Method for transcriptional activation of homologous genes of a target gene and in vitro diagnostic method

Abstract

The present invention relates to a method for transcriptionally activating a gene homologous to at least one gene of interest and, optionally, inactivating at least one gene of interest, wherein the mRNA encoded by the at least one gene of interest contains a mutation compared to a control, the method comprising the steps described herein. The present invention further relates to a method for diagnosing a disease in vitro, the method comprising the steps of: a) inducing expression of the mRNA encoded by at least one gene of interest in a cell or tissue sample obtained from a subject; b) isolating the mRNA of step a); c) analyzing the sequence of the mRNA isolated in step b); and d) thereby detecting a mutation in the mRNA compared to a control, which is indicative of the presence of a disease.

Description

Technical Field

[0001] The present invention relates to a method for transcriptionally activating a homologous gene of at least one target gene and, optionally, inactivating at least one target gene, wherein the mRNA encoded by the at least one target gene contains a mutation as compared to a control. The method comprises a binding step of a complex comprising an undenatured or genetically recombined DNA-binding protein, at least one transcriptional activation domain of a transcriptional activator or transcription factor, and at least one guide RNA (the at least one guide RNA binds to a promoter region of a homologous gene of the at least one target gene or another element that regulates the expression of the mRNA encoded by the homologous gene of the at least one target gene, and optionally, a further guide RNA binds to a coding region, a promoter region and / or another element that regulates the expression of the mRNA encoded by the at least one target gene, and the at least one target gene is selected from the group consisting of an opsin gene, a cyclic nucleotide-gated (CNG) channel gene, a retina-specific ATP-binding cassette transporter (ABC transporter) gene, and a myosin gene), a step of inducing the expression of the mRNA encoded by the homologous gene of the at least one target gene, optionally, a step of inactivating the expression of the mRNA encoded by the at least one target gene, and thereby a step of transcriptionally activating the at least one target gene. Further, the present invention relates to a method for diagnosing a disease in vitro, the method comprising: a) a step of inducing the expression of the mRNA encoded by at least one target gene in a cell or tissue sample obtained from a subject; b) a step of isolating the mRNA of step a); c) a step of analyzing the sequence of the isolated mRNA of step b); and d) thereby detecting a mutation in the mRNA as compared to a control and suggesting the presence of the disease.

Background Art

[0002] Hereditary retinal dystrophy (IRD) includes a heterogeneous group of blindness disorders affecting millions of people worldwide. Most of these blindness disorders involve functional or structural impairments of photoreceptor cells that detect light. These cells consist of rods, which are responsible for night vision, and cones, which are responsible for day vision and color vision.

[0003] Retinitis pigmentosa (RP) is the most common hereditary retinal disease, primarily affecting rod photoreceptor cells (Daiger et al., 2013). In contrast, color blindness (ACHM) is one of the most frequent hereditary diseases affecting cones (Michalakis et al., 2017). Many genes associated with RP or ACHM encode members of the rod or cone light-induced signaling cascade (also known as the phototransduction cascade). These photoreceptor cells share functional characteristics and are often mediated by homologous proteins encoded by different genes. For example, major signaling molecules in rods and cones, such as visual pigments (opsins) and cyclic nucleotide-sensitive (CNG) channels, are encoded by genes that are different but highly homologous. Rods express only the rhodopsin gene (RHO), while human cones contain three types of cone opsins (long-wavelength L-opsin (OPN1LW), medium-wavelength M-opsin (OPN1MW), and short-wavelength S-opsin (OPN1SW)). Except for humans and primates, most mammals, including mice, express only two types of cone opsins: S-opsin (OPN1SW) and M-opsin (OPN1MW). The CNG channel is a heterotetramer complex composed of two subunits: the CNG A subunit, which determines channel function, and the CNG B subunit, which regulates channel function. Unmodified rod CNG channels contain the CNGA1 and CNGB1 subunits, while cones contain the CNGA3 and CNGB3 subunits. Previous studies have shown that rod and cone CNG A subunits, along with other photoreceptor-type CNG B subunits (CNGA1 / CNGB3 and CNGA3 / CNGB1), can also form functional units (Finn et al., 1998). Many more homologous genes that play important roles in visual detection and processing are found in photoreceptors and non-photoreceptor cells such as retinal pigment epithelial (RPE) cells.

