PRPF31 variants and uses thereof

Abstract

A PRPF31 variant comprising an exogenous nuclear localization (NLS) domain comprising an amino acid sequence as shown in any one of SEQ ID NOs. 3 to 4. The PRPF31 variant can improve the biological activity of retinal pigment epithelial (RPE) cells and can treat diseases caused by PRPF31 gene mutations.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, and in particular to PRPF31 mutants and uses thereof. [Background technology]

[0002] Retinitis pigmentosa (RP) is a progressive retinal degenerative disorder caused by degeneration of retinal photoreceptors and the retinal pigment epithelium. Clinically, it is a hereditary blinding eye disease characterized by night blindness, progressive visual field narrowing and loss, retinal pigmentation, waxy optic disc atrophy, and electroretinogram abnormalities. RP is inherited in various ways, including autosomal dominant inheritance (ADRP).

[0003] The RHO gene encodes rhodopsin, a key protein involved in the visual transduction pathway, and approximately 30-40% of ADRP patients have RHO gene mutations. The PRPF31 gene, short for pre-mRNA processing factor 31, is involved in the pre-mRNA splicing process. Approximately 8-10% of Chinese ADRP patients are caused by PRPF31 gene mutations.

[0004] Therefore, there is an urgent need to restore normal gene splicing in RP cells and thereby restore cellular function. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides a PRPF31 variant containing an exogenous nuclear localization (NLS) domain with a specific amino acid sequence, and uses thereof. The PRPF31 variant described herein further has the following properties: (1) localization in the nucleus, (2) promotion of gene mRNA splicing, (3) improvement of the phagocytic function of induced RPE cells, (4) increase in cilia length of induced RPE cells, and (5) treatment of diseases caused by PRPF31 gene mutations. The present application also provides uses of the PRPF31 variant in preparing pharmaceuticals for treating diseases caused by PRPF31 gene mutations, and in improving the biological activity and / or mRNA splicing efficiency of induced RPE cells.

[0006] In one aspect, the present application provides a PRPF31 variant comprising an exogenous nuclear localization (NLS) domain comprising the amino acid sequence set forth in any one of SEQ ID NOs. 3 to 4, or a truncated form thereof.

[0007] In some embodiments, the PRPF31 mutant does not contain the NLS domain of wild-type PRPF31.

[0008] In some embodiments, the PRPF31 variant comprises a NOSIC domain.

[0009] In some embodiments, the NOSIC domain comprises the amino acid sequence set forth in SEQ ID NO.35.

[0010] In some embodiments, the PRPF31 variant comprises a NOP domain.

[0011] In some embodiments, the NOP domain comprises the amino acid sequence set forth in SEQ ID NO.36.

[0012] In some embodiments, the PRPF31 mutant is capable of cleaving mRNA of genes, including the RHO gene.

[0013] In some embodiments, the PRPF31 variant is derived from human PRPF31.

[0014] In some embodiments, the PRPF31 variant comprises the amino acid sequence shown in any one of SEQ ID NOs. 20-21.

[0015] In another aspect, the present application provides a nucleic acid molecule encoding a PRPF31 variant described herein.

[0016] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs. 28-29.

[0017] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0018] In some embodiments, the vector comprises a viral vector.

[0019] In some embodiments, the vector comprises an AAV vector.

[0020] In some embodiments, the vector comprises a lentiviral vector.

[0021] In some embodiments, the serotype of the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 and / or AAV10.

[0022] In some embodiments, the lentivirus comprises a human immunodeficiency virus (HIV), a simian immunodeficiency virus (SIV), or a non-primate lentivirus such as a feline immunodeficiency virus (FIV), an equine infectious anemia virus (EIAV), a bovine immunodeficiency virus (BIV), and / or a visna-maedi virus (VMV).

[0023] In another aspect, the present application provides use of a PRPF31 variant described herein, a nucleic acid molecule described herein, and / or a vector described herein in the preparation of a medicament for treating a disease.

[0024] In some embodiments, the disease comprises a disease caused by a PRPF31 gene mutation.

[0025] In some embodiments, the disease comprises retinitis pigmentosa (RP).

[0026] In some embodiments, the disease comprises autosomal dominant retinitis pigmentosa (ADRP).

[0027] In another aspect, the present application provides a method for improving the biological activity of retinal pigment epithelial (RPE) cells, comprising administering to the RPE cells a PRPF31 variant described herein, a nucleic acid molecule described herein, and / or a vector described herein.

[0028] In some embodiments, the biological activity comprises phagocytic activity of the RPE cells and / or cilia length of the RPE cells.

[0029] In some embodiments, the induced RPE cells are derived from pluripotent and / or totipotent stem cells.

[0030] In some embodiments, the induced RPE cells are derived from human iPS cells.

[0031] In some embodiments, the PRPF31 mutant does not contain the NLS domain of wild-type PRPF31.

[0032] In some embodiments, the PRPF31 variant comprises the amino acid sequence shown in any one of SEQ ID NOs. 20-21.

[0033] In some embodiments, the administering comprises administering a vector comprising a nucleic acid molecule encoding the PRPF31 mutant.

[0034] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs. 28-29.

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

[0036] In another aspect, the present application provides a method for improving the splicing efficiency of mRNA, the method comprising administering to a cell containing the mRNA a PRPF31 mutant comprising an exogenous NLS domain comprising the amino acid sequence set forth in any one of SEQ ID NOs. 3 to 4.

[0037] In some embodiments, the PRPF31 mutant does not contain the NLS domain of wild-type PRPF31.

