Gene-editing drug vector for autosomal dominant retinitis pigmentosa

Abstract

The present invention relates to a vector containing a Cas protein coding sequence and an sgRNA for specifically targeting an RHO pathological allele. The present invention further relates to a CRISPR-Cas system, a cell, a pharmaceutical composition, and a kit containing the vector, and the use thereof in the treatment of retinitis pigmentosa caused by RHO gene mutations.

Description

A gene editing drug carrier for autosomal dominant retinitis pigmentosa TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. More particularly, the present application relates to a gene editing drug carrier for RHO-adRP patients. BACKGROUND

[0002] Autosomal dominant Retinitis Pigmentosa (adRP) is a group of inherited blinding eye diseases characterized by progressive loss of photoreceptor and / or retinal pigment epithelial cell function. The clinical manifestations of RP are initially night blindness, most patients develop in childhood or adolescence, and as the disease progresses, the visual field gradually narrows to a tubular visual field, and most patients develop into legally blind people at the age of 40-50. There is currently no effective treatment for RP patients (traditional drugs and surgical treatment are basically ineffective). Since adRP is autosomal dominant, there are normal proteins and abnormal proteins in the cells of patients, and the dominant negative effect of abnormal proteins will cause cell apoptosis, so the conventional gene replacement therapy is not suitable for adRP.

[0003] RHO (Rhodopsin) is the earliest discovered RP pathogenic gene, about 25%-30% of autosomal dominant RP (adRP) is caused by this gene, and it is the most important pathogenic gene of adRP [1, 2]. The protein encoded by RHO gene is a G protein-coupled receptor with 7 transmembrane structures, which is composed of 348 amino acids, mainly expressed in the outer segment disc of rod cells (Rods), and exists in the form of highly ordered dimers or multimers, accounting for more than 85% of the total disc membrane protein, and plays an important role in phototransduction by combining with opsin [3]. RHO gene mutation leads to the production of abnormal proteins, and abnormal proteins cause diseases through dominant negative effect [4]. Since the patient with RHO mutation still has a normal allele in one DNA strand, and the protein encoded by one DNA strand is enough to maintain the normal function of RHO protein. Therefore, if specific gene editing can be performed on the mutation site, the translation of the mutant chain is silenced, and the expression of mutant proteins is inhibited, while the normal allele is kept intact, which is the most ideal treatment plan for this gene [5].

[0004] In early 2013, the discovery of the third generation of artificial endonuclease CRISPR / Cas9 brought a revolutionary leap to genome site-directed editing technology[5,6]. Gene editing is to use endonuclease to produce double-strand breaks at a specific location in the genome, and through the body's endogenous DNA repair process to repair the break point. In the repair process, the genome can be modified to obtain the desired genotype. Compared with the first generation of artificial endonuclease ZFN and the second generation of artificial endonuclease TALEN, the CRISPR / Cas9 editing system has the advantages of simple target design, easy operation, low cost and high editing efficiency, and is widely used in cell line modification, disease animal model establishment and gene therapy.

[0005] Among the thousands of RP patients who visited Peking University Third Hospital in recent years, multiple RP families with clear genetic diagnosis of RHO gene mutations were collected, and the mutation hotspot c.403C>T; p.Arg135Trp (i.e. RHO-R135W mutation) of RHO gene was found. This mutation site makes the binding ability of RHO to V-Arrestin stronger, causing RHO aggregation in endocytic vesicles, resulting in abnormal endocytosis of cells, and ultimately leading to severe RP phenotype[4,7]. In the previous research, the applicant screened different combinations of Cas9 and sgRNA (CN 116334141A), and delivered them to target cells through AAV to effectively edit the mutated RHO DNA sequence. In CN 116334141A, the promoters of Cas protein sequences are EFS and hGRK1. Although the EFS promoter has high editing efficiency, it is not an eye-specific promoter, and there is a potential off-target risk and safety risk. Literature research has shown[8,9] that the hGRK1 promoter has good specificity and expression efficiency in photoreceptor cells, but due to the high expression abundance of RHO in photoreceptor cells, whether the hGRK1 promoter can effectively improve the retinal pigmentosa phenotype caused by RHO mutation still needs further research.

[0006] Because gene editing therapy targets patient DNA and AAV has the characteristics of long-term expression, the vector structure used in clinical treatment needs to be further optimized to ensure the editing efficiency and safety of gene editing drugs in vivo.

[0007] SUMMARY

[0008] The inventors further improved the editing efficiency of gene editing vector drugs by optimizing the promoters for expressing Cas9 and sgRNA, reduced the amount of vector used, and thus limited the widespread distribution of the vector, thereby reducing the potential off-target risk and safety risk. Research results show that the optimized promoter combined with candidate Cas9 and sgRNA can effectively improve the phenotype of mouse models and has good safety.

[0009] Specifically, the inventors have found, through in-depth research, that, relative to the Cas protein promoters EFS and hGRK1 used in CN 116334141 A, the use of the optimized new constitutive promoters RK1 or RK2 can not only significantly improve the expression efficiency and editing efficiency of the RHO-R135W gene therapy drug, but also surprisingly does not affect its specificity (only expressed in retinal outer nuclear layer cells), achieving an excellent balance between editing efficiency and safety.

[0010] Therefore, according to one aspect of the present application, a vector is provided, comprising: (1) a nucleotide sequence encoding a Cas protein and a first promoter operably linked thereto, wherein the first promoter is an RK1 promoter or an RK2 promoter; and (2) a nucleotide sequence encoding a gRNA and a second promoter operably linked thereto, wherein the gRNA targets or specifically binds to a RHO gene or a fragment thereof.

[0011] In some embodiments, the RK1 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 5, and the RK2 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 6.

[0012] In some embodiments, the Cas protein comprises a Cas9 protein.

[0013] In some embodiments, the Cas9 protein comprises KKH-SaCas9, SauriCas9, SlugCas9 or SlugCas9-HF.

[0014] In some embodiments, the nucleotide sequence encoding the Cas protein comprises the nucleotide sequence set forth in SEQ ID NO: 16, 17, 18 or 19.

[0015] In some embodiments, the gRNA targets the mutation hotspot c.403C>T; p.Arg135Trp of the RHO gene.

[0016] In some embodiments, the nucleotide sequence encoding the gRNA is a single guide RNA (sgRNA), preferably comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some embodiments, the second promoter is a U6 promoter, preferably the U6 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 20.

[0018] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0019] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0020] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0021] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2

[0022] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0023] In some embodiments, the first promoter is an RK1 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0024] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1;

[0025] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1;

[0026] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0027] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0028] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2; or

[0029] In some embodiments, the first promoter is an RK2 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0030] In some embodiments, the vector further comprises a nuclear import signal, preferably the nuclear import signal comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 21-23.

[0031] In some embodiments, the vector comprises, in order, the following elements: a first promoter, a Kozak sequence, a nuclear localization signal (NLS), a nucleotide sequence encoding a Cas protein, a nuclear localization signal, a transcription terminator sequence, a second promoter, a nucleotide sequence encoding a gRNA, and a gRNA scaffold sequence.

[0032] In some embodiments, the Kozak sequence comprises the nucleotide sequence set forth in SEQ ID NO: 24.