[0004] Recent studies on mouse models have shown that rhodopsin and cone opsin are functionally equivalent (Fu et al., 2008, Kefalov 2012, Sakurai et al., 2007, Shi et al., 2007). This suggests that activating the gene encoding rod-pyramid opsin can compensate for rhodopsin deficiency in the corresponding mouse model. The same applies to rod and cone CNG channel subunits, which have been shown to functionally compensate for each other in heterologous expression systems (Finn et al., 1998, Gerstner et al., 2000, Sautter et al., 1998).

[0005] Mutations in the rhodopsin gene (RHO) are the primary cause of autosomal dominant rhodopsytropin (adRP). In contrast, mutations in the genes encoding the Cohn CNG channel subunits (CNGA3 and CNGB3) are the most frequent cause of acute hypertension of the brain (ACHM). Mouse models lacking rhodopsin (Rho- / -) or Cnga3 (Cnga3- / -) strongly reflect the clinical phenotypes of adRP and ACHM, respectively (Biel et al., 1999; Humphries et al., 1997).

[0006] Over the past few decades, many different approaches have been developed to combat IRD, such as RP or ACHM (Scholl et al., 2016). From a clinical standpoint, the most popular and ultimate approach today is classical gene replacement therapy, which has been successfully applied in various mouse models of retinal degeneration (Boye et al., 2013, Koch et al., 2012, Michalakis et al., 2010). These studies have used recombinant adeno-associated virus (rAAV) vectors to efficiently introduce each gene into the retina and express it for extended periods. AAV is a small virus derived from parvovirus that acts as a vehicle to precisely copy and deliver the target gene. While AAV offers many advantages (i.e., high delivery efficiency, long-term expression without genome integration, non-toxic or very low toxicity, and good immune resistance), it also has several significant drawbacks that have hindered its widespread application in conventional gene replacement therapy. A major drawback of rAAV vectors is their limited genome packaging capacity (approximately 4.7 kb including the promoter and reverse-terminal repeat (ITR) (Wu et al., 2010)). However, many IRDs are caused by genes with coding sequences that far exceed the AAV packaging limits, such as USH2A, MYO7A, ABCA4, CACNA1F, CDH23, GPR98, EYS, RP1, or PRPH8. Thus, the need for developing methods to overcome this significant limitation of AAV remains unmet.

[0007] CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 genome editing technology is a pioneering approach for treating genetic disorders. The DNA-targeted endonuclease Cas9 is recruited to specific gene loci in the genome via a short complementary RNA molecule called guide RNA (gRNA). Previous studies have developed endonuclease-deficient Cas9 variants (called "dead" Cas9, or dCas9) to further expand the applications of the Cas9 enzyme (Sander & Joung 2014, Wang et al., 2016). In particular, the applications of these modified Cas9 variants include efficient gene activation both in vivo and in vitro (Sander & Joung 2014). For this purpose, dCas9 is C-terminally fused to the transcriptional activation domain of a transcription factor. Recent studies have compared the efficiencies of different CRISPR / Cas9 gene activation domains for several genes in various different cell types. In this study, a specific single-gene activation domain (VPR, a hybrid VP64-p65-Rta triplicate activator (Chavez et al., 2015)) was shown to exhibit the highest gene activation efficiency across all experiments and all species tested. Furthermore, this study demonstrated that the gRNA binding site in the promoter region of each gene also affects the efficiency of gene activation. Finally, it was shown that increasing the number of gRNAs improved gene activation (Chavez et al., 2016).

[0008] However, despite its highly promising and potent in vitro capabilities, the application of dCas9 VPR-based therapy in the retina and other tissues is hindered by the lack of efficient delivery techniques. Due to its size (5.8 kb), dCas9-VPR far exceeds the DNA packaging capacity of rAAV vectors. In the past, several methods have been developed to circumvent this limitation of rAAV (Chamberlain et al., 2016, Flotte 2000). These methods are based on pre-transcriptional or post-transcriptional rearrangement of fragmented rAAV transgenes at the DNA, mRNA, or protein level.