[0038] In some embodiments, the PRPF31 variant comprises the amino acid sequence shown in any one of SEQ ID NOs. 20-21.

[0039] In some embodiments, the administering comprises administering a vector comprising a nucleic acid molecule encoding the PRPF31 mutant.

[0040] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs. 28-29.

[0041] In some embodiments, the vector comprises a viral vector.

[0042] 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, given the content of the present application, they may make changes to the particular embodiments disclosed without departing from the spirit and scope of the invention covered by the present application. Accordingly, the drawings and descriptions herein are illustrative only, and not restrictive. [Brief explanation of the drawings]

[0043] Particular features of the present invention are set forth in the appended claims. The features and advantages of the present invention can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings, a brief description of which follows. [Figure 1] Figure 1 shows the main distribution results of detecting the expression of human PRPF31 in cells using fluorescent labeling. [Figure 2] Figure 2 shows the main distribution results of intracellular human PRPF31 expression detected using fluorescent labeling and DAPI staining. [Figure 3] FIG. 3 shows the effect of human PRPF31 on mRNA splicing of the RHO reporter gene. [Figure 4] Figure 4 shows the results of verification of cells transfected with human PRPF31. [Figure 5] Figure 5 shows the main distribution results of using fluorescent labeling to detect the expression of the PRPF31 mutants described in this application in cells. [Figure 6] Figure 6 shows the main distribution results of detecting the expression of the PRPF31 mutants described in this application in cells using fluorescent labeling. [Figure 7] FIG. 7 shows the results of validation of cells transfected with the PRPF31 mutants described in this application. [Figure 8] FIG. 8 shows the effect of the PRPF31 mutants described in this application on mRNA splicing of the RHO reporter gene. [Figure 9] FIG. 9 shows the splicing efficiency of the PRPF31 mutants described in this application for the mRNA of the RHO reporter gene. [Figure 10] FIG. 10 shows the effect of the PRPF31 mutants described in this application on the phagocytic function of human RPE cells. [Figure 11] FIG. 11 shows the effect of the PRPF31 mutants described in this application on the phagocytic function of human RPE cells. [Figure 12] FIG. 12 shows the effect of the PRPF31 mutants described in this application on the phagocytic function of human RPE cells. [Figure 13] FIG. 13 shows the effect of the PRPF31 mutants described in this application on cilia length in human RPE cells. [Figure 14] FIG. 14 shows the effects of the PRPF31 mutants described herein on the phagocytic function and cilia length of RPE cells derived from human IPSCs. DETAILED DESCRIPTION OF THE INVENTION

[0044] Hereinafter, embodiments of the present invention will be described using specific examples, but those familiar with the technology will be able to easily understand other advantages and effects of the present invention from the disclosure of this specification.

[0045] Definition of Terms In this application, the term "PRPF31" generally refers to a pre-mRNA processing factor, a component of the U4 / U6.U5 trisnRNP (small nuclear ribonucleoprotein) subunit of the spliceosome. PRPF31 is responsible for pre-mRNA splicing in the nucleus. The PRPF31 gene is 16 kb long, contains 14 exons, and is located on chromosome 19q13.4. The protein encoded by PRPF31 is 499 amino acids in length and has a molecular weight of 61 kDa. PRPF31 contains three domains: the NOSIC domain, the NOP domain, and the NLS domain. The NOSIC and NOP domains are thought to be RNA-binding domains, while the NLS domain localizes translated PRPF31 in the cytoplasm to the nucleus to complete the pre-mRNA splicing function. The NOSIC and NOP domains are more conserved. The NOSIC domain can be amino acids 93-144 of human PRPF31, and the NOP domain can be amino acids 190-334 of human PRPF31. Deletion of PRPF31 causes abnormal mRNA splicing in cells, severely affecting photoreceptor cells and retinal pigment epithelial cells (RPE).

[0046] As used herein, the term "variant" generally refers to a polypeptide or protein that differs in one or more length or sequence changes compared to the polypeptide or protein from which it is derived. The peptide or protein from which a peptide or protein variant is derived may also be referred to as the parent peptide or protein (e.g., PRPF31 protein). The term "variant" can also include "fragments" or "derivatives" of the parent polypeptide or protein. Generally, a "fragment" may be smaller in length or size than the parent molecule, and a derivative may exhibit one or more sequence differences compared to the parent molecule. As used herein, the term "variant" can also include, but is not limited to, modified molecules such as post-translationally modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins). Variants may be artificially constructed, for example, by genetic techniques, and the parent polypeptide or protein may be a wild-type polypeptide or protein. However, naturally occurring variants are also understood to be encompassed by the term "variant" as used herein. The variants described herein can also be derived from homologs, orthologs, or paralogs of the parent molecule, or from artificially constructed variants, provided that the variant exhibits at least one biological activity, i.e., functional activity, of the parent molecule. In the present application, the variants can have a degree of sequence identity with the parent polypeptide or protein from which they are derived. For example, the variants can have at least 80% sequence identity with the parent polypeptide. For example, the variants can have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity.

