; SaCas9) in the intronic region between exon 6 and exon 7 of CYP4V2.

[0099] Based on the scores, a total of seven sgRNAs (five for NNGRRT and two for NNGRR) were designed, the sequences of which are shown in Table 1 .

[0100] [Table 1]

[0101] 2. Synthesis of sgRNA Based on the sequence of the enzyme cleavage site Bbs1, Bbs1 enzyme cleavage sites were added upstream and downstream of the designed sgRNA, and corresponding primers were designed within 400 bp upstream and downstream of each sgRNA. The corresponding oligonucleotide sequences and primer designs are shown in Table 2.

[0102] [Table 2]

[0103] 3. The specific steps of constructing the sgRNA vector and extracting the plasmid are as follows:

[0104] 1. The synthesized sgRNA was annealed by the following steps. Each sgRNA forward primer (F) and reverse primer (R) synthesized above were diluted to 50 μmol, and 5 μL of each sgRNA (F, R) was taken to prepare sgRNA mixtures 1-7. T4 polynucleotide kinase (PNK) and 10× T4 ligation buffer were thawed on ice until ready for use. The following reaction system was prepared.

[0105] [Table 3]

[0106] The reaction system prepared above was placed in a PCR device, and the following reaction program was started.

[0107] [Table 4]

[0108] The reaction product was collected.

[0109] 2. The process for enzymatically cleaving the vector is as follows: The plasmid used for constructing the sgRNA vector is pX601 vector (Addgene, 61591). The plasmid map is shown in Figure 1.

[0110] The binding site of sgRNA was cleaved and released using BSaI enzyme, and the following enzyme cleavage reaction system was prepared in a 1.5 mL PCR tube.

[0111] [Table 5]

[0112] After that, enzymatic digestion was performed for 1-2 hours (or overnight), and the product was collected and purified, the concentration was measured, and diluted to 50 ng / μL.

[0113] 3. sgRNA was ligated to the vector recovered in step 2 Using the vector recovered in step 2 and the annealed sgRNA, the following ligation system (200 μL PCR tube) was prepared.

[0114] [Table 6]

[0115] The ligation reaction system was placed at 37°C and ligated for approximately 1-2 hours to complete the construction of the sgRNA vector.

[0116] 4. Plasmid Transformation (1) Take out TransStbl3 chemically competent cells (CD-521-02, Beijing Global Gold Biotechnology Co., Ltd.) and thaw them on ice; (2) Add 1 μL of the ligation product to 50 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42 degrees for 90 seconds, and leave on ice for 2 minutes; (3) Add 500 μL of non-antibacterial culture medium and shake at 37°C and 200 rpm for 1 h; (4) Centrifuge at 800 rpm / min for 5 min, remove the supernatant to obtain about 100 μL, and screen positive clones using ampicillin-containing LB agar medium; (5) The next day, the screened positive clone bacteria (500 μL of culture medium) were shaken for 3-4 hours, and a 200 μL sample was sequenced.

[0117] 5. Extraction of plasmid (using Omega's Endofree Plasmid Maxi Kit) (1) The seven plasmids pX601-SaCas9-CYP4V2-SgRNA1-7, which were determined to be accurate by sequencing, were shaken overnight (50-200 mL), and then shaken at 37°C for 12-16 h to amplify the plasmids, which were then extracted the next day (shaking time was less than 16 h).

[0118] (2) 50-200 mL of the bacterial liquid was taken and centrifuged at 4,000 × g for 10 minutes at room temperature to collect the bacterial cells.

[0119] (3) The medium was discarded. 10 mL of a mixture of Solution I / RNA enzyme A was added to the pellet, and the cells were completely resuspended by pipetting or vortexing.

[0120] (4) Add 10 mL of Solution II, close the lid, and gently invert the centrifuge tube 8-10 times to obtain a clear lysate. If necessary, the lysate may be allowed to stand at room temperature for 2-3 min.

[0121] (5) Add 5 mL of pre-cooled N3 buffer, close the lid, and gently invert the centrifuge tube upside down 10 times until a white aggregate precipitate forms. You can leave it at room temperature for 2 minutes to incubate.

[0122] (6) Prepare a syringe filter, pull out the piston from the syringe, place the syringe vertically in an appropriate test tube rack, and place a collection tube at the outlet of the lower end of the injector with the opening of the syringe facing upwards. The lysate was immediately poured into the filter syringe. The cell lysate was left to stand in the syringe for 5 min. Then, white aggregates began to float on the surface of the lysate. The cell lysate may have flowed out from the outlet of the filtration injector. The cell lysate was collected in a new 50 mL test tube. Carefully insert the injector piston lightly into the syringe and slowly push the piston so that the lysate flows into the collection tube.

[0123] (7) 0.1-fold volume of ETR solution (blue) was added to the flow-through filtered lysate, the test tube was inverted 10 times, and the tube was placed in an ice bath for 10 minutes.

[0124] (8) The above lysate was placed in a water bath at 42°C for 5 minutes. The lysate became cloudy again. Then, it was centrifuged at 25°C and 4000×g for 5 minutes, and the ETR solution formed a blue layer at the bottom of the test tube.

[0125] (9) The supernatant was transferred to another new 50 mL test tube, and 0.5 volumes of room temperature absolute ethanol was added. The test tube was gently inverted 6-7 times and allowed to stand at room temperature for 1-2 minutes.

[0126] (10) A HiBind® DNA Maxi Binding column was placed in a 50 mL collection tube, and 20 mL of the filtrate was added to the HiBind® DNA Maxi Binding column. The column was centrifuged at 4000 × g for 3 minutes at room temperature. The filtrate was discarded.

[0127] (11) A HiBind® DNA Maxi Binding column was placed in the same collection tube, and step 10 was repeated and centrifuged under the same conditions until all of the remaining filtrate was bound to the HiBind® DNA Maxi Binding column.

[0128] (12) A HiBind® DNA Maxi binding column was placed in the same collection tube, 10 mL of HBC buffer was added to the HiBind® DNA Maxi binding column, and the column was centrifuged at 4,000 × g for 3 minutes at room temperature, and the filtrate was discarded.

[0129] (13) A HiBind® DNA Maxi Binding column was placed in the same collection tube, and 15 mL of DNA Wash Buffer (diluted with absolute ethanol) was added to the HiBind® DNA Maxi Binding column. The column was centrifuged at 4,000 × g for 3 min at room temperature, and the filtrate was discarded.

[0130] (14) A HiBind® DNA Maxi Binding column was placed in the same collection tube, and 10 mL of DNA Wash Buffer (diluted with absolute ethanol) was added to the HiBind® DNA Maxi Binding column. The column was then centrifuged at 4,000 × g for 3 min at room temperature, and the filtrate was discarded.

[0131] (15) Spin at maximum speed (<6000 × g) for 10 min to dry the matrix of the HiBind® DNA Maxi binding column.