[0009] To date, more than 60 IRD genes have been identified. While genetic diagnosis has improved significantly, a large number of IRD patients remain undiagnosed (up to 40%) (Audo et al., 2012; Shanks et al., 2013). Possible reasons for this lack of genetic diagnosis include technical limitations and the presence of pathogenic mutations in unknown genes in the patients. Furthermore, among patients with autosomal recessive IRD who have not received a confirmed genetic diagnosis, a high proportion have mutations in only one gene, such as those associated with Leber congenital amaurosis (LCA), Usher syndrome (USH), and Stargard disease (STGD). These patients are likely to have a second mutation in the non-coding region of the same gene that would not be detected by standard diagnostic panels.

[0010] Next-generation sequencing technologies such as whole-genome sequencing (WGS) and whole-exome sequencing (WES) have advanced the diagnosis of genetic diseases. However, both WGS and WES have key limitations. WES does not cover non-exon regions (introns, promoters, and other transcriptional regulatory elements) that are crucial for mRNA stability and processing. WGS is still expensive in terms of both time and cost, and interpreting the big data obtained in this process is difficult and requires trained biochemical information engineers. Furthermore, even if disease-causing mutations are identified in the exon or non-exon regions of candidate genes by WGS, it is essential to experimentally verify how these mutations affect the mRNA level.

[0011] Single nucleotide variants can affect mRNA through various mechanisms. The most common mechanism is alteration of mRNA splicing (Baralle & Buratti 2017, Kim et al., 2018). Classical splice variants affect the consensus sequence of known splice sites. These variants are typically detected by the methods described above (WGS and / or WES) and classified as splice variants by standard splice prediction software. However, with the exception of those affecting the nucleotides of the first two introns adjacent to an exon (GT of AG), classifying a variant as a splice variant usually requires experimental validation using mRNA levels in affected cells or minigene-based tests expressing the corresponding gene fragment in commonly available cell lines. In addition to false-positive results, splice prediction software can generate an uncertain number of false-negative records. Generally, these "false-negative" splice mutations are located in deep exon coding regions where splice prediction is considerably difficult (Grodecka et al., 2017; Ohno et al., 2018). Typically, (deep) exon point mutations predicted to affect conserved and / or functionally important amino acids are classified as missense mutations. However, regardless of their type, disease-causing mutations can also lead to abnormal splicing, which is largely an unexplored option. Furthermore, identifying splice mutations is crucial for developing appropriate treatments for affected individuals. Since splice mutations associated with IRD can be treated, for example, with antisense oligonucleotides (Bergsma et al., 2018; Godfrey et al., 2017), identifying these mutations will have a strong impact on the development of future treatments.

[0012] Some publications have used WGS to identify deep intron (splice) mutations in IRD patients (Bax et al. 2015, Braun et al. 2013, Carss et al. 2017, Khan et al. 2017, Liquori et al. 2016, Mayer et al. 2016, Naruto et al. 2015, Rio Frio et al. 2009, Vache et al. 2012, Webb et al. 2012). However, as explained above, experimental validation of these mutations at the mRNA level is quite advanced and difficult to apply to a large number of patients.

[0013] A recent study analyzed the potential impact of two deep intronic mutations in the ABCA4 gene on mRNA splicing in photoreceptor progenitor cells induced from patient fibroblasts (Albert et al., 2018). This procedure has two key limitations: i) it is elaborate and time-consuming, making it largely unsuitable for routine diagnostics; and ii) the induced progenitor cells do not express all IRD genes, making it unsuitable for genetic diagnosis of many IRD patients.

[0014] Thus, there is a need to develop improved and easily accessible technologies that can investigate pathogenic gene mutations at the transcript level. The most convenient way to analyze the transcripts of corresponding genes is to use patient tissue. However, biopsies (such as retinal resections) are often impractical, and many disease genes are expressed in a tissue-specific manner (for example, most IRD-related genes are expressed only in retinal cells) but not in cells that can be easily isolated (e.g., blood cells, fibroblasts, and urinary cells). [Overview of the project] [Problems that the invention aims to solve]

[0015] In particular, the present invention enables the avoidance of these disorders by activating a single (or more) gene in a patient's cells using a dCas9-VPR-based transcriptional activation method and examining the pathogenic changes of the corresponding mRNA by target RNA sequencing and / or conventional RT-PCR analysis.