[0047] As used herein, the term "nuclear localization domain (NLS)" generally refers to an amino acid sequence that facilitates protein entry into the nucleus, for example, via nuclear transport. The nuclear localization domain can be a domain or domains that mediate the import of proteins or polynucleotides into the nucleus or their retention within the nucleus. The nuclear localization domain can be one known to those skilled in the art. For example, NLS sequences can be found in WO / 2001 / 038547. Representative examples of NLSs include, but are not limited to, single-component nuclear localization signals, two-component nuclear localization signals, and N- and C-terminal motifs. N-terminal basic domains generally conform to the consensus sequence KK / RXK / R, which was originally discovered in the SV40 large T antigen and represents a single-component NLS. A non-limiting example of an N-terminal basic domain NLS is PKKKRKV. Two-component nuclear localization signals are also known, which contain two clusters of basic amino acids separated by a gap of approximately 10 amino acids. N- and C-terminal motifs include, for example, the acidic M9 domain of hnRNP A1, the sequence KIPIK in the yeast transcriptional repressor Matα2, and the complex signal of U snRNP. Most of these NLSs are directly recognized by specific receptors of the importin β family.

[0048] In this application, the term "RHO" generally refers to rhodopsin 2, Opsin-2, Opsin2, OPN2, CSNBAD1, or RP4. Rhodopsin protein is located in the rod outer segment (ROS) and is necessary for normal vision, especially for the perception of low-light stimuli. Rods are a type of retinal photoreceptor cell responsible for scotopic vision. Another type of retinal photoreceptor cell is the cone cell, which is responsible for scotopic and color vision. In ROS, rhodopsin generally binds to 11-cis retinal (11cRAL), a derivative of vitamin A. ROS absorb photons to convert rhodopsin into active rhodopsin (R*), isomerizing 11cRAL to all-trans retinal (atRAL). AtRAL is immediately reduced to all-trans retinol (atROL) after separating from R*. Interphotoreceptor retinoid-binding protein (IRBP) transports atROL to RPE cells. In RPE cells, atROL is converted to all-trans retinyl ester by lecithin retinol acyltransferase (LRAT), and then further converted to 11-cis retinyl ester, which is then converted to 11-cis retinol (11-cis retinol) by the hydrolytic isomerase RPE65. 11-cis retinol is isomerized to 11cRAL by RDHs, which then binds to IRBP and transports it to the photoreceptor cells for recycling. R* converts GDP to GTP on the α subunit of transducin G (Gt) in the downstream membrane disc, separates the α subunit from the βγ subunit, activates cyclic guanosine monophosphate phosphodiesterase 6 (cGMP-PDE6), hydrolyzes cGMP, and the intracellular cGMP concentration decreases, closing the cGMP-gated cation channel in the OS and increasing Ca in the photoreceptor cells. 2+The concentration of Rhods decreases, the cell membrane becomes hyperpolarized, and the light signal is converted into a visible electrical signal. After the phototransduction is completed, the photoreceptor cells undergo a series of chemical reactions to return to the non-light state. At this time, R* is phosphorylated and binds to inhibitory proteins, inhibiting the downstream signal transduction pathway. PDE6 is in an inactive state. At the same time, cGMP is synthesized in the Rods. The increased cGMP concentration opens the cation channel, allowing Ca 2+ These nerve impulses, triggered by the opening and closing of cGMP-gated cation channels, are transmitted through connections between the synaptic terminals of photoreceptor cells and neurons at all levels of the retina and through the optic nerve to the visual centers in the cerebral cortex, forming vision.

[0049] The human RHO gene is located at 22.1 of the long arm of chromosome 3 (3q22.1), with the molecule located at base pairs 129,528,639–129,535,344 on chromosome 3 (Homo sapiens, Annotation release, version 109.20200228, GRCh38.p13, NCBI). The nucleotide sequence of the RHO gene can be found in NCBI GenBank Accession No. NG_009115.1. The RHO gene has five exons. Table 1 shows the exon identifiers and exon start / stop sites of the RHO gene in the Ensembl database.

[0050] [Table 1]

[0051] As used herein, the term "nucleic acid molecule" generally refers to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple loci defined by binding 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. Polynucleotides can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0052] As used herein, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and which can then express that protein. Vectors can be expressed by transforming, transducing, or transfecting a host cell, resulting in the expression of the genetic material contained in the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), artificial chromosomes such as bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and viral vectors. Vectors can contain various elements that control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors can also contain origins of replication. Vectors include, but are not limited to, components that facilitate cell entry, such as viral particles, liposomes, and protein coats.

[0053] As used herein, the term "viral vector" generally refers to a non-wild-type recombinant viral particle that functions as a gene delivery vector and contains a recombinant viral genome packaged within a viral capsid. Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAVs), herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and polyoma vacuolating viruses (e.g., SV40).

[0054] In this application, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated viruses are small, single-stranded DNA viruses that replicate exclusively intracellularly, with some functions provided by a coinfecting helper virus. Currently, there are 13 characterized AAV serotypes, as shown in Table 1. General information and reviews on AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, because the various serotypes are known to be very closely related, both structurally and functionally, even at the genetic level, it is fully expected that these same principles will also apply to other AAV serotypes. For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all retain three related capsid proteins, such as those expressed in AAV6. The degree of relatedness is further substantiated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats (ITRs)." Similar infection patterns also indicate that the replication functions of each serotype are under similar control.

[0055] [Table 2]

[0056] As used herein, the term "AAV vector" generally refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). When present in a host cell transfected with a vector encoding and expressing the rep and cap gene products, such AAV vectors are replicated and packaged into infectious viral particles. The terms "AAV virion" or "AAV viral particle" or "AAV vector particle" refer to a viral particle consisting of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is generally referred to as an "AAV vector particle" or simply as an "AAV vector." Thus, the production of AAV vector particles necessarily includes the production of AAV vectors in which the vector is contained within the AAV vector particle.