[0132] (16) (Optional) Further air-dry the HiBind® DNA Maxi Binding Column. (Optional) Further dry the HiBind® DNA Maxi Binding Column before eluting the DNA (if required) by one of the following methods:

[0133] a) Place the HiBind(R) DNA Maxi binding column in a vacuum chamber for 15 min to dry off the ethanol: At room temperature, transfer the column to a vacuum chamber and connect all the devices in the vacuum chamber. Seal the vacuum chamber and apply vacuum for 15 min. Transfer the HiBind(R) DNA Maxi binding column to the next step.

[0134] b) Dry the column in a vacuum oven or at 65°C for 10-15 min. Remove the HiBind® DNA Maxi Binding column and proceed to the next step.

[0135] (17) Place the HiBind® DNA Maxi Binding Column in a clean 50 mL centrifuge tube and add 1-3 mL of endotoxin-free elution buffer directly to the matrix of the HiBind® DNA Maxi Binding Column (the amount depends on the concentration of the final product) and incubate at room temperature for 5 min.

[0136] (18) The DNA was eluted by centrifugation at 4,000 × g for 5 minutes.

[0137] (19) The column was discarded, and the DNA product was stored at -20 °C.

[0138] (20) The extracted plasmids pX601-SaCas9-CYP4V2-SgRNA1 to pX601-SaCas9-CYP4V2-SgRNA7 were sequenced again to confirm that the constructed plasmids were accurate.

[0139] 4. Verification of sgRNA efficacy in 293T cells 1. Cultivation of 293T cells (1) Thawing of frozen cells a) The temperature of the thermostatic water bath was adjusted to 37°C, and the frozen preserved cells were removed from the liquid nitrogen, and, holding the lid with tweezers, were quickly shaken in water.

[0140] b) The cryopreservation solution was transferred to a 15 mL graduated centrifuge tube, 10 mL of cell culture solution was slowly added, and the tube was gently shaken to mix the liquid. The tube was then capped, heated, and centrifuged at 1000 rpm / min for 3 minutes.

[0141] c) After heating, an appropriate amount of culture medium was added, and the cell precipitate at the bottom was gently pipetted, after which the cells were transferred to a culture flask and cultured in an incubator. The medium used for 293T cells was high glucose DMEM supplemented with 10% fetal bovine serum and 100U / mL double antibody, and the cells were cultured at 37℃ and 5% CO2.

[0142] (2) Cell passage a) The morphology and density of the cells were observed under an inverted microscope, and when the confluency of the cells in the culture flask reached 80%-90%, the cells were subcultured.

[0143] b) Wash out the old culture medium from the cell culture bottle and wash it with PBS three times. Add 500μL of EDTA-containing trypsin to the culture bottle and incubate it in an incubator for about 1min. When the gaps between the cells become larger and the cells become round, immediately add 1mL of culture medium to the culture bottle to stop digestion. Gently pipette the cells with a suction tube until all the cells float from the bottom of the bottle. Transfer the liquid in the culture bottle to a centrifuge tube and centrifuge at 1000rpm / min for 2min.

[0144] c) The supernatant was removed, 2 mL of medium was added to the centrifuge tube, and the precipitated cells were resuspended. The cell suspension was placed in four new culture bottles, 4 mL of culture medium was added to each, and the culture bottles were gently shaken to mix the cells and spread them evenly in the culture bottles, and then the bottles were placed in a cell incubator for culture.

[0145] d) One day before transfection, 100w of 293T cells with 80% density, cell spreading, and uniform cell spacing were added per well, and the next day, when the cells reached 80-90% confluency, plasmid transfection was performed.

[0146] 2. Transfection into 293T cells using the PEI (polyethyleneimine) method (1) Take 1.5mL EP tubes, add 250μL of DMEM medium (without serum) to each tube, add 1.5μg of pX601-CYP4V2-sgRNA1 plasmid and 1μg of pLent-GFP plasmid (co-transfection, rough indication of whether pX601-CYP4V2-sgRNA1 plasmid was introduced or not), mix thoroughly by vortexing, add 7.5μL of PEI transfection reagent to each tube, mix by vortexing, and leave at room temperature for 20min before transfection. In the same way, add the plasmid transfection system from pX601-CYP4V2-sgRNA2 to pX601-CYP4V2-sgRNA7, the transfection system without pX601 plasmid, and the blank control to the other wells of the 6-well plate.

[0147] (2) The medium was dropped (6-well plate medium, serum-free DMEM = 2 mL / well), cultured in an incubator, and half of the medium was replaced every 12-18 hours. The next day, the expression status of GFP was observed under a fluorescent microscope to evaluate the transfection efficiency, and if the transfection efficiency was good, the culture of the transfected plasmid was continued.

[0148] (3) Screening for resistant cells a) Preparation of solution: Puromycin diluted from 10 mg / mL (mother solution) to 1 μg / mL, screened cells

[0149] b) Fluid replacement: Two days after transfection, add 3mL of puromycin-containing 293T cell medium per well (including the negative control), and begin screening for positively transfected cells. After that, observe cell viability every day, replace the fluid every two days, and add the corresponding amount of puromycin when replacing the fluid. If the cells in the negative control wells are completely dead, while the cells in the experimental and control groups are alive (indicating successful transfection), discontinue antibiotic screening and use normal medium instead.

[0150] 3. Extraction of 293T cell genomic DNA (1) After the cells in the 6-well plate reached 80-90% confluence, they were passaged and cultured in a 6 cm culture dish.

[0151] (2) After the cells reach 80-90% confluency, harvest the cells and prepare for genomic DNA extraction. The whole process takes about 7-10 days. For genome extraction, we use a cell extraction kit from Nanjing Vazyme Biotech. The experimental steps are as follows:

[0152] a) Cells were collected by centrifugation at 400 × g for 5 min, and the supernatant was removed. 220 μL of PBS (phosphate buffer), 10 mL of RNase solution, and 20 μL of PK working solution were added to the sample to resuspend the cells. The sample was left to stand at room temperature for 15 min or more; b) Add 250 μL of GB buffer to the cell resuspension, mix by vortexing, incubate at 65°C for 15-30 min in a water bath, and purify on a column; c) Add 250 μL of absolute ethanol to the digestion solution and mix by vortexing for 15-20 sec; d) A gDNA adsorption column was set in a 2 mL collection tube. The mixture (including precipitate) obtained in the previous step was transferred to the adsorption column. It was centrifuged at 12,000 × g for 1 min. If the column was clogged, it was centrifuged at 14,000 × g for 3-5 min. If the mixture exceeded 750 μL, it was applied to the column in several times; e) The filtrate was discarded, and the adsorption column was placed in a collection tube. 500 μL of washing buffer A was added to the adsorption column. Centrifuge at 12000 × g for 1 min; f) The filtrate was discarded and the adsorption column was placed in a collection tube. 650 μL of washing buffer B was added to the adsorption column. Centrifuge at 12000×g for 1 min; g) repeat step 4; h) The filtrate was discarded, and the adsorption column was placed in the collection tube. Centrifuge at 12,000×g for 2 min in an empty tube; i) The adsorption column was placed in a new 1.5 mL centrifuge tube. 30-100 μL of elution buffer preheated to 70° C. was added to the center of the membrane of the adsorption column and left at room temperature for 3 minutes. Centrifuge at 12,000×g for 1 minute; NOTE: For tissues with high DNA content, elution can be repeated by adding an additional 30-100 µL of elution buffer; j) The adsorption column was removed, and the DNA was stored at 2-8°C, the concentration was measured and recorded, and then stored long-term at -20°C.