[0016] In this invention, the inventors introduce transcriptional activation of a (homologous) gene using dCas9-VPR for therapeutic (see Figure 1) and diagnostic (see Figure 2) applications, in order to overcome the above-mentioned drawbacks and meet the intended needs. [Means for solving the problem]

[0017] The present invention relates to a method for transcriptionally activating a homologous gene of at least one target gene and, optionally, inactivating at least one target gene, wherein the mRNA encoded by the at least one target gene contains mutations compared to a control. The method comprises a conjugation step of a complex comprising an unmodified or recombinant DNA-binding protein, a transcription activator or at least one transcription activation domain of a transcription factor, and at least one guide RNA. In this step, at least one guide RNA binds to the promoter region of the homologous gene of at least one target gene or to other elements that regulate the expression of mRNA encoded by the homologous gene of at least one target gene, optionally, further guide RNAs bind to the coding region, promoter region and / or other elements that regulate the expression of mRNA encoded by at least one target gene, and further, the at least one target gene is selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes. This method further includes the steps of inducing the expression of mRNA encoded by a homologous gene of at least one target gene, optionally inactivating the expression of mRNA encoded by at least one target gene, and thereby transcriptionally activating at least one target gene. The optional inactivation of at least one target gene preferably includes the inactivation of at least one further target gene in addition to the inactivation of at least one target gene whose homologous gene has been transcriptionally activated. Here, the target gene and further target gene are genes whose function is impaired by mutation, in other words, the mRNA encoded by the target gene contains mutations.

[0018] In one embodiment of the transcriptional activation method, the method further comprises the steps of inducing the expression of a protein encoded by the mRNA of a homologous gene of at least one target gene, and analyzing the sequence, expression level, localization, or function of the at least one protein encoded by the mRNA.

[0019] In one embodiment of the transcriptional activation method, at least one homologous gene of the target gene is selected from the group consisting of ABCA1 (SEQ ID NO: 1), ABCA2 (SEQ ID NO: 3), ABCA7 (SEQ ID NO: 7), ABCA12 (SEQ ID NO: 9), ABCA13 (SEQ ID NO: 11), CNGA1 (SEQ ID NO: 13), CNGA2 (SEQ ID NO: 15), CNGA3 (SEQ ID NO: 17), CNGA4 (SEQ ID NO: 19), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), MYO7B (SEQ ID NO: 33), MYO5A (SEQ ID NO: 25), MYO5B (SEQ ID NO: 27), MYO5C (SEQ ID NO: 29), MYO10 (SEQ ID NO: 35), MYO15B (SEQ ID NO: 39), MYO15A (SEQ ID NO: 37), OPN1LW (SEQ ID NO: 41), OPN1MW (SEQ ID NO: 43), and OPN1SW (SEQ ID NO: 45).

[0020] In one embodiment of the transcription activation method, the undenatured or recombinant DNA-binding protein is selected from the group consisting of Cas-enzymes (preferably Cas9 (SEQ ID NO: 92), dCas9-enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), or Cas12b (SEQ ID NO: 94)), zinc finger nucleases, and transcription activator-like nucleases, where at least one transcription activation domain of the transcription activator or transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75), or a combination thereof. Preferably, the nucleic acid sequences of the undenatured or recombinant DNA-binding protein and at least one transcription activation domain of the transcription activator or transcription factor are separated into two fragments. More preferably, the undenatured DNA-binding protein is the Cas9 enzyme (SEQ ID NO: 92) from Streptococcus pyogenes. More preferably, the recombinant DNA-binding protein is selected from the group consisting of dCas9 having mutations D10A and H840A according to SEQ ID NO: 96, and dCas9 having mutations D10A, D839A, H840A, and N863A according to SEQ ID NO: 97. However, in principle, all Cas enzymes from any known organism can be used in this method of the present invention.

[0021] In one embodiment of the transcriptional activation method, the nucleotide sequences of an undenatured or recombinant DNA-binding protein and at least one transcriptional activation domain of a transcription activator or transcription factor are present in two separate plasmids and / or vectors.

[0022] In one embodiment of the transcriptional activation method, the method further includes using a recombinant AAV vector of natural or artificial origin, preferably an AAV vector variant having retinal cell tropism and enhanced retinal transduction efficiency.