[0033] In some preferred embodiments, the transcription terminator sequence is a BGH sequence (bGH poly(A)) or a SV40 poly(A). More preferably, the BGH sequence is a BGH sequence, most preferably comprising the nucleotide sequence set forth in SEQ ID NO: 25.

[0034] In some embodiments, the gRNA scaffold sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27.

[0035] In some embodiments, the vector comprises a viral vector.

[0036] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0037] In some embodiments, the adeno-associated viral vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variant or mixtures, preferably an AAV8 vector.

[0038] According to another aspect of the application, there is provided a CRISPR-Cas system comprising a vector according to the application.

[0039] According to another aspect of the application, there is provided a cell comprising a vector according to the application.

[0040] According to another aspect of the application, there is provided a pharmaceutical composition comprising a vector, a CRISPR-Cas system, and / or a cell according to the application.

[0041] According to another aspect of the application, there is provided a kit comprising a vector, a CRISPR-Cas system, and / or a cell according to the application.

[0042] According to another aspect of the application, there is provided the use of a vector, a CRISPR-Cas system, and / or a cell according to the application for the manufacture of a medicament for the treatment of retinitis pigmentosa caused by a mutation in the RHO gene (R135W).

[0043] In some embodiments, the medicament is formulated in a form suitable for injection.

[0044] In some embodiments, the medicament is formulated in a form suitable for subretinal cavity injection.

[0045] According to another aspect of the application, there is provided a method of treating retinitis pigmentosa caused by a mutation in the RHO gene (R135W), the method comprising administering to a subject a therapeutically effective amount of a vector, a CRISPR-Cas system, and / or a cell according to the application.

[0046] The present application is based on the research results of gene editing drugs designed for RHO-R135W mutant patients in the early stage. By screening different promoters expressing Cas9 in gene editing drugs, the specificity and expression efficiency of Cas9 in target cells are improved. The editing efficiency of different promoters on target cells is analyzed by gene editing efficiency detection, and a promoter with high specificity and high editing efficiency is screened. Further, the different promoters are verified in humanized mouse models to recover the function of the disease phenotype in the mouse model, and the in vivo effectiveness of the candidate promoter is confirmed. In addition, the safety of gene editing drugs is crucial for clinical application. Further, the non-target distribution and non-target editing efficiency of different promoters in mice are compared in vivo, and a vector molecule with good effectiveness and safety is screened to ensure the safety and effectiveness of the drug molecule in clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0047] The above features and advantages of the present application will be more apparent from the following detailed description in conjunction with the accompanying drawings, in which:

[0048] Figure 1. Structure diagram of gene editing treatment vector for adRP caused by RHO mutation;

[0049] Figure 2. T7E1 detection of sgRNA editing efficiency in 293A cells;

[0050] Figure 3. Hi-tom sequencing detection of editing efficiency of different promoters in cells;

[0051] Figure 4. In vivo EGFP expression efficiency detection of different promoters;

[0052] Figure 5. ERG detection of the effect of different vector combinations on the function of the model retina (error bar = mean ± SEM, n = 12);

[0053] Figure 6. FP and OCT detection of the effect of different vector combinations on the function of the model retina;

[0054] Figure 7. Target expression product mRNA level detection of target tissue;

[0055] Figure 8. T7E1 enzyme digestion detection of editing efficiency in model mice;

[0056] Figure 9. Hi-tom sequencing detection of editing efficiency in model mice;

[0057] Figure 10. Non-target expression product mRNA level detection of target tissue; and

[0058] Figure 11. Liver tissue DNA editing detection. DETAILED DESCRIPTION

[0059] The terms used herein have their ordinary technical meanings as understood by one of ordinary skill in the art unless otherwise defined. For definitions and terms of the art, the skilled artisan is particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0060] Definitions of Terms

[0061] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made by known or novel methods. The sequence of a polynucleotide can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation to an labeling component.

[0062] In the present application, the "vector" generally refers to a nucleic acid molecule that is capable of self-replication in a suitable host, to transfer an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-described functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0063] In the present application, the term "viral vector" generally refers to a non- wild type recombinant viral particle that serves as a gene delivery vehicle and comprises a recombinant viral genome packaged within a viral capsid. The animal viral species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40).

[0064] In the present application, the term "promoter" generally refers to a segment of deoxyribonucleic acid (DNA) sequence that enables transcription of a particular gene. The promoter can be recognized by RNA polymerase and initiates transcription to synthesize RNA. In the synthesis of ribonucleic acid (RNA), the promoter can interact with transcription factors that regulate gene transcription, control the initiation time of gene expression (transcription), and the extent of expression. The promoter comprises a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression, upstream of the transcription initiation site of the gene (5' direction of the DNA anti-sense strand), and has no coding function itself. According to its mode of action and function, it is divided into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or developmental stage-specific), and inducible promoters (regulated by external chemical or physical signals).

[0065] In the present application, the term "operably linked" generally refers to the placement of regulatory sequences necessary for expression of a coding sequence in the appropriate position relative to the coding sequence so as to effect expression of the coding sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. In certain embodiments, this can mean the arrangement of coding sequences and transcriptional control elements in an expression vector. The control elements can include promoters, enhancers, and termination elements. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In certain embodiments, "operably linked" can also mean that a gene of interest is linked to a vector such that the transcriptional and translational control sequences within the vector function to direct the transcription and translation of the gene of interest.

[0066] In the present application, the term "cell" can generally be or have been a single cell, a cell line, or a cell culture that is the recipient of a nucleic acid molecule or a vector. The cell can include a nucleic acid molecule described in the present application or a vector described in the present application. The cell can include progeny of a single cell. The progeny can not necessarily be identical to the original parent cell (in morphology of total DNA complement or on the genome) due to natural, accidental, or deliberate mutation. The cell can include a cell transfected in vitro with a vector described in the present application. The cell can be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NSO cell, or a myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293T cell.

[0067] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, a human patient. For example, a pharmaceutical composition described in the present application, which can include a vector described in the present application and / or a cell described in the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition can also include one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable ingredients of the composition can be non-toxic to the recipient at the used dose and concentration. The pharmaceutical composition of the present application includes but is not limited to liquid, frozen and lyophilized compositions.

[0068] In the present application, the term "treatment" generally refers to a clinical intervention aimed at altering the natural course of a clinical pathology in the individual or cell being treated. It can include improving the disease state, eliminating the lesion, or improved prognosis.

[0069] In the present application, the term "subject" generally refers to any subject for which diagnosis, treatment or therapy is desired. For example, in the present application, the RHO gene of the subject in need comprises a c.C403T mutation site. In certain cases, the subject can include a mammal. For example, the subject can include but is not limited to a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, or a monkey. In certain cases, the subject can include a human. In certain cases, the subject can include an East Asian.

[0070] In the present application, the term "administering" can involve introducing cells and / or vectors into a subject, or a desired site of a subject, by a method or route. The cells and / or vectors can express nucleic acid molecules of the present application (e.g., sequences encoding gRNAs and / or gRNAs) at the desired site (e.g., site of injury or repair), thereby producing a desired effect. Cells (or their differentiated progeny) and / or vectors can be administered by any suitable route that can deliver the cells (or their differentiated progeny) and / or vectors to a desired site in a subject, and at least a portion of the implanted cells (or cellular components) and / or vectors remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, a few days, up to several years, or even consistent with the life span of the patient. In certain instances, the administration involves injection. For example, the vectors can be administered by systemic routes of administration such as intraperitoneal or intravenous routes. For example, the administration can involve subretinal cavity injection.