[0153] 4. T7E1 enzyme cleavage experiment (1) Genomic DNA extracted from 293T cells transfected with the above-mentioned pX601-sgRNA1 to pX601-sgRNA7 plasmids was used as a template to amplify the extracted genomic DNA near the target site using upstream and downstream primers designed and synthesized corresponding to each of the above-mentioned sgRNAs, and PCR of the DNA fragments was performed (7 sets in total, referred to as J1-J7, respectively).

[0154] (2) Using a liquid recovery kit (OMEGA Gel Extraction Kit (200) D2500-02), liquid DNA was extracted from the PCR product. The DNA extraction process is as follows:

[0155] a) Add an equal volume of membrane binding solution to the PCR reaction product, and for recovery from gel cut, add 1 μL of membrane binding solution per 1 mg, heat at 50-60°C for 7 min until all gel is completely dissolved, mix by vortexing, and recover on a column; b) Put the above liquid into a recovery column, centrifuge at 10,000×g for 1 min, and remove the filtrate; c) add 700 μL of wash buffer, centrifuge at >13,000 × g for 1 min, and remove the filtrate; d) repeating step c); e) Centrifuge the empty tube at >13,000 x g for 10 min; f) Transfer the centrifuge column to a new 1.5mL EP tube, mark it, add 20-30μL of elution buffer or ddH2O, and leave it at room temperature for 2min; g) The mixture was centrifuged at 13,000 x g for 1 min, the adsorption column was removed, and the DNA was stored at 2-8°C. The concentration was measured and recorded, and the DNA was stored long-term at -20°C.

[0156] (3) Verification of sgRNA efficiency by T7E1 enzyme cleavage experiment The PCR or gel cut recovered products obtained above were subjected to T7E1 enzyme digestion reaction.

[0157] a) T7E1 enzyme cleavage annealing system (19.5 μL):

[0158] [Table 7]

[0159] b) Program for T7E1 enzyme cleavage and annealing: 95℃ 2min 95℃ to 85℃ Temperature is reduced at 2℃ / s 85℃ to 25℃ Temperature is decreased at 0.1℃ / s 16℃∞.

[0160] c) T7E1 enzyme cleavage reaction system

[0161] [Table 8]

[0162] The reaction system was incubated at 37° C. for 20 min.

[0163] d) Electrophoresis of enzyme cleavage products Gel preparation: 2.5% gel, 2x dye Electrophoresis program: voltage 140V, 20min to 30min

[0164] e) Confirmation of electrophoresis results. The results of the enzymatic cleavage are shown in FIG. 2. Lane 1 is the result of electrophoresis of the marker, lane 2 is the result of electrophoresis of the negative control (i.e., no sgRNA was added), lane 3 is the result of the length of the fragment cleaved by sgRNA1, lane 4 is the result of the length of the fragment cleaved by sgRNA5, lane 5 is the result of electrophoresis of the negative control, lane 6 is the result of the length of the fragment cleaved by sgRNA2, lane 7 is the result of the length of the fragment cleaved by sgRNA6, lane 8 is the result of the length of the fragment cleaved by sgRNA7, lane 9 is the result of electrophoresis of the negative control, lane 10 is the result of the length of the fragment cleaved by sgRNA3, and lane 11 is the result of the length of the fragment cleaved by sgRNA4.

[0165] The amplification primers for each fragment, the fragment length and the length of the cleaved fragment are summarized in Table 3.

[0166] [Table 9]

[0167] 5. Gene sequencing of PCR fragments containing cleavage sites in genomic DNA, multi-peak test The DNA amplified fragments J1 to J7 were sequenced, and the sequencing spectra are shown in Figures 3A-3D. It was found that sgRNA1.2.3.4 had a relatively obvious cleavage effect.

[0168] Example 2 Verification of the effect of sgRNA target site on splicing First, we designed pMD19-T minigene plasmids corresponding to the four sgRNAs, as well as negative and positive plasmids as controls.

[0169] Due to the characteristic that PAM+3bpsgRNA / 18bpsgRNA fragments remain after insertion by the donor HITI method, minigenes 1.2.3.4 were designed, which correspond to sgRNA1.2.3.4. The minigene fragments are shown in Figure 4.

[0170] 2. The minigene plasmid construction process is as follows:

[0171] 1. Minigene DNA fragments and positive control (normal wild-type intron, does not affect splicing) and negative control (patient mutant intron, affects splicing) DNA fragments were synthesized.

[0172] 2. Enzyme-cleaved plasmid vector The plasmid vector was PMD-19T-MCS, the plasmid map of which is shown in FIG.

[0173] (1) The plasmid was cut with two enzymes, KpnI and MluI. The minigene was incubated in CutSmart buffer with KpnI and MluI enzymes at 37°C for 30 min. A 2.5% electrophoresis gel was prepared, 2x dye was added, and electrophoresis was performed on the enzyme cleavage products. The voltage was set to 140V and the time was set to 20 to 30 min. After electrophoresis, the gel was cut and purified.

[0174] (2) The linearized vector after enzyme digestion was subjected to gel recovery. Using a liquid recovery kit (OMEGA Gel Extraction Kit (200) D2500-02), perform liquid DNA recovery for the above PCR products; the DNA recovery steps are as follows:

[0175] a) Add an equal volume of membrane binding solution to the PCR reaction product, and the recovery from the gel cut requires adding 1 μL of membrane binding solution per 1 mg, heat at 50-60°C for 7 min until all the gel is completely dissolved, mix by vortexing, and recover on a column; b) Transfer the liquid to a collection column, centrifuge at 10,000×g for 1 min, and remove the filtrate; c) add 700 μL of wash buffer, centrifuge at >13,000 × g for 1 min, and remove the filtrate; d) repeating step c); e) Centrifuge the empty tube at >13,000 x g for 10 min; f) Transfer the centrifuge column to a new 1.5mL EP tube, mark it, add 20-30μL of elution buffer or ddH2O, and leave it at room temperature for 2min; g) The mixture was centrifuged at 13,000 x g for 1 min, the adsorption column was removed, and the DNA was stored at 2-8°C. The concentration was measured and recorded, and the DNA was stored long-term at -20°C.

[0176] (3) Ligation The following ligation system was prepared using the vector recovered in the previous step, the synthesized minigene 1-6 DNA fragment, and the corresponding positive control (prepared in a 200 μL PCR tube):

[0177] [Table 10]

[0178] The ligation reaction system from the previous step was placed at 37°C for approximately 1-2 hours for ligation to complete the construction of the sgRNA vector.