[0057] The AAV "rep" and "cap" genes refer to the genes encoding replication and capsid proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes studied to date and are described herein and in the cited references. In wild-type AAV, the rep and cap genes are generally adjacent to each other within the viral genome (i.e., they are "coupled" to each other as contiguous or overlapping transcription units) and are generally conserved among AAV serotypes. The AAV rep and cap genes, individually or collectively, are sometimes referred to as "AAV packaging genes." The AAV cap gene encodes the Cap protein, which, in the presence of rep and adenoviral helper functions, is capable of packaging an AAV vector and binding to target cell receptors. In some cases, the AAV cap gene encodes a capsid protein derived from a specific AAV serotype, such as the serotypes listed in Table 1.

[0058] Different AAV serotypes have remarkably homologous genome sequences at both the amino acid and nucleic acid levels, serve a similar set of genetic functions, produce essentially physically and functionally equivalent virions, and are replicated and assembled by nearly identical mechanisms.

[0059] In this application, the term "retinitis pigmentosa (RP)" generally refers to a type of hereditary blinding ocular fundus disease caused by abnormal function of retinal photoreceptors, and is mostly a single-gene genetic disease. The main inheritance patterns of RP include autosomal dominant inheritance (ADRP), autosomal recessive inheritance (ARRP), and X-linked inheritance. P11 is an ADRP caused by a gene mutation in PRPF31, and the probability that ADRP contains RP11 is approximately 10%.

[0060] In this application, the term "retinal pigment epithelium (RPE) cells" generally refers to the layer of pigment cells located just outside the sensory neurons of the retina. The RPE is composed of a single layer of hexagonal cells containing densely packed pigment granules. The RPE is closely associated with the underlying choroid and the retinal neurons above it. Its main functions include regulating fluid and nutrients in the subretinal space, acting as a blood-retinal barrier, synthesizing growth factors and regulating local structure, absorbing light and regulating electrical balance, regenerating and synthesizing visual pigments, phagocytosis and digestion of photoreceptor outer segments, maintaining retinal attachment, and regenerating and repairing after injury. In general, the RPE is considered a critical tissue for maintaining photoreceptor function and is also affected by many pathologies of the choroid and retina.

[0061] As used herein, the term "retinal pigment epithelium (RPE) atrophy" generally refers to degenerative changes in the retinal pigment epithelium (RPE), manifesting as cell death or dysfunction. Age-related macular degeneration or retinitis pigmentosa (RP) is often accompanied by retinal pigment epithelial atrophy. Retinitis pigmentosa (RP), also known as retinal pigment disease, typically refers to a type of hereditary eye disease. There are three inheritance patterns: autosomal recessive, dominant, and X-linked recessive, as well as double-gene and mitochondrial inheritance. Initial symptoms often include night blindness and narrowing of the visual field, resulting in slight blurring of vision to the left and right while still being able to see the view ahead, and gradually leading to complete loss of vision. RP includes unicular primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, achromatic retinitis pigmentosa, albeit punctate retinitis degeneration, crystalline retinal degeneration, venous-lipid retinitis pigmentosa, arteriolar pigment epithelium-sparing retinitis pigmentosa, Leber congenital amaurosis, and retinitis pigmentosa in other syndromes.

[0062] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, i.e., a human patient. For example, a pharmaceutical composition described herein can include a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and any pharmaceutically acceptable adjuvant. The pharmaceutical composition can also include one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of a composition can be non-toxic to a recipient at the dosages and concentrations used. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0063] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. Such polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include modified amino acid polymers. These modifications may include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations (such as conjugation to a labeling moiety). The term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and D- and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0064] As used herein, the term "pluripotent stem cells" generally refers to stem cells that are capable of eventually forming the cells and tissues found in a complete organism, but are unable to do so. For example, pluripotent stem cells can be proliferated in vitro for extended periods or virtually indefinitely while maintaining an undifferentiated state and exhibiting a normal karyotype (chromosomes). Under appropriate conditions, pluripotent stem cells can have the ability to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm). For example, pluripotent stem cells can include ES cells isolated from early embryos and / or isofunctional EG cells isolated from fetal primordial germ cells.

[0065] In this application, the term "totipotent stem cells" generally refers to stem cells with complete differentiation versatility, i.e., stem cells that have the ability to develop into multiple cell types in any fetal or adult mammalian body. For example, totipotent stem cells can differentiate into three germ layers: endoderm, mesoderm, and ectoderm, thereby giving rise to any fetal or adult cell type. The totipotent stem cells can include embryonic stem (ES) cells, embryonic germ (EG) cells, and embryonic carcinoma (EC) cells.

[0066] In this application, the term "and / or" should be understood to mean either or both of the options.

[0067] In this application, the terms "comprise" or "contain" generally mean the inclusion of explicitly specified features but not the exclusion of other elements.

[0068] As used herein, the term "about" generally refers to a variation within 0.5% to 10% above and below a particular 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 and below a particular value. Detailed Description of the Invention

[0069] In one aspect, the present application provides a PRPF31 variant comprising an exogenous nuclear localization (NLS) domain comprising the amino acid sequence set forth in any one of SEQ ID NOs. 3-4.

[0070] In the present application, the PRPF31 mutant may not contain the NLS domain of wild-type PRPF31. For example, the PRPF31 mutant may not contain the complete NLS domain of wild-type PRPF31. In some cases, the PRPF31 mutant may not have the biological function of the NLS domain of wild-type PRPF31 (e.g., the function of exogenous nuclear localization). In some cases, the NLS domain of wild-type PRPF31 in the PRPF31 mutant is replaced, or at least partially replaced, with the exogenous NLS domain. For example, the PRPF31 mutant may contain the complete exogenous NLS domain.