[0071] In the present application, the term "comprising" generally means including, but not excluding other elements.

[0072] RP and RHO genes

[0073] In one aspect, the present application provides a method of treating retinitis pigmentosa.

[0074] In the present application, the term "Retinitis Pigmentosa (RP)" generally refers to a genetic disease that causes degeneration of the retina. There are more than 80 genes identified to be associated with RP, and these genes are involved in autosomal recessive (50-60%), autosomal dominant (autosomal dominant: AD, 30-40%), and X-linked inheritance (5-15%). RP is characterized by progressive vision loss due to dysfunction of the receptor cells (cone and rod cells) and / or retinal pigment epithelial cells of the retina. The clinical manifestations of RP can include night blindness, progressive visual field defects, central visual acuity reduction after macular involvement, and eventually blindness, with Electroretinogram (ERG) showing decreased rod function or even extinguished. The main fundus changes of RP are pigment disorders in the equatorial retina, with osteocyte-like pigmentation, gradually developing towards the posterior pole and ora serrata. The RPE, photoreceptor cells, and choroidal capillary layer gradually atrophy, with visible choroidal large vessels, a bluish-gray retina, retinal arterioles, and a waxy yellow atrophic optic disc. Among them, retinal vascular stenosis, waxy yellow discoloration of the optic disc, and osteocyte-like pigmentation are the typical triad of RP (Hartong DT et al., 2006). Methods for assessing retinal function and morphology can include Best Corrected Visual Acuity (BCVA), fundus autofluorescence, visual field examination, ERG, fundus color photography, optical coherence tomography (OCT), and fluorescein angiography (FFA), etc. Among them, methods for assessing visual function can include BCVA, visual field, etc.

[0075] The vectors or methods described in the present application can provide a subject in need with a functional RHO gene.

[0076] In some cases, the RP described in the present application can be caused by mutations in the RHO gene. There are various RHO gene mutations associated with RP, which can cause the RHO gene to encode a functionally abnormal rhodopsin. The mutations can include, but are not limited to, missense, nonsense, insertion, deletion, etc. For example, the mutation site can comprise mutation site c.C403T. For another example, the mutation site can cause an amino acid change, and the amino acid mutation can comprise a p.Arg135Trp change. In the present application, the method can comprise providing a subject in need with a RHO gene that does not have a heterozygous mutation site, which can be selected from c.C403T.

[0077] Any one or more mutations can be repaired to provide a subject in need with a functional RHO gene. For example, the pathological variant c.C403T can be removed, restored, or corrected.

[0078] In the present application, the term "c.403C>T" or "c.C403T" generally refers to a mutation of the base at position 403 of the coding sequence of the RHO gene (from the 5' end to the 3' end, the "A" in the start ATG of the coding sequence is at position 1) from cytosine (C) to thymine (T) as compared to the nucleotide sequence of the wild-type RHO gene, which mutation can result in a mutation of the amino acid encoded by the RHO gene, for example, such that the amino acid is mutated from arginine (Arg) to tryptophan (Trp). In the present application, the term "p.Arg135Trp" generally refers to a mutation of the amino acid at position 135 of the RHO protein from arginine (Arg) to tryptophan (Trp).

[0079] In vivo or in vitro methods

[0080] The methods described herein can comprise knocking out the mutation site and / or reducing the expression level of the mutation site. Methods of knocking out a gene or reducing the expression level of a gene can comprise performing gene knockout, conditional gene knockout methods (e.g., using Cre / LoxP and / or FLP-frt systems), inducible gene knockout methods (e.g., Cre / loxp system-based knockouts, including tetracycline induction, interferon induction, hormone induction, adenovirus induction, etc.), gene knockout using random insertion mutations (e.g., gene trap methods), gene knockout using RNAi, zinc finger nucleases (ZNF)-mediated gene editing technology, transcription activator-like effector nucleases (TALEN)-mediated gene editing technology, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated proteins (Cas) (CRISPR / Cas)-mediated gene editing technology, and / or NgAgo-gDNA gene editing technology. For any genome editing strategy, the gene editing can be confirmed by sequencing or PCR analysis.

[0081] In some cases, the methods described herein can be in vivo cell-based methods. In some cases, the methods comprise editing the genomic DNA of a cell of a subject. For example, the methods can comprise editing a mutation in the RHO gene in a cell of a subject (e.g., a photoreceptor cell and / or a retinal progenitor cell). For example, the genetic mutation can be a c.C403T mutation. While certain cells can be ideal targets for ex vivo methods or ex vivo therapies, the use of effective delivery methods can also allow for the direct delivery of the desired reagents to such cells in vivo. In some cases, the methods can comprise targeting and editing to the relevant cells. Lysis of other cells can also be prevented by the use of promoters that are active only in certain cells and / or at certain stages of development.

[0082] The exogenous promoters are inducible, thus, if the nucleic acid molecule is delivered in a plasmid vector, the time of delivery can be controlled. The time that the delivered nucleic acid or protein remains resident within the cell can also be adjusted by methods that alter the half-life. In vivo methods can save certain processing steps, but require higher editing efficiency. In vivo therapy can eliminate the problems and losses associated with ex vivo therapy and implantation.

[0083] In vivo methods can facilitate the production and administration of therapeutic products. The same therapeutic method or therapy will have the potential to be used to treat more than one subject, e.g., many subjects having the same or similar genotype or allele.

[0084] The methods described herein can comprise ex vivo methods. In some cases, a subject-specific induced pluripotent stem cell (iPSC) can be obtained. The genomic DNA of these iPSC cells can then be edited using the methods described herein. For example, the methods can comprise editing within or near a mutation site of the RHO gene of the iPSC such that it does not have the amino acid mutation of p.Arg135Trp, e.g., the genetic mutation can be a c.C403T mutation. Next, the genetically edited iPSCs can be differentiated into other cells, e.g., photoreceptor cells or retinal progenitor cells. Finally, the differentiated cells (e.g., photoreceptor cells or retinal progenitor cells) can be implanted into a subject.

[0085] In other cases, photoreceptor cells or retinal progenitor cells can be isolated from a subject. Next, the genomic DNA of these photoreceptor cells or retinal progenitor cells can be edited using the methods described herein. For example, the methods can comprise editing within or near a mutation site of the RHO gene of the photoreceptor cells or retinal progenitor cells such that it does not have the amino acid mutation of p.Arg135Trp, e.g., the genetic mutation can be a c.C403T mutation. Finally, the genetically edited photoreceptor cells or retinal progenitor cells can be implanted into a subject.

[0086] In other cases, mesenchymal stem cells can be isolated from bone marrow or peripheral blood in other cases. Next, the genomic DNA of these mesenchymal stem cells can be edited using the methods described herein. For example, the methods can include editing within or near the mutation site of the RHO gene of the mesenchymal stem cell such that it does not have the amino acid mutation of p.Arg135Trp, e.g., the genetic mutation can be a c.C403T mutation. Next, the genetically edited mesenchymal stem cells can be differentiated into any type of cell, e.g., photoreceptor cells or retinal progenitor cells. Finally, the differentiated cells, e.g., photoreceptor cells or retinal progenitor cells, can be implanted into the subject.