[0179] (4) Plasmid transformation a) Remove TransStbl3 chemically competent cells and thaw on ice; b) Take 1 μL of the ligation product of step 3, add it to 50 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42 degrees for 90 seconds, and leave it on ice for 2 minutes; c) adding 500 μL of non-antibacterial culture medium, shaking at 37°C, 200 rpm for 1 h, centrifuging at 800 rpm / min for 5 min, removing the supernatant to obtain about 100 μL, and screening positive clones using LB agar medium containing ampicillin; d) The next day, the screened positive clone bacteria (500 μL of medium) were shaken for 3-4 hours, and a 200 μL sample was subjected to sequencing.

[0180] (5) Extraction of plasmid A total of six plasmids, including minigenes 1-4 and positive and negative controls, which were determined to be accurate by sequencing, were shaken overnight (50-200mL), and the plasmids were amplified by shaking at 37°C for 12-16h, and extracted the next day (shaking time was less than 16h). Plasmids were extracted using Omega's Endofree Plasmid Maxi Kit.

[0181] 3. Transfection of 293T cells with minigene plasmid 1. Cultivation of 293T cells

[0182] 2. Transfection of 293T cells using the PEI (polyethyleneimine) method (1) Take 1.5mL EP tubes, number them in the above order, add 250μL of DMEM medium (without serum) to each tube, add 1.5μg of minigene 1-4 plasmid and positive and negative control plasmids in the order of the above table, mix thoroughly by vortexing, add 7.5μL of PEI transfection reagent to each tube, mix by vortexing, and leave at room temperature for 20min before transfection. Another well of the 6-well plate was used as a blank control.

[0183] (2) The medium was dropped (6-well plate medium, serum-free DMEM = 2 mL / well), cultured in an incubator, and half of the medium was replaced every 12-18 hours. The next day, the expression status of GFP was observed under a fluorescent microscope to evaluate the transfection efficiency, and if the transfection efficiency was good, the culture of the transfected plasmid was continued.

[0184] (3) Screening for resistant cells a) Preparation of solution: Puromycin diluted from 10 mg / mL (mother solution) to 1 μg / mL, screened cells b) Fluid replacement: Two days after transfection, add 3mL of puromycin-containing 293T cell medium per well (including the negative control), and begin screening for positively transfected cells. After that, observe cell viability every day, replace the fluid every two days, and add the corresponding amount of puromycin when replacing the fluid. If the cells in the negative control wells are completely dead, while the cells in the experimental and control groups are alive (indicating successful transfection), discontinue antibiotic screening and use normal medium instead.

[0185] 3. Extraction of 293T cell genomic DNA (1) After the cells in the 6-well plate reached 80-90% confluence, they were passaged and cultured in a 6 cm culture dish.

[0186] (2) After the cells reach 80-90% confluency, the cells are harvested and prepared for genomic DNA extraction. The whole process takes about 7-10 days. For genome extraction, we used a cell extraction kit from Nanjing Vazyme Biotech.

[0187] (3) Validate RNA levels in transfected 293T cells 1) RNA extraction RNA was extracted using the TIANGEN RNA prep Pure cultured cell / bacteria total RNA extraction kit (DP430). The experimental steps are as follows:

[0188] a) Cell collection i. Harvesting of suspended cells (the number of cells harvested was 1 x 10 7 The number of cells was estimated (it should be less than 100%), centrifuged at 300 x g for 5 min, the cells were collected in a centrifuge tube, and all the medium supernatant was carefully removed by aspiration.

[0189] ii. Harvesting of adherent monolayer cells (the number of harvested cells was 1 × 10 7 The cell suspension was then aspirated, the medium was washed with PBS, the PBS was aspirated, PBS-treated cells containing 0.10-0.25% trypsin was added to the cells, and once the cells had detached from the surface of the vessel, medium containing serum was added to inactivate the trypsin, the cell solution was added to an RNAse-free centrifuge tube, centrifuged at 300 x g for 5 min, the cell pellet was collected, and all the supernatant was carefully aspirated.

[0190] b) Lysate processing The bottom of the centrifuge tube was gently tapped to loosen the cell pellet, and an appropriate amount of lysate RL (check whether β-mercaptoethanol had been added) was added according to the number of cells, and the tube was shaken by vortexing.

[0191] c) The entire solution was transferred to a filtration column CS (the filtration column was set in the CS collection test tube), centrifuged at 12000 rpm (~13400 x g) for 2 min, and the filtrate was collected. One volume of 70% ethanol (usually 350 μL or 600 μL) was added to the filtrate, mixed (a precipitate may appear), and the resulting solution and precipitate were transferred to an adsorption column CR3 (the adsorption column CR3 was set in the collection test tube), centrifuged at 12000 rpm (~13400 x g) for 30-60 sec, the waste liquid in the collection test tube was discarded, and the adsorption column CR3 was returned to the collection test tube.

[0192] d) 350 μL of deproteinized solution RW1 was added to adsorption column CR3 and centrifuged at 12,000 rpm (~13,400 × g) for 30-60 seconds. The waste liquid in the collection tube was discarded, and adsorption column CR3 was returned to the collection tube.

[0193] e) Preparation of DNase I working solution: 10 μL of DNase I stock solution was placed in a new centrifuge tube without RNA enzyme, 70 μL of RDD buffer was added and mixed gently.

[0194] f) 80 μL of DNase I working solution was added to the center of the adsorption column CR3, and the column was allowed to stand at room temperature for 15 minutes.

[0195] g) Add 350 μL of deproteinization solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm (~13,400 × g) for 30-60 seconds, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube. Add 500 μL of rinse solution RW (check whether ethanol has been added) to the adsorption column CR3, leave it at room temperature for 2 min, centrifuge at 12000 rpm (~13,400 × g) for 30-60 seconds, discard the waste liquid in the collection tube, return the adsorption column CR3 to the collection tube, and repeat this process.

[0196] h) Centrifugation was performed at 12,000 rpm (~13,400 x g) for 2 minutes, and the waste liquid was discarded. The adsorption column CR3 was left to stand at room temperature for several minutes to thoroughly dry the rinsing liquid remaining on the adsorbent.

[0197] i) The adsorption column CR3 was transferred to a new centrifuge tube without RNA enzyme, 30-100 μL of ddH2O without RNA enzyme was added, and the tube was left to stand at room temperature for 2 minutes, and centrifuged at 12,000 rpm (~13,400 × g) for 2 minutes to obtain an RNA solution.

[0198] 2) Reverse transcription of cDNA For reverse transcription of cDNA, we used the TRAN Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AT311). The experimental steps are as follows:

[0199] a) cDNA synthesis and gDNA removal. Add the following components and mix gently.

[0200] [Table 11]

[0201] b) The reverse transcription program is as follows: 42℃ 30min 85℃ 5sec

[0202] c) The sample was collected

[0203] 3) PCR was performed on the cDNA.