[0071] In the present application, the PRPF31 mutant may comprise a NOSIC domain. For example, the NOSIC domain may comprise amino acids 93 to 144 of the amino acid sequence of the PRPF31 mutant. For example, the NOSIC domain may comprise the amino acid sequence set forth in SEQ ID NO. 35.

[0072] In the present application, the PRPF31 mutant may comprise a NOP domain. For example, the NOP domain may comprise amino acids 190 to 334 of the amino acid sequence of the PRPF31 mutant. For example, the NOP domain may comprise the amino acid sequence set forth in SEQ ID NO. 36.

[0073] For example, the PRPF31 variant can include the exogenous NLS domain, the NOSIC domain, and the NOP domain.

[0074] In the present application, the PRPF31 mutant is capable of cleaving the mRNA of genes, which may include the RHO gene.

[0075] In the present application, the PRPF31 mutant can cleave the mRNA of a gene. For example, the PRPF31 mutant can be a pre-mRNA splicing factor gene that can be involved in mRNA splicing. The PRPF31 mutant can affect the splicing process of the pre-mRNA of the RHO gene. The PRPF31 mutant can interact with the RHP gene to affect retinitis pigmentosa (RP). In the present application, the RHO gene can include a wild-type RHO gene. The RHO gene can include any mutant RHO gene. For example, the RHO minigene reporter gene can include the nucleotide sequence set forth in SEQ ID NO. 33.

[0076] In the present application, the PRPF31 variant is derived from human PRPF31. For example, the PRPF31 variant may be a PRPF31 variant that includes the exogenous NLS domain in addition to human PRPF31.

[0077] In the present application, the PRPF31 mutant comprises an amino acid sequence shown in any one of SEQ ID NOs. 20 to 21.

[0078] In the present application, the nucleic acid molecule can encode an exogenous nuclear localization (NLS) domain comprising the amino acid sequence shown in any one of SEQ ID NOs. 3 to 4 described in the present application.

[0079] For example, the nucleic acid molecule can comprise the nucleotide sequence set forth in any one of SEQ ID NOs. 10-11.

[0080] In another aspect, the present application provides a nucleic acid molecule encoding a PRPF31 variant described herein.

[0081] In the present application, the nucleic acid molecule may comprise a nucleotide sequence shown in any one of SEQ ID NOs. 28-29.

[0082] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0083] In the present application, the vector can include a plasmid, for example, a linear plasmid.

[0084] In the present application, the vector may include a viral vector, which may include a lentiviral vector, a retroviral vector, an adenoviral vector, an adenovirus-associated viral vector, and / or a herpes simplex viral vector.

[0085] For example, the lentiviral vector can include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and non-primate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), and / or visna-maedi virus (VMV).

[0086] In the present application, the vector may include an AAV vector.

[0087] In the present application, the serotype of the AAV vector may include AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 and / or AAV10.

[0088] In another aspect, the present application provides use of a PRPF31 variant described herein, a nucleic acid molecule described herein, and / or a vector described herein in the preparation of a medicament for treating a disease.

[0089] The present application provides a PRPF31 mutant described herein, a nucleic acid molecule described herein, and / or a vector described herein for treating a disease.

[0090] The present application provides methods for treating diseases, comprising administering a PRPF31 mutant described herein, a nucleic acid molecule described herein, and / or a vector described herein.

[0091] In this application, the disease may include a disease caused by a PRPF31 gene mutation. In some cases, the disease may be associated with a functional defect of the retinal pigment epithelium (RPE). In some cases, the disease may be associated with a functional defect of retinal photoreceptor cells.

[0092] In the present application, the disease may include retinitis pigmentosa (RP). In the present application, the disease may include inherited retinal degeneration (IRD).

[0093] In the present application, the disease can include autosomal dominant retinitis pigmentosa (ADRP).

[0094] In another aspect, the present application provides a method for improving the biological activity of retinal pigment epithelial (RPE) cells, comprising administering to the RPE cells a PRPF31 variant described herein, a nucleic acid molecule described herein, and / or a vector described herein.

[0095] In this application, the biological activity can include the phagocytic activity of the RPE cells and / or the cilia length of the RPE cells, for example, the biological activity can include maintaining selective transport of nutrients and metabolites, secreting various growth factors, participating in the optic circulation, maintaining the blood-retinal barrier, and / or phagocytosis of outer segment disc membranes detached from photoreceptor cells.

[0096] In the present application, the phagocytic activity can include phagocytosis of disc membranes detached from digested photoreceptor outer segments. The phagocytic activity can also include nonspecific phagocytosis. For example, the phagocytic activity can include phagocytosis of substances that do not have a specific recognition moiety, such as polyethylene microspheres, melanin droplets, or trypan blue dye.

[0097] In this application, the cilia of the RPE cells can be primary cilia on RPE cells (RPE, which can be small tube-like projections on RPE cells), and the cilia of the RPE cells can participate in the phototransduction cascade.

[0098] In the present application, the induced RPE cells can be derived from pluripotent stem cells and / or totipotent stem cells.

[0099] In the present application, the pluripotent stem cells can include stem cells that have the ability to eventually form cells and tissues found in a complete organism, but are unable to form a complete organism. For example, the pluripotent stem cells can be proliferated in vitro for a long period of time or virtually indefinitely while maintaining an undifferentiated state and exhibiting a normal karyotype (chromosomes). The pluripotent stem cells can have the ability to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) under appropriate conditions. For example, the pluripotent stem cells can include mesenchymal stem cells. For example, the pluripotent stem cells can include induced pluripotent stem cells, i.e., iPS cells.