[0023] The present invention further provides a complex comprising an unmodified or recombinant DNA-binding protein, at least one transcriptional activation domain of a transcription activator or transcription factor, and at least one guide RNA, for use in a method of treating hereditary retinal dystrophy (IRD) caused by mutations in at least one target gene selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes. The method comprises transcriptionally activating a homologous gene of at least one target gene and optionally deactivating at least one target gene (e.g., the mRNA encoded by at least one target gene contains mutations compared to a control), wherein at least one guide RNA binds to the promoter region of the homologous gene of at least one target gene or other elements that regulate the expression of mRNA encoded by the homologous gene of at least one target gene, and optionally further guide RNAs bind to the coding region, promoter region, and / or other elements that regulate the expression of mRNA encoded by at least one target gene, thereby activating the homologous gene of at least one target gene. Expression of mRNA encoded by the offspring is induced, and optionally, expression of mRNA encoded by at least one target gene is inactivated. Preferably, the complex is provided as a nucleotide sequence of an undenatured or recombinant DNA-binding protein, a transcription activator or at least one transcription activation domain of a transcription factor, and at least one guide RNA. Optionally, the nucleotide sequences of the undenatured or recombinant DNA-binding protein and at least one transcription activation domain of a transcription activator or transcription factor are located on two separate plasmids and / or vectors, preferably the two separate vectors are recombinant AAV vectors. The AAV vectors may be of natural or engineered origin, and more preferably, the AAV vectors may be AAV vector variants having improved retinal cell type targeting and / or retinal delivery efficiency.

[0024] The present invention further includes a method for diagnosing a disease in vitro, the method comprising: a) inducing the expression of mRNA encoded by at least one target gene in a cell or tissue sample obtained from a subject; b) separating the mRNA of step a); c) analyzing the sequence of the mRNA separated in step b); and d) thereby detecting mutations in the mRNA as compared to a control and suggesting the presence of the disease.

[0025] In one embodiment of the method for diagnosing a disease in vitro, the method further comprises inducing the expression of a protein encoded by mRNA and analyzing the sequence, expression level, localization or function of at least one protein encoded by mRNA in a cell or tissue sample.

[0026] In one embodiment of the method for diagnosing a disease in vitro, step a) comprises specifically binding a complex comprising an unmodified or recombinant DNA-binding protein and at least one transcriptional activation domain of a transcriptional activator or transcription factor to a promoter region of at least one target gene or to another element that regulates the expression of at least one target gene.

[0027] In one embodiment of the method for diagnosing a disease in vitro, the unmodified or recombinant DNA-binding protein is selected from the group consisting of Cas enzymes (preferably Cas9 (SEQ ID NO: 92), dCas9-enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93) or Cas12b (SEQ ID NO: 94)), zinc finger nucleases (ZFN), and transcriptional activator-like nucleases (TALEN). More preferably, the unmodified DNA-binding protein is the Cas9 enzyme of Streptococcus pyogenes (SEQ ID NO: 92). More preferably, the recombinant DNA-binding protein is selected from the group consisting of dCas9 having mutations D10A and H840A according to SEQ ID NO: 96, and dCas9 having mutations D10A, D839A, H840A and N863A according to SEQ ID NO: 97. However, in principle, all Cas enzymes of any known organism can be used in this method of the present invention.

[0028] In one embodiment of a method for diagnosing a disease in vitro, the native or genetically engineered DNA-binding protein is a Cas enzyme (preferably Cas9 (SEQ ID NO: 92), dCas9-enzyme (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93) or Cas12b (SEQ ID NO: 94)), the complex further comprises at least one guide RNA, and this guide RNA can bind to the promoter region of at least one target gene or other elements that regulate the expression of at least one target gene.

[0029] In one embodiment of a method for diagnosing a disease in vitro, the DNA-binding protein is fused at the C-terminus or N-terminus to at least one transcriptional activation domain of a transcriptional activator or transcription factor, preferably at least one transcriptional activation domain of a transcriptional activator or transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75) or combinations thereof.