[0204] 4) The PCR products were sequenced.

[0205] (5) Protein level verification after transfection into 293T cells 1) Protein extraction a) Obtaining 293T cell lysate. After washing the cells with PBS, add 100-200 μL of RIPA lysate (for 6-well plates) and add protease inhibitor cocktail, phosphatase inhibitor PMSF and RNA enzyme (if necessary), scrape the cells and place them in EP tubes. Place on ice and vortex every 5 minutes, repeating three times.

[0206] b) Lysed cells were centrifuged at 12000 rpm for 10 min at 4°C. 50 μL of the supernatant was taken as an input control and boiled for 5 min with 10 μL of 6x loading buffer. Boiled samples can be stored at 4°C for short periods or at -20°C for long periods.

[0207] 2) Western blot (protein hybridization experiment) verification a) After aligning the glass plates, they were set on the rack so that the short glass was on the outside and the long glass was on the inside, and clamped with both grips. The rack was hung vertically to prepare for pouring the gel. During the work, it was necessary to align both sides of the glass to prevent the gel from leaking, and water was added before pouring the gel to check that there was no leakage. When preparing the gel, TEMED was added in the last step, and then it was immediately shaken to make it uniform, and the gel was poured immediately. The gel was poured with a tip attached to the corner to prevent air bubbles from entering, and the distance between the surface of the gel and the short glass should be 1-2 cm. Anhydrous ethanol was added and layered.

[0208] b) When the boundary between the absolute ethanol and the gel becomes clear, it indicates that the gel has solidified. Wait for 3 minutes until the gel has solidified sufficiently, discard the absolute ethanol on the top layer of the gel, and wipe it off with paper. Prepare the concentrated gel according to the ratio specified in the instructions, insert the comb, and wait until the gel has solidified. Note: When inserting the comb, the comb should be kept horizontal.

[0209] c) Sample loading.

[0210] d) Electrophoresis: The loading amount per lane of the minigel is usually less than 20 μg of total protein. As much as possible, the ionic strength is kept uniform in each sample, including the marker and the blank lane without sample. During electrophoresis, the voltage for running the stacking gel is 80 V, and the voltage for running the separating gel is 120 V. The higher the voltage, the faster the separation. The electrophoresis time is generally 1-2 h, but the electrophoresis can be stopped when the bromophenol blue is about to flow out of the gel.

[0211] e) Transfer i The layer arrangement from the protein gel to the membrane is: black side of the grip - sponge - filter paper - gel - membrane - filter paper - sponge - white side of the grip. Then, the whole device was immersed in the buffer. However, the PVDF membrane was immersed in methanol, and finally, it was immersed in the transfer solution.

[0212] ii. Place the grip into the transfer slot so that the black side of the grip faces the black side of the slot and the white side of the grip faces the red side of the slot.

[0213] iii Ice had to be placed in the transfer slot for cooling, and transfer was performed at a voltage of 120 V and a constant current of 200 mA. The larger the protein molecule, the higher the voltage / current and the longer the transfer time required.

[0214] f) Blocking The membrane was removed and blocked with TBS solution containing 5% skim milk for 1 hour or more. Note: The surface of the membrane that contacts the gel faces upwards.

[0215] g) Antibody incubation and testing Incubation of primary antibody: Dilute the antibody in TBST containing 2.5% skim milk at a ratio of 1:1000 and leave it at room temperature for 1.5-2h or overnight at 4℃. Washing of membrane: Wash three times with 0.1% TBST for 10min each time. Incubation of secondary antibody: Dilute the antibody in TBST solution containing 2.5% skim milk at an appropriate ratio and incubate at room temperature for 2h. Washing of membrane: Wash three times with 0.1% TBST for 10min each time, wash with TBS for 10min, and wipe the moisture appropriately on the side of the membrane with filter paper. The membrane was placed on a cling film soaked in ECL color development solution mixture (1mL), and the cling film was folded. Finally, it was examined using a gel imaging system.

[0216] The results of Western blot are shown in Figures 6A-6B. Figure 6A shows the expression of β-actin in the cells of each group. Lane 1 is marker, lane 2 is minigene 1, lane 3 is minigene 2, lane 4 is minigene 3, lane 5 is minigene 4, lane 6 is negative control, and lane 7 is positive control. In each group, β-actin is expressed and the expression level is almost the same, and the loading amount is the same, so the results can be compared. Figure 6B shows the expression of green fluorescent proteins (GFP) in the cells of each group. The group represented by each lane is the same as Figure 6A. According to the results of each group, the expression of minigenes 1-4 is consistent with the positive control (normal human intron fragment, does not affect splicing), GFP is expressed, and the negative control (patient intron fragment, affects splicing) is weakly expressed. Figure 6C shows the expression of tag protein (Flag) in the cells of each group. Each lane represents the same group as in Figure 6A, and the results show that flag expression was not observed in the negative control group, but was expressed in each experimental group and the positive control group, indicating that sgRNA1-4 did not affect splicing.

[0217] Example 3 Donor design and screening 1. Vector construction: The sequence between intron 6 and exons 7-11 of the wild-type gene of CYP4V2 was used as the Donor sequence; the Donor sequence and the EGFP reporter gene were constructed in the pX601 vector (pX601-sgRNA1 to pX601-sgRNA4 vectors of sgRNA1-4) to obtain the pX601-donor(1-4)-EGFP vector; the pX601-sgRNA(1-4) vector in Example 1 was used as the sgRNA vector. The plasmid map of pX601 is shown in FIG.

[0218] Among them, the length of intron 6 was adjusted according to the sgRNA cleavage site. The exon 7-11 donor sequence is shown in SEQ ID NO:39; the EGFP sequence is shown in SEQ ID NO:40.

[0219] II. Transfection of iPSCs using PEI method 1.The specific steps are as follows:

[0220] Take the sgRNA1 group as an example.

[0221] (1) Take four 6-well plates, seed iPSC cells, and transfect them when the cell density reaches 80%; (2) Group 1 (blank control group): 1.5 μg of pX601 plasmid was added to a 1.5 mL tube; Second group (experimental group): add 1.5 μg of the above constructed pX601-sgRNA1 plasmid (donor SaCas9) and 1.5 μg of pX601-Donor-EGFP plasmid into a 1.5 mL tube; Add 250 μL serum-free DMEM medium to each of the two tubes, mix, then add 12 μg PEI (1 mg / mL), mix thoroughly immediately, and incubate at room temperature for 20 min; (3) The mixture was added dropwise to the medium and cultured in an incubator, and half of the medium was replaced every 24 hours.

[0222] The experimental method for the sgRNA2-4 group was the same as that for the sgRNA1 group.

[0223] 3. Screening for resistant cells Preparation of solutions: Puromycin 10 mg / mL (mother solution) diluted to 1 μg / mL;

[0224] Liquid replacement: 3mL of medium containing 1μg / mL puromycin was added per well, and the liquid was replaced daily, and the cell death status was continuously observed. The transfection effects of the experimental group and the blank group were similar, so a representative image of the transfection was selected. The transfection effect is shown in Figure 7. The left side of Figure 7 is the blank control, and the right side is the transfected cells. It was observed that the cells expressed the fluorescent protein, and the transfection effect was excellent.