[0100] As used herein, the totipotent stem cells can include cells that can form all cells and tissues found in an entire organism, for example, the totipotent stem cells can form an entire organism.

[0101] For example, the induced RPE cells can be derived from human iPS cells.

[0102] In the present application, the PRPF31 mutant may not contain the NLS domain of wild-type PRPF31.

[0103] In the present application, the PRPF31 mutant may comprise an amino acid sequence shown in any one of SEQ ID NOs. 20 to 21.

[0104] In the present application, the administration can include administering a vector containing a nucleic acid molecule encoding the PRPF31 mutant.

[0105] In the present application, the nucleic acid molecule may comprise a nucleotide sequence shown in any one of SEQ ID NOs. 28-29.

[0106] In the present application, the vector may include a viral vector.

[0107] In another aspect, the present application provides a method for improving the splicing efficiency of mRNA, the method comprising administering to a cell containing the mRNA a PRPF31 mutant comprising an exogenous NLS domain comprising an amino acid sequence set forth in any one of SEQ ID NOs. 20 to 21.

[0108] In the present application, the PRPF31 mutant may not contain the NLS domain of wild-type PRPF31.

[0109] In the present application, the PRPF31 mutant may comprise an amino acid sequence shown in any one of SEQ ID NOs. 20 to 21.

[0110] In the present application, the administration can include administering a vector containing a nucleic acid molecule encoding the PRPF31 mutant.

[0111] In the present application, the nucleic acid molecule may comprise a nucleotide sequence shown in any one of SEQ ID NOs. 28-29.

[0112] In the present application, the vector may include a viral vector.

[0113] Without intending to be bound by any theory, the following examples are intended only to illustrate the present PRPF31 mutants, preparation methods, and uses, and are not intended to limit the scope of the present invention. [Example]

[0114] Example 1 Nuclear localization of Mcherry-PRPF31 To facilitate localization of PRPF31, human PRPF31 cDNA (SEQ ID NO. 25) was cloned into vector pMcherry-N1 (purchased from Clontech) to obtain vector pMcherry-PRPF31. Among these, vector pMcherry-N1 can express mcherry, a red fluorescent protein that can be used as a tracer. The nucleotide sequence of the nucleic acid molecule encoding mcherry is shown in SEQ ID NO. 34.

[0115] HEK293 cells were transfected with the vector pMcherry-PRPF31 (i.e., mcherry-PRPF31) and the control vector pMcherry-N1 (i.e., mcherry), and the expression of mCherry red fluorescence was observed 24 and 48 hours after transfection. The results are shown in Figure 1.

[0116] The location of mCherry red fluorescent expression was observed using DAPI staining, and the situation 48 hours after transfection was confirmed. The results in Figures 1 and 2 show that the red fluorescent protein expressed by the vector pMcherry-PRPF31 was expressed primarily in the nucleus and showed good colocalization with DAPI, while the red fluorescent protein expressed by the control vector pMcherry-N1 was expressed throughout the cell, including the nucleus and cytoplasm.

[0117] Example 2 Promotion of mRNA splicing of the RHO reporter gene by Mcherry-PRPF31 To detect the pre-mRNA splicing function of PRPF31, a RHO minigene reporter gene was constructed (its nucleotide sequence is shown in SEQ ID NO. 33).

[0118] The exon 3-4 region of the RHO gene was cloned from the HEK293 cell genome. When the reporter gene was normally cleaved, the intron between E3 and E4 was excised, resulting in a 229-bp mRNA fragment. Conversely, when normal splicing of the reporter gene was inhibited, the intron between E3 and E4 was retained, resulting in a 345-bp mRNA fragment. PRPF31 function could be characterized by comparing the ratio of the 345-bp fragment to the 225-bp fragment.

[0119] The RHO minigene reporter gene was cloned into the vector pcDNA4 (purchased from Thermo Fisher) to obtain the vector p4-RHO.

[0120] The results are shown in Figure 3. When vector p4-RHO was cotransfected with the control vector pMcherry-N1 (i.e., mcherry), the 345bp / 225bp ratio was 7.3. This means that most of the mRNA contained introns and was not properly cleaved. In contrast, when vector p4-RHO was cotransfected with vector pMcherry-PRPF31 (i.e., m31), coexpression of the RHO gene and Mcherry-PRPF31 resulted in a 345bp / 225bp ratio of 1.9. This shows that the cleavage ratio increased by approximately fourfold. This indicates that the presence of PRPF31 can promote mRNA splicing of the RHO minigene gene.

[0121] Figure 4 verifies that the vector pMcherry-PRPF31 can successfully express the Mcherry-PRPF31 fusion protein.

[0122] Example 3: Detection of the effects of different NLS sequences on PRPF31 localization To investigate whether the NLS affects the function of PRPF31, the nuclear localization signal of the PRPF31 protein was replaced; that is, the nuclear localization sequence of the PRPF31 protein (the amino acid sequence of which is shown in SEQ ID NO. 15) was replaced with SV40 (the amino acid sequence of which is shown in SEQ ID NO. 1), A1 (the amino acid sequence of which is shown in SEQ ID NO. 2), D (the amino acid sequence of which is shown in SEQ ID NO. 4), M (the amino acid sequence of which is shown in SEQ ID NO. 5), NP (the amino acid sequence of which is shown in SEQ ID NO. 6), SRY (the amino acid sequence of which is shown in SEQ ID NO. 7), or TAT (the amino acid sequence of which is shown in SEQ ID NO. 3).