[0087] The methods can include a comprehensive analysis of the therapeutic agent prior to administration. For example, the entire genome of the corrective cells is sequenced to ensure that any off-target effects, if any, can be at genomic locations associated with minimal risk to the subject. In addition, populations of specific cells, including clonal cell populations, can be isolated prior to implantation.

[0088] Using the methods described herein can not affect the expression level and / or function of the wild-type RHO gene in the subject.

[0089] Genetic editing

[0090] The methods described herein can include methods of using site-directed nucleases to cleave DNA at precise target locations in the genome, thereby creating single- or double-stranded DNA breaks at specific locations within the genome. Such breaks can be periodically repaired by endogenous cellular processes, such as HDR and non-homologous end joining (NHEJ). These two major DNA repair processes consist of a series of alternative pathways. NHEJ directly ligates the DNA ends resulting from double-stranded breaks, sometimes with loss or addition of nucleotide sequences, which can disrupt or enhance gene expression. HDR utilizes a homologous sequence or donor sequence as a template to insert a specific DNA sequence at the breakpoint. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus. These exogenous nucleic acids can comprise a region of high homology to the nuclease-cleaved locus, and in addition can comprise additional sequences or sequence alterations, including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ (ANHEJ)", which can result in small deletions and insertions at the cleavage site, with genetic outcomes similar to NHEJ. MMEJ can utilize a few base pairs of homology flanking the DNA break site to drive a more favorable DNA end joining repair outcome. In some cases, it can be possible to predict the likely repair outcome based on analysis of potential microhomologies at the DNA break site.

[0091] These gene editing mechanisms can all be used for the removal of a desired genetic mutation site according to the present application. The methods described herein can include creating one or two DNA breaks at a location in the target locus proximal to the intended mutation site, which can be a double-stranded break or two single-stranded breaks. In some cases, the removal can include making a double-stranded break in the RHO allele comprising the mutation. The break can be achieved by a site-directed polypeptide. Site-directed polypeptides, such as DNA endonucleases, can introduce double-stranded breaks or single-stranded breaks in a nucleic acid, such as genomic DNA. Double-stranded breaks can stimulate the cell's endogenous DNA repair pathways, such as, for example, HDR, NHEJ, or MMEJ. NHEJ can repair the cleaved target nucleic acid without the need for a homologous template.

[0092] In some cases, a foreign polynucleotide sequence can be inserted into the target nucleic acid cleavage site using homologous recombination. The foreign polynucleotide sequence can be referred to as a donor polynucleotide (or donor, or donor sequence, or polynucleotide donor template). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be a foreign polynucleotide sequence, i.e., a sequence that is not naturally occurring at the target nucleic acid cleavage site.

[0093] HDR occurs when a homologous repair template or donor is available. The homologous donor template can comprise at least a portion of a wild-type RHO gene or cDNA. The at least a portion of a wild-type RHO gene or cDNA can be exon 1, exon 2, exon 3, exon 4, exon 5, an intronic region, a fragment or combination of the foregoing, or an entire RHO gene or cDNA. The donor template can be a single- or double-stranded polynucleotide. The donor template can be delivered by AAV. The homologous donor template can comprise sequences homologous to sequences flanking the target nucleic acid cleavage site. For example, the donor template can have arms homologous to the 3q22.1 region. The donor template can also have arms homologous to the pathological variant c.C403T. The sister chromatid can be used by the cell as a repair template. However, for the purposes of gene editing, the repair template can be provided as an exogenous nucleic acid, such as a plasmid, a double-stranded oligonucleotide, a single-stranded oligonucleotide, or a viral nucleic acid. With the exogenous donor template, additional nucleic acid sequences (e.g., transgenes) or modifications (e.g., single- or multi-base alterations or deletions) can be introduced between the homologous flanking regions, such that additional or altered nucleic acid sequences can also be incorporated into the target locus. MMEJ can utilize a few base pairs of homologous sequence located on either side of the cleavage site to drive a favored end-joining DNA repair outcome. In some cases, possible repair outcomes can be predicted based on analysis of potential microhomologies in the nuclease target region.

[0094] CRISPR / Cas system

[0095] In the present application, the term "CRISPR / Cas system" or "CRISPR-Cas system" generally refers to a nuclease system composed of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (i.e., Cas proteins) that is capable of cleaving almost all genomic sequences adjacent to a protospacer-adjacent motif (PAM) in eukaryotic cells. "CRISPR / Cas system" can be used to refer collectively to transcripts involving CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or in directing their activity, which can include sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions thereof), tracr-mate sequences (encompassing "direct repeats" and processed partial direct repeats in the context of endogenous CRISPR / Cas systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR / Cas systems), or other sequences and transcripts from CRISPR loci. Five types of CRISPR systems have been identified (e.g., Type I, Type II, Type III, Type U, and Type V).

[0096] In the present application, the term "Cas protein" also referred to as "CRISPR-associated protein" generally refers to a class of enzymes that are complementary to CRISPR sequences, capable of using CRISPR sequences as guides to recognize and cleave specific DNA strands. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or their homologs, or modified versions thereof. In some embodiments, the Cas protein is a Cas9 protein.

[0097] In the present application, the term "Cas9 protein" or "Cas9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in type II CRISPR / Cas system that are involved in both crRNA biosynthesis and destruction of invading DNA. Cas9 proteins generally include a RuvC nuclease domain and a HNH nuclease domain, which cut two different strands of a double-stranded DNA molecule, respectively. Cas9 proteins have been described 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, Streptococcus pyogenes Cas9 protein, the amino acid sequence of which can be found in SwissProt database accession number Q99ZW2; Neisseria meningitides Cas9 protein, the amino acid sequence of which can be found in UniProt database number A1IQ68; Streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, the amino acid sequence of which can be found in UniProt database number J7RUA5.

[0098] The CRISPR / Cas system can include a number of short repeat sequences, referred to as "repeats". When expressed, the repeat sequences can form secondary structures (e.g. hairpins) and / or comprise unstructured single-stranded sequences. The repeat sequences usually occur in clusters and often diverge between species due to evolution. These repeat sequences are regularly spaced with unique intervening sequences referred to as "spacers", thereby forming a repeat-spacer-repeat locus structure. The spacers are identical or have high homology to known foreign invader sequences. The spacer-repeat units encode a crispRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. The crRNA comprises a "seed" or spacer sequence that targets a target nucleic acid (naturally occurring form in prokaryotes, the spacer sequence targets a foreign invader nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0099] The CRISPR / Cas system can also include a polynucleotide sequence encoding a CRISPR-associated protein (Cas protein). Cas genes encode nucleases involved in the biogenesis and interference stages of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0100] In nature, crRNA biogenesis in Type II CRISPR systems requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNase III and then hybridizes to the crRNA repeat sequences in the pre-crRNA. Endogenous RNase III can be recruited to cleave the pre-crRNA. The cleaved crRNAs can be trimmed by exonucleases to produce mature crRNA forms (e.g., 5' end trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex can direct the complex to a target nucleic acid that can hybridize to the crRNA. Hybridization of the crRNA to the target nucleic acid can activate the Cas9 to cleave the target nucleic acid. The target nucleic acid in Type II CRISPR systems is referred to as a protospacer adjacent motif (PAM). In fact, the PAM is essential to promote binding of the site-directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) can be further subdivided into Type II-A (CASS4) and Type II-B (CASS4a). CRISPR / Cas9 systems useful for RNA-programmable gene editing can be found in Jinek et al., Science, 337(6096):816-821 (2012). International Patent Application Publication No. WO 2013 / 176772 provides numerous examples and applications of CRISPR / Cas endonuclease systems useful for site-specific gene editing.