[0225] 4. Extract the cellular DNA, sequence it, and verify the sequence repair results. Genome extraction was performed using a cell extraction kit from Nanjing Vazyme Biotech Co., Ltd. Sequencing results showed that the pX601-sgRNA-Donor1.2.3.4 vector was effective in repair.

[0226] Example 4 In vitro validation of gene editing efficiency of sgRNA using 3D (three-dimensional) retinal tissue from patients 1. Extraction and culture of renal epithelial cells from patients The experiment was carried out using a renal epithelial cell isolation and culture kit provided by Beijing Speil. The experimental steps are as follows:

[0227] 1.UrinEasy (R) Complete isolation medium, supplements, gelatin, and washing solution were brought into the cell culture room.

[0228] 2. Turn on the ultraviolet lamp and prepare a 12-well plate, a 50 mL centrifuge tube, a 15 mL centrifuge tube, an electronic pipette, a pipette, an aspirator, a 5 mL micropipette and tips, a 1 mL micropipette and tips, and a 37°C water bath.

[0229] 3. Urine collection: Wearing gloves, disinfecting, midstream urine was collected and sealed in sealing film.

[0230] Gelatin was applied to the bottom of the dish (3 wells) at 4.750 μL / well and incubated at 37° C. for >30 min.

[0231] The outer surface of the urinal was disinfected with 5.75% alcohol, and the sample was divided into 50 mL conical-bottom centrifuge tubes, sealed, and centrifuged at 400 × g for 10 min.

[0232] 6. UrinEasy (R) Separation complete medium and supplements were removed and prepared (mix 5 mL of basal medium per 0.5 mL of supplement).

[0233] 7. Using minimal speed, move the pipette along the upper liquid surface to slowly aspirate off 1 mL of supernatant.

[0234] 8. The cells were resuspended and placed in a 15 mL centrifuge tube, 10 mL of washing solution was added, mixed, and centrifuged at 200 x g for 10 minutes.

[0235] 9. Remove the 12-well plate, remove the gelatin, wash once with washing solution (500 μL), and add 750 μL of UrinEasy per well. (R) Complete isolation medium was added and the mixture was left to stand at 37°C.

[0236] 10. The 15 mL centrifuge tube was removed leaving a 0.2 mL cell pellet.

[0237] 11. UrinEasy (R) The cell pellet was resuspended in complete isolation medium: one well for males and two wells for females, designated as day 0 (D0).

[0238] 12. Observations: D1: Observation of the presence or absence of contamination; D2: Replenishment of isolation medium-Female: 500 μL / well; Male: 250 μL / well; D4: In the absence of attachment: half the volume of the medium was replaced, changing the liquid every 2 days and adding 1 mL of complete isolation medium along the surface.

[0239] 13. After attachment: Once the cells have attached to the surface (3-7 days or 9-10 days),(R) The cells were cultured in complete amplification medium, and after 2 days of culture, the entire volume was replaced with 500 μL of culture medium. After attachment to the surface, approximately 9 to 12 days (less than 14 days), when the cells reached 80 to 90% confluency, they were passaged into 6-well plates, 6 cm culture dishes, and 10 cm culture dishes, and then frozen and stored for later use.

[0240] 2. Induction of iPSCs Patient-derived (c.802-8_810del17bpinsGC) kidney epithelial cells were induced into iPSCs by the following steps:

[0241] 1. When the somatic cells reach 70-90% confluence, they can be digested and subcultured. The cells are seeded into a 96-well plate; the seeding density is controlled to be 5000-15000 cells / well, and three density gradients can be set according to the cell conditions, with triple wells set up for each gradient. The day of cell seeding is designated as day -1.

[0242] Day 2.0: The cells were observed under a microscope for confluence and their condition, digestion and counting were performed for the multi-wells with different gradients, and reprogramming was performed for the wells where the amount of cells reached 10,000-20,000 cells. Reprogramming medium A was prepared according to the table below.

[0243] [Table 12]

[0244] 3. After centrifuging the initialization additive II, 97 μL of initialization medium A was added to the test tube of initialization additive II and mixed evenly to prepare initialization medium B. 100 μL of initialization medium B was added to wells of the 96-well plate that met the selected conditions, and the culture plate was returned to the incubator.

[0245] 4. Days 1-2: Observe under a microscope and record the changes in cell morphology with photographs. If the cell morphology has obviously changed, remove the initialization medium B and continue culturing with the initialization medium A instead; if the morphological change is not obvious, there is no need to change the liquid.

[0246] 5. Day 3: If the morphology of the cells has obviously changed in the first two days and the cell proliferation rate is fast, they can be subcultured by trypsin digestion. According to the condition of the cells and their quantity, the cells are subcultured into two to six wells of a 6-well plate, and reprogramming medium C is added to form single cell attachment as much as possible. Reprogramming medium C is prepared according to the table below.

[0247] [Table 13]

[0248] 6. Day 4: Observe the attachment of cells to the vessel. If most of the cells are well attached to the vessel, place fresh Reproeasy (R) The medium was replaced with somatic cell medium (Seikai Seibutsusha, CA5001050), and the culture was continued.

[0249] Day 7.5: When small cluster clones (a group of clones with four or more cells) are formed under microscope observation, change the somatic cell medium to Reproeasy (R) Change to human somatic cell reprogramming medium. If small cluster clones have not yet formed, continue observation for 1-2 days and then reproeasy (R) The medium can be replaced with human somatic cell reprogramming medium.

[0250] 8. Days 6-8: Observe under the microscope. If the small clusters of clones grow and each clone has more than 10 cells, Reproeasy (R) Human somatic cell reprogramming medium PSCeasy (R)The medium can be changed directly to human pluripotent stem cell medium (or PGM1 human pluripotent stem cell medium). If many dead cells are observed before changing the medium, the medium can be changed after washing with PBS equilibrated at room temperature.

[0251] 9. Days 9-20: The cells were observed under a microscope and the changes in morphology were recorded by photographs. Fresh PSCeasy 200 cells were equilibrated at room temperature. (R) The human pluripotent stem cell medium was replaced daily.

[0252] 10. Day 21: Under microscope observation, if a single cell clone filled the entire 10x field of view, cut the clone with a 1mL syringe needle (or an instrument such as a glass needle) and pick it into a 48-well plate pre-coated with Matrigel (if the clone is in good condition, the cells are thick and grow quickly, it can be picked directly into a 24-well plate).

[0253] 11. After picking clones, use PSCeasy (R) Human pluripotent stem cells were seeded in thawing medium, and after cell attachment, they were incubated with PSCeasy (R) The medium was replaced with human pluripotent stem cell medium, and the culture was continued until the required number of generations had been reached.