[0123] The nucleotide sequences encoding these substituted nuclear localization sequences are respectively designated as follows: SV40 (the nucleotide sequence of which is shown in SEQ ID NO. 8), A1 (the nucleotide sequence of which is shown in SEQ ID NO. 9), D (the nucleotide sequence of which is shown in SEQ ID NO. 11), M (the nucleotide sequence of which is shown in SEQ ID NO. 12), NP (the nucleotide sequence of which is shown in SEQ ID NO. 13), SRY (the nucleotide sequence of which is shown in SEQ ID NO. 14), or TAT (the nucleotide sequence of which is shown in SEQ ID NO. 10).

[0124] The nucleic acid molecules encoding the substituted PRPF31 mutants were each cloned into the vector pMcherry-N1 according to the procedure described in Example 1 to obtain the vector pMcherry-SV40 (can also be abbreviated as SV40), vector pMcherry-A1 (can also be abbreviated as A1), vector pMcherry-D (can also be abbreviated as D), vector pMcherry-M (can also be abbreviated as M), vector pMcherry-NP (can also be abbreviated as NP), vector pMcherry-SRY (can also be abbreviated as SRY), and vector pMcherry-TAT (can also be abbreviated as TAT), respectively.

[0125] The intracellular expression distribution of the nucleic acid molecules contained in the vectors detected above was then detected according to the procedure described in Example 1. The results are shown in Figures 5 and 6. The left column shows the expression of red fluorescence, and the right column shows the result when the red fluorescence and bright field are superimposed.

[0126] Example 4: Detection of the effect of different NLS sequences on mRNA splicing of the RHO reporter gene According to the method described in Example 2, vector p4-RHO containing the RHO minigene reporter gene (SQE ID NO33) was co-transfected into cells with vector pMcherry-SV40 (can also be abbreviated as SV40), vector pMcherry-A1 (can also be abbreviated as A1), vector pMcherry-D (can also be abbreviated as D), vector pMcherry-M (can also be abbreviated as M), vector pMcherry-NP (can also be abbreviated as NP), vector pMcherry-SRY (can also be abbreviated as SRY), and vector pMcherry-TAT (can also be abbreviated as TAT) prepared in Example 3, or vector p4-RHO was co-transfected into cells with control vector pMcherry-N1 (i.e., mcherry) or vector pMcherry-PRPF31 (i.e., mcherry-PRPF31) prepared in Example 1, and cells not transfected with the plasmid (mock) xx were used as blank controls.

[0127] Figure 7 demonstrates that vectors SV40, A1, D, M, NP, SRY, and TAT can all successfully express the corresponding Mcerry-PRPF31 fusion protein variants.

[0128] The effects of vectors SV40, A1, D, M, NP, SRY, and TAT on RHO reporter gene mRNA splicing are shown in Figures 8 and 9. The results in Figures 8 and 9 indicate that PRPF31 mutants with different NLS sequences have different splicing efficiencies for the RHO reporter gene mRNA. Among these, PRPF31 mutants with NLS sequences D or TAT spliced ​​more efficiently for the reporter gene mRNA, while the PRPF31 mutant with NLS sequence D spliced ​​slightly more efficiently than PRPF31.

[0129] Furthermore, the splicing efficiency of PRPF31 mutants with other NLS sequences was slightly lower than that of PRPF31.

[0130] Example 5 Promotion of PRPF31 or its mutants on the phagocytic function of RPE cells derived from human IPSCs Based on the vector pMcherry-PRPF31 prepared in Example 1, vector D prepared in Example 3, and the control vector pMcherry-N1, a nucleic acid molecule encoding Mcherry-PRPF31 (wherein the PRPF31 nucleotide sequence is shown in SEQ ID NO. 25), a nucleic acid molecule encoding an Mcherry-PRPF31 mutant (wherein the NLS sequence is sequence D, and the nucleotide sequence of the mutant is shown in SEQ ID NO. 29), and a nucleic acid molecule encoding Mcherry (the nucleotide sequence of which is shown in SEQ ID NO. 34) were obtained.

[0131] The nucleic acid molecules were cloned into the viral expression vector pFG12 (Addgene, #14884). The three plasmids were co-transfected with lentiviruses packaged in 293T cells, concentrated, and then used to infect human iPSC-derived RPE cells. Eight days after infection, FITC-conjugated rat POS was added to the RPE cells to observe their phagocytosis.

[0132] The results are shown in Figures 10 to 12. The results in Figures 10 to 12 demonstrate that either PRPF31 or its NLS mutant can promote the phagocytic function of RPE cells induced from human IPSCs.

[0133] Example 6 Promotion of PRPF31 or its mutants on cilia length of RPE cells derived from human IPSCs Based on the vector pMcherry-PRPF31 prepared in Example 1, vector D prepared in Example 3, and the control vector pMcherry-N1, a nucleic acid molecule encoding Mcherry-PRPF31 (wherein the PRPF31 nucleotide sequence is shown in SEQ ID NO. 25), a nucleic acid molecule encoding an Mcherry-PRPF31 mutant (wherein the NLS sequence is sequence D, and the nucleotide sequence of the mutant is shown in SEQ ID NO. 29), and a nucleic acid molecule encoding Mcherry (the nucleotide sequence of which is shown in SEQ ID NO. 34) were obtained.