[0101] gRNA

[0102] In the present application, the term "sequence encoding a gRNA" generally refers to a DNA sequence from which the gRNA can be obtained by transcription.

[0103] The gRNAs described herein can bind to a sequence in a target nucleic acid of interest. Nucleic acids (or portions thereof) that target a genome can interact with a target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The gRNAs described herein can be single guide RNAs (sgRNAs), and the nucleotide sequence of the sgRNAs can vary depending on the sequence of the target nucleic acid of interest.

[0104] In the CRISPR / Cas system of the present application, the gRNA sequence can be designed to hybridize to a target nucleic acid proximal to a PAM sequence recognizable by the Cas protein used in the system. The gRNA can be perfectly matched or mismatched to the target sequence. Cas proteins generally all have a specific PAM sequence that can be recognized in the target DNA.

[0105] For example, the Cas9 protein can be from S. pyogenes, which recognizes a PAM comprising the sequence 5'-NRG-3' in the target nucleic acid, wherein R comprises A or G, and wherein N can be any nucleotide. For another example, the Cas9 protein can be from Staphylococcus aureus, which recognizes a PAM comprising the sequence 5'-NNGRR(T)-3' in the target nucleic acid, wherein R comprises A or G, and wherein N can be any nucleotide. In some more specific cases, the PAM sequence recognized by SaCas9 can comprise 5'-NNGRR-3', wherein R comprises A or G, and wherein N can be any nucleotide. A mutant of SaCas9, KKH-SaCas9 (PAM sequence: 5'-NNNRRT-3'), can successfully generate high efficiency mutations in the host. A miniaturized Cas9 protein, such as SauriCas9 (Hu Z, Wang S, Zhang C, Gao N, Li M, et al. (2020) A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope. PLoS Biol 18: e3000686), SlugCas9, or SlugCas9-HF (Hu Z, Zhang C, Wang S, Gao S, Wei J, et al. (2021) Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity. Nucleic Acids Res 49: 4008-4019) can also be used.

[0106] The gRNAs for use in the CRISPR system described herein can be synthesized chemically, e.g., by high performance liquid chromatography. For example, two or more RNA molecules are ligated together. Longer RNAs, such as those encoding Cas9, can be obtained by enzymatic reactions. In the art, various types of RNA modifications can be introduced during or after chemical and / or enzymatic synthesis of the RNA, e.g., modifications to enhance stability, reduce innate immune responses, and / or enhance other properties.

[0107] gRNA scaffold

[0108] The CRISPR / Cas9-based gene editing system includes at least one gRNA scaffold. The gRNA scaffold facilitates the binding of Cas9 to the gRNA and the endonuclease activity. The gRNA scaffold is a polynucleotide sequence immediately following the gRNA targeting sequence. The gRNA targeting sequence and the gRNA scaffold together form a polynucleotide. In some embodiments, the gRNA scaffold comprises the nucleotide sequence set forth in SEQ ID NO: 27.

[0109] Vector

[0110] Vectors are provided herein. The vectors described herein can comprise the nucleic acid molecules (e.g., sequences encoding gRNAs and / or gRNAs) described herein. The polynucleotides can be delivered by non-viral delivery vectors, including but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes.

[0111] The vectors can also be polynucleotide vectors, e.g., plasmids, cosmids, or transposons. Vectors suitable for use have been widely described and are well known in the art. Those skilled in the art will appreciate that vectors comprising the nucleic acid molecules described herein can also comprise additional sequences and elements that can be desirable for replication of the vector in prokaryotic and / or eukaryotic cells. For example, the vectors described herein can include a prokaryotic replicon, i.e., a nucleotide sequence that has the ability to direct the host itself to replicate and maintain in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well known in the art. In certain instances, the vectors can comprise a shuttle element, which makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In addition, the vectors can also include a gene that is capable of expressing a detectable marker (e.g., a drug resistance gene). The vectors can also have a reporter gene, e.g., a gene that encodes a fluorescent or other detectable protein.

[0112] In some cases, the vector can comprise a viral vector, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus. Methods for producing viral vectors comprising a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are well known in the art and can be performed by one of skill in the art without undue experimentation. In other cases, the vector can be a recombinant AAV virion packaged with a nucleic acid molecule described herein. Methods of producing recombinant AAV can include introducing a nucleic acid molecule described herein into a packaging cell line, producing AAV helper functions, AAV cap and rep genes, and recovering the recombinant AAV from the supernatant of the packaging cell line. Various types of cells can be used as packaging cell lines. For example, packaging cell lines that can be used include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

[0113] In some cases, the vector can be an adenovirus-associated vector (AAV). In the present application, the term "adenovirus-associated vector" generally refers to vectors derived from naturally occurring and available adeno-associated viruses as well as artificial AAVs. The AAVs can include different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any AAV variants or mixtures. The AAV genome usually has terminal inverted repeats (ITRs) at both ends, and the term "ITR" or "terminal inverted repeat" refers to a segment of nucleic acid sequence present in AAV and / or recombinant AAV that can form a T-shaped palindromic structure required for completion of the AAV lytic and latent life cycle. Techniques for producing AAV vectors are standard in the art, which include providing a cell with a polynucleotide to be delivered, a rAAV genome, AAV rep and cap genes, and helper virus functions to be packaged into the rAAV genome. Production of AAV vectors generally requires the presence of the following components within a single cell (herein referred to as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (e.g., not within) the rAAV genome, and helper virus. The AAV rep and cap genes can be from any AAV serotype, or from a different AAV serotype than the ITRs of the AAV genome, including but not limited to the AAV serotypes described herein. The AAV vector in the present application can comprise a gRNA targeting a mutation site of the RHO gene.

[0114] In some cases, the sequence encoding the gRNA can be located in the same vector as the nucleic acid encoding the Cas9 protein. In other cases, the sequence encoding the gRNA can be located in a different vector from the nucleic acid encoding the Cas9 protein.

[0115] The AAV vectors of the present application can be from a variety of species. For example, the AAV can be an avian AAV, a bovine AAV, or a goat AAV. In certain embodiments, the vector is AAV8.

[0116] The methods of the present application can include the production of packaging cells, i.e., the production of a cell line that can be used to stably express all the necessary components of AAV. For example, the AAV genome, lacking AAV rep and cap genes, the AAV rep and cap genes isolated from the AAV genome, and a plasmid (or multiple plasmids) with a selectable marker such as a neomycin resistance gene are integrated into the genome of the cell. The AAV genome can have been introduced into a bacterial plasmid by methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. U.S.A., 79:2077-2081). The packaging cell line can then be infected with a helper virus (e.g., adenovirus). In addition to plasmids, adenovirus or baculovirus can also be used to introduce the AAV genome and / or rep and cap genes into the packaging cell.