[0030] In one embodiment of a method for diagnosing a disease in vitro, the disease is selected from the group consisting of neurodegenerative diseases, epilepsy, mental diseases (preferably depression, mania, bipolar disorder, schizophrenia, or autism), or retinal diseases (preferably hereditary retinal dystrophy, more preferably hereditary retinal dystrophy, age-related macular degeneration (AMD), genetically induced age-related macular degeneration (AMD), autosomal dominant, autosomal recessive, X-linked or digenic retinitis pigmentosa, color vision disorders, Stargardt disease, Best disease, Leber congenital amaurosis, retinoblastoma, congenital stationary night blindness, choroidemia, early onset retinal dystrophy, cone-rod-cone dystrophy, pattern dystrophy, syndromic ciliopathy such as Ascher syndrome, more preferably Bardet-Biedl syndrome, Usher syndrome, Senior-Loken syndrome or Alstrom syndrome). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [Figure 1] Figure 1 illustrates dCas9-VPR-mediated transcriptional activation of homologous genes as a novel treatment option for genetic disorders. Figure 1A shows that gene A is active in a given cell type or tissue but is deficient due to a disease-causing mutation. Gene B is a homolog of gene A, and is structurally and functionally very similar to gene A, but is not expressed (inactive) in diseased cell types and tissues. Figure 1B shows transcriptional activation therapy that combines the dCas9-VPR module with gene B-specific guide RNA (gRNA) to activate gene B in the appropriate tissue (or cell type). Gene B compensates for the deficient function (gene A) and provides a therapeutic effect to the patient. TSS stands for transcription start site.

[0032] [Figure 2] Figure 2 shows gene transcription activation as a novel diagnostic tool.

[0033] [Figure 3-1] Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figure 3A is an illustration of the hybrid VP64-p65-Rta tripartite activator (dCas9-VPR, SEQ ID NO: 95). VP64 (SEQ ID NO: 73) is a transcriptional activator consisting of four tandem copies of VP16 (herpes simplex virus protein 16) linked by a glycine-serine linker. dCas9-VPR (SEQ ID NO: 95) consists of dCas9 fused to the activation domains VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), and Rta (SEQ ID NO: 75), with each activation domain separated by a short amino acid linker. Figure 3B shows the binding sites (in bp) of a single Cnga1-specific gRNA (g1-g3) (target sequences of g1-g3 of Cnga1 (sequences 76-78) including the PAM sequence) (black arrows) to the transcription start site (TSS) within the mouse Cnga1 promoter. The promoter and TSS were obtained from the website http: / / epd.vital-it.ch / mouse / mouse_database.php.

[0034] [Figure 3-2] Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figure 3C shows a doxycycline-inducible cassette expressing dCas9-VPR (SEQ ID NO: 95) along with Cnga1 gRNA (VPR-A1, top panel) (target sequences g1-g3 of Cnga1 (SEQ ID NOs: 76-78) containing PAM sequences) or lacZ (VPR-lacZ, bottom panel) gRNA (target sequence of lacZ (SEQ ID NO: 125) containing PAM sequences). Each gRNA is driven by the U6 promoter.

[0035] [Figure 3-3] Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figure 3D shows representative results for 661w cells expressing one of the cassettes shown in Figure 3C and co-immunolabeled with a Cnga1-specific antibody.

[0036] [Figure 3-4] Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figures 3E and 3F show qRT-PCR quantifying Cnga1 (Figure 3E) or dCas9 (Figure 3F) mRNA levels in 661w cells expressing the VPR-A1 cassette at different doxycycline concentrations, as shown. Figures 3G-L show inside-out patch-clamp recordings excised from 661w cells expressing the VPR-A1 (CNGA1) or VPR-lacZ (LacZ) cassette.

[0037] [Figure 3-5]Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figures 3G-L show inside-out patch-clamp recordings excised from 661w cells expressing VPR-A1 (CNGA1) or VPR-lacZ (LacZ) cassettes. Figures 3G-J show absolute current changes (Figures 3G and 3I) and normalized current changes (Figures 3H and 3J) obtained from each cell in the presence of cGMP alone (Figures 3G and 3H) and Ca2+ / Mg2+ alone or in combination with cGMP (Figures 3I and 3J). Figures 3K and 3L show representative outputs from membrane patches of 661w cells expressing VPR-A1 (CNGA1, Figure 3K) or VPR-lacZ (LacZ, Figure 3L) under basal conditions, after the addition of cGMP and / or Ca2+ / Mg2+. Statistical analysis was performed using an independent Student's t-test for comparison between the two groups. ***, p < 0.001.