[0134] The nucleic acid molecules were cloned into the viral expression vector pFG12 (Addgene, #14884), and the three plasmids were co-transfected into 293T cells with the packaged lentivirus. After enrichment, the resulting vectors were used to infect RPE cells derived from patient iPSCs.

[0135] Eight days after infection, cells were fixed and immunostained using anti-ARL13B antibody (Proteintech). The results in Figure 13 demonstrate that either PRPF31 or its NLS mutant can promote cilia length in RPE cells derived from patient IPSCs.

[0136] Example 7 Promotion of AAV expressing PRPF31 or its mutants on phagocytic function and cilium length of RPE cells derived from human IPSCs The CDS of PRPF31 or the CDS portion of mutant D was cloned into the pAV-CAG expression vector, and the three plasmids were co-transfected with recombinant adeno-associated virus (AAV) packaged in 293T cells. After enrichment, RPE cells derived from patient iPSCs were infected.

[0137] Twenty-one days after infection, a portion of the AAV-infected iPSC-RPE cells was treated with FITC-conjugated rat POS (prepared in-house) to observe the phagocytosis of rat POS by the RPE. Another portion of the AAV-infected iPSC-RPE cells was fixed and immunostained using an anti-ARL13B antibody (Proteintech). The results in Figure 14 demonstrate that AAVs expressing PRPF31 or its mutants can promote the phagocytic function of RPE cells derived from human iPSCs and enhance cilia length in RPE cells derived from patient iPSCs.

[0138] The foregoing detailed description has been provided by way of illustration and example and is not intended to limit the scope of the appended claims. Many variations of the presently recited embodiments will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A PRPF31 variant comprising an exogenous nuclear localization (NLS) domain comprising the amino acid sequence set forth in SEQ ID NO. 4 or a truncated form thereof.

2. The PRPF31 mutant of claim 1, which does not contain the NLS domain of wild-type PRPF31.

3. The PRPF31 mutant according to any one of claims 1 to 2, comprising a NOSIC domain.

4. The PRPF31 mutant according to any one of claims 1 to 3, comprising an NOP domain.

5. The PRPF31 mutant according to any one of claims 1 to 4, which is capable of cleaving the mRNA of a gene including the RHO gene.

6. The PRPF31 mutant according to any one of claims 1 to 5, wherein the PRPF31 mutant is derived from human PRPF31.

7. The PRPF31 mutant according to any one of claims 1 to 6, comprising the amino acid sequence shown in SEQ ID NO.

21.

8. A nucleic acid molecule encoding the PRPF31 mutant according to any one of claims 1 to 7.

9. 9. The nucleic acid molecule of claim 8, comprising the nucleotide sequence set forth in SEQ ID NO.

29.

10. A vector comprising the nucleic acid molecule according to any one of claims 8 to 9.

11. The vector of claim 10 , comprising a viral vector.

12. The vector according to any one of claims 10 to 11, comprising a lentiviral vector and an AAV vector.

13. 13. The vector of claim 12, wherein the lentivirus comprises a human immunodeficiency virus (HIV), a simian immunodeficiency virus (SIV), and a non-primate lentivirus such as a feline immunodeficiency virus (FIV), an equine infectious anemia virus (EIAV), a bovine immunodeficiency virus (BIV), and / or a visna-maedi virus (VMV).

14. The vector according to any one of claims 12 to 13, wherein the serotype of the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 and / or AAV10.

15. Use of a PRPF31 mutant described in any one of claims 1 to 7, a nucleic acid molecule described in any one of claims 8 to 9, and / or a vector described in any one of claims 10 to 14 in the preparation of a medicament for treating a disease.

16. The use according to claim 15, wherein the disease comprises a disease caused by a PRPF31 gene mutation.

17. The use according to any one of claims 15 to 16, wherein the disease comprises retinitis pigmentosa (RP).

18. The use according to any one of claims 15 to 17, wherein the disease comprises autosomal dominant retinitis pigmentosa (ADRP).

19. Use of a PRPF31 mutant described in any one of claims 1 to 7, a nucleic acid molecule described in any one of claims 8 to 9, and / or a vector described in any one of claims 10 to 14 for improving the biological activity of retinal pigment epithelial (RPE) cells in vitro.

20. 20. The use of claim 19, wherein the biological activity comprises phagocytic activity of the RPE cells and / or cilia length of the RPE cells.

21. The use according to any one of claims 19 to 20, wherein the RPE cells are derived from pluripotent stem cells and / or totipotent stem cells.

22. The use according to any one of claims 19 to 21, wherein the RPE cells are derived from human iPS cells.

23. The use according to any one of claims 19 to 22, wherein the PRPF31 mutant does not contain the NLS domain of wild-type PRPF31.

24. The use according to any one of claims 19 to 23, wherein the PRPF31 mutant comprises the amino acid sequence shown in SEQ ID NO.

21.

25. The use according to any one of claims 19 to 24, wherein the vector comprises a viral vector.

26. Use of a PRPF31 variant and / or a vector comprising a nucleic acid molecule encoding said PRPF31 variant for improving mRNA splicing efficiency in vitro, wherein said PRPF31 variant comprises an exogenous nuclear localization (NLS) domain comprising the amino acid sequence set forth in SEQ ID NO.

4.

27. The use of claim 26, wherein the PRPF31 mutant does not contain the NLS domain of wild-type PRPF31.

28. The use according to any one of claims 26 to 27, wherein the PRPF31 mutant comprises the amino acid sequence shown in SEQ ID NO.

21.

29. 27. The use of claim 26, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO.

29.

30. The use according to any one of claims 26 to 29, wherein the vector comprises a viral vector.

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