[0254] 3. Induction of 3D retina in patients The specific steps are as follows:

[0255] [Table 14]

[0256] 4. Construction and coating of AAV8 virus 1. Plasmid amplification: The constructed AAV vector, packaging plasmid and auxiliary plasmid need to be extracted in large quantities to remove endotoxins, so the Qiagen Maxi kit was used to extract large quantities of plasmids. The process was the same as above; 2. AAV8-293T cell transfection: Observe the cell density on the day of transfection, when it reaches 80-90%, the vector plasmid, packaging plasmid and auxiliary plasmid can be transfected; 3. Harvesting AAV8 virus: viral particles are present in packaging cells and culture supernatant simultaneously. To obtain the best yield, cells and culture supernatant can be harvested; 4. After AAV8 purification, it was stored for a long period at -80℃.

[0257] 5. Verification of sgRNA gene editing efficiency in vitro using 3D retinal tissue 1. Infection of 3D retinal tissue with AAV8: Observe the condition of the tissue, and when the growth condition of the 3D retinal tissue is good, it can be infected with AAV8 virus carrying the desired plasmid.

[0258] 2. Verification of gene editing efficiency. (1) T7E1 enzyme cleavage experiment The steps are shown in the T7E1 enzyme cleavage experiment in Example 1.

[0259] (2) TOPO PCR cloning To quantify the editing efficiency of pX601-CYP4V2-sgRNA and PMD19-T-donor and perform statistical analysis of the cleavage efficiency in the experimental and control groups, we used Invitrogen's Zero Blunt™ PCR kit. (R) TOPO (R) The experiment was carried out using a PCR cloning kit, and after bacterial cell selection, sequencing was carried out using Sanger.

[0260] a) 3D retinal tissue was infected with AAV virus, and the DNA obtained was used as an amplification template using Invitrogen's Platinum SuperFi TM PCR reactions were carried out using the DNA polymerase series in the following reaction system (50 μL reaction system):

[0261] [Table 15]

[0262] b) Touch down PCR program: The specific steps are the same as the sgRNA annealing PCR step in step 3 of Example 1.

[0263] c) The reaction system for TOPO PCR cloning is as follows:

[0264] [Table 16]

[0265] The above reaction system was prepared, mixed gently, and allowed to stand at room temperature for 5 minutes; The mixture was placed on ice, the competent cells were removed, and the mixture was prepared for transformation.

[0266] d) Transformation of competent cells by TOPO PCR cloning reaction Take 50 or 100 μL of competent cells, add 2 μL of the above TOPO PCR cloning reaction solution, and leave on ice for 5-30 min; heat shock at 42°C for 30 sec without shaking; quickly transfer the reaction system to ice and add 250 μL of SOC medium; shake and thaw at 200 rpm at 37°C for 1 h; take a corresponding amount of LB culture plate (containing 25 μg / mL bleomycin), apply 100 μL of the above bacterial solution to the plate, and culture overnight in a 37°C incubator.

[0267] e) Sanger sequencing of selected organisms. The next morning, the cell plates were removed and 80 cells were selected from each plate; shaken at 200 rpm at 37° C. for 3-4 h; and 200 μL of each cell sample was taken for Sanger sequencing.

[0268] f) Sanger sequencing results were analyzed and statistical analysis was performed.

[0269] Example 5: Verification of gene editing efficiency in a humanized mouse model 1. Construction of humanized mice: Construction of humanized mice was performed by Biocytogen Pharmaceuticals (Beijing) Co., Ltd.

[0270] 1. Protocol for creating humanized mice Regarding the human mutation site, we requested Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to construct a CYP4V2 humanized mouse model.

[0271] 2. Details of the technology: (1) Design and construction of sgRNA to recognize the target sequence; (2) Construction of CRISPR / Cas9 vectors that cause cleavage of target genes; (3) sgRNA / Cas9 activity testing; (4) Design and construct a knock-in targeting vector and replace exons 6-8 (including intron sequences) according to the design; (5) Obtaining sgRNA / Cas9 mRNA by in vitro transcription; (6) Injection of sgRNA / Cas9 mRNA and targeting vector into mouse fertilized eggs; (7) Examination and propagation of CYP4V2 knock-in F0 mice; (8) Obtaining CYP4V2 knock-in F1 heterozygous mice and genotyping them (southern blotting DNA hybridization assay, including one exogenous and one endogenous probe).

[0272] 3.Technical methods: (1) Amplify and sequence the target sequence in the mouse genome, design and construct a CRISPR / Cas9 vector plasmid targeting the target sequence, and perform activity testing: (2) Extract mouse genomic DNA and amplify sgRNA that recognizes the target sequence; (3) Design and construct a CRISPR / Cas9 vector plasmid to induce cleavage of the target gene; (4) Use Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to perform activity testing on sgRNA / Cas9 using a test kit independently developed by the company; (5) Select highly active sgRNA / Cas9 target site sequence information and design and construct a CYP4V2 knockout targeting vector; (6) sgRNA / Cas9mRNA and targeting vector were injected into mouse fertilized egg pronuclei to obtain F0 / F1 positive mice. This experiment mainly includes the following: In vitro transcription of sgRNA / Cas9mRNA; Collection of mouse fertilized eggs; injection of RNA and targeting vectors into mouse fertilized eggs; Transfer of fertilized eggs into the surrogate mother's fallopian tubes; Genotyping and propagation of F0 mice with point mutations in the CYP4V2 gene humanization; Obtaining and genotyping F1 mice carrying point mutations in the humanized CYP4V2 gene (including PCR and southern blotting hybridization assays).

[0273] 2. Breeding and rearing After obtaining both kinds of humanized mice, they were intercrossed with F1 heterozygous mice to obtain a sufficient number of F2 or F3 homozygous humanized mice for AAV virus injection as soon as possible.

[0274] 3. AAV virus injection into the subretinal space of mice The pX601-sgRNA (SaCas9 donor) and pX601-Donor-EGFP vectors were packaged into adeno-associated viruses and injected into the retina of CYP4V2 mutant model mice to verify the in vivo editing and repair efficiency of the designed sgRNA and Donor. Taking pX601-sgRNA1+pX601-Donor1-EGFP as an example, the specific steps are as follows. The experimental methods for the sgRNA2-4 groups are similar.

[0275] 1. Experimental Design (1) Blank control group: saline.

[0276] (2) Experimental group: pX601-sgRNA1 (donor of SaCas9) and pX601-Donor-EGFP.

[0277] 2. Selection of AAV (adeno-associated virus) serotype The AAV2 / 8 serotype was chosen due to its better retinal preference.

[0278] 3. Viral Packaging The pX601-Donor1-EGFP and pX601-sgRNA1 vectors were packaged into adeno-associated virus (AAV) with AAV2 / 8 and AAV-helper, respectively.

[0279] 4. Mouse Injection Experiments The experiment was conducted by dividing 20 CYP4V2 mutant model mice into four groups of five mice each.

[0280] The experimental design is as follows. (1) Blank control group: Mice were injected with 2 μL of saline per eye.