[0117] The previous research results of the present inventors in CN 116334141 A show that Sauri-sg2 (SauriCas9+sg2), Slug-sg2 (SlugCas9+sg2), and KKH-SaCas9-sg3 (KKH-SaCas9+sg3) can effectively knock down the expression of RHO mutant proteins in vitro. And by injecting a virus vector containing expressed Cas9 and sgRNA into the subretinal space of a model mouse, the DNA sequence of the RHO mutant of the model mouse can be effectively edited, wherein Sauri-sg2 can effectively edit the mutant DNA sequence in vivo.

[0118] Since the RHO protein has significant distribution specificity, it is only distributed in the outer segment region of photoreceptor cells. On this basis, the inventors further added enhancers and different introns to the promoter region to further improve the editing efficiency and safety of the RHO-R135W gene therapy drug. First, based on the hGRK1-Sauri-sg2 and hGRK1-KKH-SaCas9-sg3 vectors, different introns were inserted into the promoter region to form new constitutive promoters RK1, RK2, and RK3. In addition, enhancers were inserted into the newly constructed promoter region, and were named enRK1 and enRK2, respectively. After obtaining the above new vectors, the expression efficiency and editing efficiency of different promoters were first verified in vitro cell lines, and the experimental results showed that the in vitro expression efficiency and editing efficiency of enRK1 and enRK2 were significantly higher than those of RK1 and RK2, and further higher than those of the original vector and RK3.

[0119] To further explore whether the constitutive promoter can effectively improve the editing efficiency of target cells and ensure its expression specificity, the inventors further verified the expression efficiency of different vector molecules in a humanized mouse model. To more intuitively determine the expression position of different promoters in mouse retinal cells, the inventors constructed a vector expressing EGFP under different promoters and packaged it into AAV8, which was injected subretinally into wild-type mice. Eight weeks after administration, the mouse retinal GFP signal was detected by sectioning, and the results showed that the GFP signal of enRK1 and enRK2 vectors was stronger than that of RK1 and RK2, which was stronger than that of hGRK1 and RK3. However, the GFP fluorescence signal expression range of enRK1 and enRK2 was more extensive, indicating that its specificity was poor, and therefore, based on the target cell editing specificity, RK1, RK2, RK3, and hGRK1 were preferentially selected.

[0120] Further, to determine whether the optimized promoter can effectively improve the editing efficiency of mutant RHO gene and improve the phenotype of mouse models in vivo, the inventors packaged Sauri-sg2 and KKH-SaCas9-sg3 expressed by different promoters into AAV8 virus and administered them subretinally to model mice. Six months after administration, the changes in retinal function of the mouse model were detected by ERG, and RK1 and RK2 showed that the phenotype of the model mice was significantly better than that of hGRK1. After the functional detection was completed, the mouse retinal tissue and liver tissue were collected to detect the editing efficiency of the mutant DNA of the model mice. The experimental results showed that RK1, RK2, and hGRK1 could effectively edit the mutant DNA sequence of the mouse retina. The editing efficiency of RK1 and RK2 was better than that of hGRK1, and RK1 and RK2 showed better recovery ability for the retinal function of the mouse model. In addition, RK1 and RK2 did not show potential non-target editing.

[0121] The present application is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present application. The reagents and materials used in the following experiments are commercially available products unless otherwise specified.

[0122] Example

[0123] Example 1 Vector construction

[0124] The sgRNA in CN 116334141 A, i.e. Sauri-sg2 (SEQ ID NO: 1), Slug-sg2 (SEQ ID NO: 1), KKH-SaCas9-sg3 (SEQ ID NO: 2), the expression vector schematic diagram is shown in Figure 1, and the sequence information can also be seen in Table 1 of CN 116334141 A, wherein Sauri represents that the Cas9 enzyme used is SauriCas9 (the amino acid sequence encoded by SEQ ID NO: 17), Slug represents that the Cas9 enzyme used is SlugCas9 (the amino acid sequence encoded by SEQ ID NO: 18), and KKH-SaCas9 represents that the Cas9 enzyme used is KKH-SaCas9 (the amino acid sequence encoded by SEQ ID NO: 16).

[0125] By replacing the EFS promoter (SEQ ID NO: 3) of the Cas9 protein in it with the hGRK1 (SEQ ID NO: 4), RK1 (SEQ ID NO: 5), RK2 (SEQ ID NO: 6), RK3 (SEQ ID NO: 7), enRK1 (SEQ ID NO: 8) and enRK2 (SEQ ID NO: 9) promoters respectively, new gene editing vectors are constructed, and expression plasmids hGRK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK2-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK3-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, enRK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, enRK2-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3 are obtained respectively. In addition, in order to more intuitively detect the expression specificity and efficiency of different promoters in vivo, the above-mentioned promoters are used to construct EGFP expression vectors, and are packaged into AAV8 viruses in subsequent in vivo studies, and the virus packaging is completed by Guangzhou Piaozhen Biotechnology Co., Ltd.

[0126] Example 2 Detection of in vitro DNA editing efficiency

[0127] To detect the editing effect of different promoter expression vectors, the inventors constructed a 293A-RHO-Mut cell line by infecting HEK 293A cells with a lentivirus expressing RHO-Mut, wherein the RHO-Mut lentivirus plasmid was constructed by inserting the RHO-Mut sequence (SEQ ID NO: 26) into the Fugw plasmid (#14883), and the lentivirus was packaged by transfecting the RHO-Mut plasmid and Pax2 and VSVG plasmids in 293T cells. In the 293A-RHO-Mut cell line, the above-constructed expression plasmids: hGRK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK2-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, RK3-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, enRK1-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3, enRK2-Sauri-sg2 / -Slug-sg2 / -KKH-SaCas9-sg3 were transiently transfected, and 48 hours (48h) after transfection, the cell genomic DNA was collected, and primers were designed near the editing site to amplify the edited DNA, and the editing effect was detected by T7E1 and Hi-tom sequencing. The T7E1 and Hi-tom sequencing detection methods can be found in CN 116334141 A.

[0128] T7E1 endonuclease can recognize incompletely matched double-stranded DNA, and Cas9 cuts target DNA under the guidance of sgRNA to cause DNA double-strand breaks (DSB), and the cell repairs DSB through the DNA repair mechanism of non-homologous end joining (NHEJ), ultimately forming Indel (Insertion and Deletion) mutations at the cutting site. By designing PCR primers near the mutation site, CDS-F / R can be used to detect the RHO-135 sequence with a mutation site overexpressed by an exogenous (primer sequences are shown in Table 1 below), and CDS-F / R can PCR obtain a 1045bp band; after obtaining the edited gDNA fragment by PCR, in vitro annealing was performed, and the R135W mutant gDNA edited by T7E1 can be cut into two DNA fragments of 395bp and 650bp.

[0129] Table 1. T7E1 detection primer sequence information

[0130] The results of the T7E1 experiment are shown in Figure 2. The hGRK1 and RK3 promoter constructs did not show significant positive cleavage bands in the 293A cell line, while the modified RK1, RK2, enRK1 and enRK2 promoter constructs all showed positive cleavage bands, indicating that the modified vectors can effectively improve the in vitro editing activity of the gene editing drug molecules targeting RHO mutations.

[0131] To further confirm the editing efficiency, the editing efficiency of each vector was verified by Hi-tom sequencing experiment according to the method reported in the literature

[0010] . The results of the Hi-tom sequencing are shown in Figure 3. Compared with the EFS promoter, the in vitro editing efficiency of the hGRK1, RK1 and RK2 promoter constructs was significantly lower than that of the EFS promoter construct, and the editing efficiency of the enRK1 and enRK2 promoter constructs was comparable to that of the EFS. The editing efficiency of the RK1, RK2, enRK1 and enRK2 promoter constructs was significantly higher than that of the hGRK1 and RK3 promoter constructs. The above results show that the editing efficiency of the specific promoter in the 293A cell line is significantly lower than that of the broad-spectrum promoter, and the optimization of the specific promoters RK1, RK2, enRK1 and enRK2 can effectively improve the editing efficiency of the gene editing molecules in cells.

[0132] Example 3: In vivo detection of specific expression of different promoters in target cells

[0133] RHO protein expression is specific. In addition, the targeting specificity of gene editing therapy drugs is crucial for the safety of drug molecules. To further detect the differences in expression specificity after optimization of the promoters, the EGFP vectors constructed in Example 1 were packaged in AAV8, and the C57BL / 6J wild-type mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were administered subretinally at a dose of 1×10 9 vg / eye. Eight weeks after administration, the mouse eye tissues were collected for frozen sectioning, and the expression position of the GFP fluorescence signal was confirmed by fluorescence microscopy to determine the targeting specificity of different promoters.

[0134] The immunofluorescence results are shown in Figure 4. The EFS, enRK1 and enRK2 promoters have a higher expression range, and can be synchronously highly expressed in the outer nuclear layer of the retina and RPE cells, and a small amount of expression in the inner nuclear layer. The hGRK1 and RK1, RK2 and RK3 promoters are only expressed in the outer nuclear layer cells, showing a more specific expression range. In addition, the expression signal intensity of each promoter is enRK1=enRK2>RK1=RK2>hGRK1=RK3=EFS. The above results show that the specific promoter has better specificity; unexpectedly, compared with the hGRK1 promoter, the optimized RK1 and RK2 promoters significantly improve the expression efficiency without affecting the specificity; the RK3 does not show an improved expression efficiency; and the enRK1 and enRK2 promoters effectively improve the expression efficiency but significantly reduce the expression specificity. Therefore, the hGRK1, RK1 and RK2 promoters are selected for subsequent in vivo gene editing drug editing efficiency and efficacy detection.

[0135] The vector combination optimized in Example 4 can effectively restore the structural and functional abnormalities of the retina in a mouse model

[0136] The hGRK1, RK1 and RK2 promoters are selected to express Sauri-sg2 and KKH-SaCas9-sg3, respectively, and the vector packaging is AAV8, and the virus is injected into the subretinal space of the model mouse at a dose of 1x10 9 vg / eye. The model mouse is a humanized retinitis pigmentosa mouse carrying a RHO gene mutation (the model mouse replaces the mouse RHO gene coding region with a human RHO coding sequence with a mutation site based on C57BL / 6J), and the model construction method and phenotype research have been reported in the literature

[0011] . Six months after administration, the mouse retinal function is detected by ERG (electroretinogram), and the structural changes of the model mouse retina are detected by FP (fundus color photography) and OCT (optical coherence tomography).

[0137] The ERG results are shown in Figure 5. Compared with the control group, the amplitude of the dark response a wave and the dark response b wave of each administration group is recovered to a certain extent. In addition, the amplitude recovery range of the RK1 and RK2 promoter constructs is significantly higher than that of the hGRK1 promoter. The FP and OCT results are shown in Figure 6. Compared with the control group, the retinal thickness of each administration group is increased to a certain extent, and the outer nuclear layer thickness of the RK1 and RK2 promoter constructs is slightly increased compared with the corresponding hGRK1 promoter construct. In summary, the RK1 and RK2 promoter constructs can significantly improve the electrophysiological function of the model mouse retina and slightly improve the effect of the gene editing drug on the structural changes of the model mouse retina, indicating that the optimized promoter can effectively promote the in vivo effectiveness of the RHO gene editing therapeutic drug.

[0138] Example 5: Optimized promoters show high expression efficiency in mouse retinal cells

[0139] After the detection of the retinal function and structure of the model mice in vivo in Example 4, some mice were sacrificed to collect the mouse retinal tissues, extract RNA, and detect the differences in the expression products of the target of the mouse retina in different administration groups by q-RT-PCR sequencing. The primer sequences are shown in Table 2.

[0140] Table 2

[0141] The Q-RT-PCR results (Figure 7) show that the distribution of the target product mRNA in the retinal region of the RK1 and RK2 groups is significantly higher than that of the hGRK1 group, but the expression levels of the RK1 and RK2 groups show no significant difference. The above results show that the RK1 and RK2 promoters can effectively improve the expression efficiency of the target expression product in the retinal cells.

[0142] Example 6: Optimized promoters can improve the editing efficiency of the retinal cells of the model mice

[0143] After the detection of the retinal function and structure of the model mice in vivo in Example 4, the mouse retinal tissues were collected after the mice were sacrificed, DNA was extracted, and the differences in the gene editing efficiency of the mouse retina in different administration groups were detected by T7E1 and Hi-tom sequencing.

[0144] According to the description in Example 2, the T7E1 experiment was performed. The T7E1 experiment results are shown in Figure 8. The control group has no positive cleavage band, and the remaining administration groups can detect positive cleavage bands. Compared with the hGRK1 group, the cleavage bands of the RK1 and RK2 groups are clearer, indicating that the vectors containing the RK1 and RK2 promoters have higher editing efficiency. Further analysis of the specific editing efficiency by Hi-tom sequencing shows that the editing efficiency of the hGRK1-KKH-sg3 group is 13.2%, the RK1 can improve the editing efficiency to 29.3%, and the RK2 promoter can improve the in vivo editing efficiency to 35.1%. The editing efficiency of the hGRK1-Sauri-sg2 group is about 15.3%, the RK1 promoter can improve the editing efficiency to 32.8%, and the RK2 promoter can improve the in vivo editing efficiency to 39.2%. The above results show that the optimized promoters can effectively improve the targeted editing efficiency of the mouse retinal cells, and the RK2 promoter shows better therapeutic effect.

[0145] Example 7: No non-target distribution and non-target editing is detected for the optimized promoters

[0146] To further determine whether the optimized promoters can improve the expression efficiency of non-target groups, the inventors extracted the RNA of multiple tissues of the control group, RK1-Sauri-sg2 and RK2-Sauri-sg2 mice in Example 4, and detected the mRNA expression of SauriCas9 in each tissue by q-RT-PCR (the primers are the same as in Example 5). The experimental results are shown in Figure 10. The expression level of SauriCas9 mRNA expression product in RK1 and RK2 groups had no significant difference with the control group. In addition, the blood of mice was collected at different time after administration, and the RNA was extracted to detect the distribution change of expression product. The expression level of target product mRNA in the blood of mice in each group was slightly improved on the third day after administration, but there was no significant difference between the promoters, indicating that the optimized promoter does not affect its tissue expression specificity.