[0281] (2) Experimental group: The packaged pX601-sgRNA1 and pX601-Donor1-EGFP viruses were mixed in equal amounts at a 1:1 ratio and injected into each mouse eye at 2 μL (1E10vg).

[0282] (3) The treatment effect was examined one month later. The experimental steps are as follows:

[0283] (4) Thirty minutes before the injection, the pupils were dilated with 1% atropine; the pupils were dilated again before anesthesia.

[0284] (5) Ketamine 80mg / kg and xylazine 8mg / kg were intraperitoneally injected into the mouse to anesthetize it, and then the mouse was placed in front of the animal experiment table of the ophthalmic surgery microscope, and a drop of 0.5% proparacaine was dropped into the mouse's eye to anesthetize it locally. AAV virus was added to the stock solution of sodium fluorescein in a ratio of 100:1, and mixed by centrifugation at low speed.

[0285] (6) A small hole was made in advance in the ciliary plane of the mouse's eyeball with an insulin injection needle, and the needle of a microinjector was passed through the small hole and entered into the vitreous cavity of the mouse's eyeball. At this time, an appropriate amount of 2% hydroxymethylcellulose was dropped onto the mouse's eyeball so that the fundus of the mouse's eyeball could be clearly seen under a microscope. The needle was then inserted into the peripheral part of the retina on the opposite side, avoiding the vitreous body, and the AAV virus containing sodium fluorescein was slowly pushed out. The injection volume per eye was 1 μL, and sodium fluorescein was used as an indicator to determine whether it had been injected into the subretinal space.

[0286] (7) After surgery, the mice were observed for any abnormalities and administered neomycin eye ointment to prevent infection.

[0287] 4. Evaluation of the effectiveness of gene editing therapy The experimental results showed that the therapeutic effects of each experimental group were basically consistent, so the effect of gene editing therapy was explained using the pX601-sgRNA+pX601-Donor-EGFP virus group as a representative.

[0288] The effects of gene editing therapy are mainly explained in the following items: Functional alterations in mice after treatment were evaluated by ERG (retinal electrophysiology), and the retinal function of the mutant mouse model was improved after treatment; After sectioning the samples, the morphological changes in the mouse retina were observed by HE staining, and the morphology was found to be similar to that of the wild-type mouse retina; We used immunofluorescence staining to observe whether the gene editing therapeutic vector was expressed in the corresponding location of the retina, and found that the gene editing therapeutic vector was expressed in the corresponding location of the retina; Morphological changes in retinal tissue were observed through a series of staining traces of a special retinal marker, and it was found that the morphology of the retinal tissue of the mice was improved after treatment.

[0289] 5. Evaluation of editing efficiency of AAV8-pX601-CYP4V2-SgRNA in humanized mice The verification method was the same as the fifth step of Example 4, “Verification of sgRNA gene editing efficiency in vitro using retinal tissue.” The gene editing efficiency of sgRNA was verified by T7E1 enzyme cleavage experiments and TOPO PCR cloning experiments, and it was found that sgRNA can achieve good gene editing efficiency.

[0290] Finally, it should be noted that the above examples are only used to explain the technical idea of ​​the present application, and are not intended to limit the same. Although the present application has been described in detail with reference to the above examples, those skilled in the art will understand that the technical idea described in the above examples can be modified or some technical features can be replaced equally without departing from the spirit and scope of the present invention.

Claims

1. A gRNA that specifically targets a cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene, the gRNA containing any of the nucleotide sequences of SEQ ID NOs: 48-51 that specifically bind to an intron region between exon 6 and exon 7 of the CYP4V2 gene.

2. The gRNA of claim 1, wherein the gRNA is a single-stranded guide RNA (sgRNA).

3. 3. One or more isolated nucleic acid molecules encoding gRNAs that specifically target the CYP4V2 gene of any one of claims 1 to 2.

4. A vector comprising the isolated nucleic acid molecule of claim 3.

5. further comprising a donor nucleic acid molecule, The vector of claim 4, wherein the donor nucleic acid molecule contains a nucleotide sequence between intron 6 and exon 11 of the CYP4V2 gene and, when introduced into a recipient cell, is capable of repairing a DNA fragment cleaved by a gRNA encoded by the isolated nucleic acid molecule.

6. The vector of claim 5 , wherein the donor nucleic acid molecule contains the nucleotide sequence set forth in SEQ ID NO:

39.

7. The vector of claim 5 , wherein the isolated nucleic acid molecule and the donor nucleic acid molecule are present on the same vector.

8. The vector according to any one of claims 4 to 7, wherein the vector is a viral vector.

9. A cell comprising an isolated nucleic acid molecule according to claim 3 and / or a vector according to any one of claims 4 to 8.

10. The cell of claim 9 , wherein the cell comprises a HEK293 cell, a renal epithelial cell and / or an induced pluripotent stem cell.

11. The cell according to claim 9 , wherein the cell has differentiation potential due to modification.

12. The cell of any one of claims 9 to 11, wherein the cell is capable of differentiating into a 3D-retinal organoid.

13. A pharmaceutical composition comprising: i) a gRNA according to any one of claims 1 to 2, one or more isolated nucleic acid molecules according to claim 3, or a vector according to claim 4; ii) a Cas nuclease, or a nucleic acid molecule or vector containing a nucleotide sequence encoding a Cas nuclease; iii) a donor nucleic acid molecule as defined in claim 5 or 6, or a vector containing said donor nucleic acid molecule; and iv) a pharma- ceutically acceptable carrier.

14. 10. A kit comprising a gRNA according to any one of claims 1 to 2, one or more isolated nucleic acid molecules according to claim 3, and / or a vector according to any one of claims 4 to 8.

15. 14. Use of a gRNA according to any one of claims 1 to 2, one or more isolated nucleic acid molecules according to claim 3, and / or a vector according to any one of claims 4 to 8 in the manufacture of a pharmaceutical composition for the treatment of a disease, wherein the disease comprises a disease caused by a mutation in the CYP4V2 gene, and the pharmaceutical composition is the pharmaceutical composition according to claim 13.

16. The use according to claim 15, wherein the mutation is located after the intron between exons 6 and 7 of the CYP4V2 gene.

17. The use according to claim 16, wherein the disease comprises Bietti crystalline dystrophy.

18. 14. A pharmaceutical composition according to claim 13 for use in the treatment of Vietti crystalline dystrophy.

19. The pharmaceutical composition of claim 18, wherein the treatment results in a CYP4V2 protein having normal function.

20. 20. The pharmaceutical composition of any of claims 18 to 19, wherein the treatment comprises an injection.

21. 21. The pharmaceutical composition of any of claims 18 to 20, wherein the treatment comprises a subretinal injection.

22. A non-therapeutic in vitro method for regulating expression of the CYP4V2 gene in a cell, comprising introducing into said cell a gRNA described in any one of claims 1 to 2, one or more isolated nucleic acid molecules described in claim 3, and / or a vector described in any one of claims 4 to 8.

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