[0147] In addition, literature research shows that AAV vectors have a certain preference for liver. The inventors further extracted the liver tissue DNA of RK1-Sauri-sg2 and RK2-Sauri-sg2 groups of mice, and further analyzed whether the non-target editing efficiency of liver tissue DNA of the optimized promoter was improved by T7E1 experiment. The experimental results are shown in Figure 11. No positive cutting band was detected, indicating that although the optimized promoter improves the editing efficiency of the target, it does not affect its tissue targeted editing specificity.

[0148] Those skilled in the art should understand that although the present application is specifically described with reference to the above examples, the present application is not limited to these specific examples. Based on the methods and technical solutions taught by the present application, those skilled in the art can make appropriate modifications or improvements without departing from the essence of the present application, and the resulting equivalent embodiments are within the scope of the present application.

[0149] References

[0150] 1. Dias, M. F., et al., Corrigendum to molecular genetics and emerging therapies for retinitis pigmentosa: Basic research and clinical perspective progress in retinal and eye research (2018) Vol 63, 107-131. Prog Retin Eye Res, 2018. 66: p. 220-221.

[0151] 2. Ferrari, S., et al., Retinitis pigmentosa: genes and disease mechanisms. Curr Genomics, 2011. 12(4): p. 238-49.

[0152] 3. Mendes, H.F., et al., Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy. Trends Mol Med, 2005. 11(4): p. 177-85.

[0153] 4. Chuang, J.Z., et al., Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes. J Clin Invest, 2004. 114(1): p. 131-40.

[0154] 5. Wu, Y., et al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell, 2013. 13(6): p. 659-62.

[0155] 6. Cong, L., et al., Multiplex genome engineering using CRISPR / Cas systems. Science, 2013. 339(6121): p. 819-23.

[0156] 7. Aguila, M., et al., Hsp90 inhibition protects against inherited retinal degeneration. Hum Mol Genet, 2014. 23(8): p. 2164-75.

[0157] 8. Young, J.E., et al., A short, highly active photoreceptor-specific enhancer / promoter region upstream of the human rhodopsin kinase gene. Invest Ophthalmol Vis Sci, 2003. 44(9): p. 4076-85.

[0158] 9. Young, J.E., et al., Conserved interactions of a compact highly active enhancer / promoter upstream of the rhodopsin kinase (GRK1) gene. Genomics, 2007. 90(2): p. 236-48.

[0159] 10. Liu, Q., et al., Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR / Cas systems. Science China Life Sciences, 2018. 62(1): p. 1-7.

[0160] 11. Xiaozhen Liu, R.J., Xiang Meng, Ying Li, Liping Yang, Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter. Experimental Eye Research, February 2022. 215.

Claims

1. A vector comprising: (1) a nucleotide sequence encoding a Cas protein and a first promoter operably linked thereto, wherein the first promoter is an RK1 promoter or an RK2 promoter; and (2) a nucleotide sequence encoding a gRNA and a second promoter operably linked thereto, wherein the gRNA targets or specifically binds to a RHO gene or a fragment thereof.

2. The vector of claim 1, wherein the RK1 promoter comprises a nucleotide sequence set forth in SEQ ID NO: 5, and the RK2 promoter comprises a nucleotide sequence set forth in SEQ ID NO:

6.

3. The vector of claim 1 or 2, wherein the Cas protein comprises a Cas9 protein.

4. The vector of claim 3, wherein the Cas9 protein comprises a KKH-SaCas9, a SauriCas9, a SlugCas9, or a SlugCas9-HF.

5. The vector of claim 1, wherein the nucleotide sequence encoding a Cas protein comprises a nucleotide sequence set forth in SEQ ID NO: 16, 17, 18, or 19.

6. The vector of any one of claims 1 to 5, wherein the gRNA targets a mutation hot spot c.403C>T; p.Arg135Trp of a RHO gene.

7. The vector of claim 6, wherein the nucleotide sequence encoding a gRNA is a single guide RNA (sgRNA), preferably comprising a nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

8. The vector of claim 6 or 7, wherein the second promoter is a U6 promoter, preferably the U6 promoter comprises a nucleotide sequence set forth in SEQ ID NO:

20.

9. The vector of any one of claims 6 to 8, wherein: (1) the first promoter is an RK1 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding a gRNA comprises a nucleotide sequence set forth in SEQ ID NO: 1; (2) the first promoter is an RK1 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding a gRNA comprises a nucleotide sequence set forth in SEQ ID NO: 1; (3) the first promoter is an RK1 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding a gRNA comprises a nucleotide sequence set forth in SEQ ID NO: 1; (4) the first promoter is an RK1 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding a gRNA comprises a nucleotide sequence set forth in SEQ ID NO: 2; ​ (5) the first promoter is an RK1 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2; (6) the first promoter is an RK1 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2; (7) the first promoter is an RK2 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1; (8) the first promoter is an RK2 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1; (9) the first promoter is an RK2 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 1; (10) the first promoter is an RK2 promoter, the Cas9 protein is a KKH-SaCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2; (11) the first promoter is an RK2 promoter, the Cas9 protein is a SauriCas9 protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO: 2; or (12) the first promoter is an RK2 promoter, the Cas9 protein is a SlugCas9 / SlugCas9-HF protein, the second promoter is a U6 promoter, and the nucleotide sequence encoding the gRNA comprises the nucleotide sequence set forth in SEQ ID NO:

2.

10. The vector of any one of claims 1 to 9, wherein the vector further comprises a nuclear import signal, preferably the nuclear import signal comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 21-23.

11. The vector of claim 10, wherein the vector comprises, in order, the following elements: a first promoter, a Kozak sequence, a nuclear import signal, a nucleotide sequence encoding a Cas protein, a nuclear import signal, a transcription terminator sequence, a second promoter, a nucleotide sequence encoding a gRNA, and a gRNA scaffold sequence.

12. The vector of claim 11, wherein the Kozak sequence comprises the nucleotide sequence set forth in SEQ ID NO:

24.

13. The vector of claim 11 or 12, wherein the transcription terminator sequence is a BGH sequence (bGH poly(A)) or a SV40 poly(A).

14. The vector of claim 13, wherein the BGH sequence comprises the nucleotide sequence set forth in SEQ ID NO:

25.

15. The vector of any one of claims 11 to 14, wherein the gRNA scaffold sequence comprises the nucleotide sequence set forth in SEQ ID NO:

27.

16. The vector of any one of claims 1 to 15, wherein the vector comprises a viral vector.

17. The vector of claim 16, wherein the viral vector is an adeno-associated virus (AAV) vector.

18. The vector of claim 17, wherein the adeno-associated virus vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, and any AAV variant or mixtures, preferably an AAV8 vector.

19. A CRISPR-Cas system comprising the vector of any one of claims 1 to 18.

20. A cell comprising the vector of any one of claims 1-18.

21. A pharmaceutical composition comprising the vector of any one of claims 1-18, the CRISPR-Cas system of claim 19, and / or the cell of claim 20.

22. A kit comprising the vector of any one of claims 1-18, the CRISPR-Cas system of claim 19, and / or the cell of claim 20.

23. Use of the vector of any one of claims 1-18, the CRISPR-Cas system of claim 19, or the cell of claim 20, for the manufacture of a medicament for the treatment of retinitis pigmentosa caused by a RHO gene mutation (R135W).

24. The use of claim 23, wherein the medicament is formulated in a form suitable for injection.

25. The use of claim 24, wherein the medicament is formulated in a form suitable for subretinal cavity injection.

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