[0038] [Figure 3-6] Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figures 3G-L show inside-out patch-clamp recordings excised from 661w cells expressing VPR-A1 (CNGA1) or VPR-lacZ (LacZ) cassettes. Figures 3G-J show absolute current changes (Figures 3G and 3I) and normalized current changes (Figures 3H and 3J) obtained from each cell in the presence of cGMP alone (Figures 3G and 3H) and Ca2+ / Mg2+ alone or in combination with cGMP (Figures 3I and 3J). Figures 3K and 3L show representative outputs from membrane patches of 661w cells expressing VPR-A1 (CNGA1, Figure 3K) or VPR-lacZ (LacZ, Figure 3L) under basal conditions, after the addition of cGMP and / or Ca2+ / Mg2+. Statistical analysis was performed using an independent Student's t-test for comparison between the two groups. ***, p < 0.001.

[0039] [Figure 3-7]Figure 3 shows dCas9-VPR-mediated transcriptional activation of mouse Cnga1 in 661w cells. Figures 3G-L show inside-out patch-clamp recordings excised from 661w cells expressing VPR-A1 (CNGA1) or VPR-lacZ (LacZ) cassettes. Figures 3G-J show absolute current changes (Figures 3G and 3I) and normalized current changes (Figures 3H and 3J) obtained from each cell in the presence of cGMP alone (Figures 3G and 3H) and Ca2+ / Mg2+ alone or in combination with cGMP (Figures 3I and 3J). Figures 3K and 3L show representative outputs from membrane patches of 661w cells expressing VPR-A1 (CNGA1, Figure 3K) or VPR-lacZ (LacZ, Figure 3L) under basal conditions, after the addition of cGMP and / or Ca2+ / Mg2+. Statistical analysis was performed using an independent Student's t-test for comparison between the two groups. ***, p < 0.001.

[0040] [Figure 4-1] Figure 4 shows the calculation of cleavage intein efficiency in HEK293 cells. Figure 4A schematically shows the dCas9 cleavage intein mutants used. Cas9 fragments were generated by cleaving dCas9 at either the aa position V713 (upper panel) or E573 (lower panel). The first dCas9 fragment (dCas9N1 or dCas9N2, numbered 1 and 3, respectively) was fused at the C-terminus to the N-terminus of the intein (IntN). The second Cas9 fragment (dCas9C1 and dCas9C2, numbered 2 and 4, respectively) contained half of the C-terminus intein (IntC) at its N-terminus. Bp represents base pairs.

[0041] [Figure 4-2] Figure 4 shows the calculation of cleavage intein efficiency in HEK293 cells. Figure 4B shows a Western blot from HEK293 cells transiently co-introduced with a single cleavage intein dCas9 combination as shown. A specific antibody against the N-terminal region of dCas9 was used for signal detection.

[0042] [Figure 4-3] Figure 4 shows the calculation of the cleavage intein efficiency in HEK293 cells. Figure 4C shows the semi-quantitative calculation of the cleavage Cas9 rearrangement efficiency obtained from four independent gene transfer experiments shown in Figure 4B. The rearrangement efficiency was determined by calculating the intensity ratio of the rearranged full-length dCas9 band to the corresponding dCas9N1 or dCas9N2 band for each lane. The mean rearrangement efficiencies were as follows: 1 + 2 = 56.9 ± 2.1%, 3 + 4 = 33.3 ± 1.1%. Statistical analysis was performed using an unpaired Student's t-test for comparison between the two groups. ****, p < 0.0001.

[0043] [Figure 5-1] Figure 5 shows dCas9-VPR (SEQ ID NO: 95) and split V713_dC9-mediated transcriptional activation of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells. Figure 5A shows full-length Cas9 cassettes combined with Cnga1 (A1), Opn1mw (O1mw), or Opn1sw (O1sw) gRNAs used for transient gene transfection in 661w cells (for Cnga1) or MEF cells (for Opn1mw and Opn1sw). Full-length Cas9 cassettes combined with lacZ gRNA were used as a control (target sequence of lacZ gRNA containing the PAM sequence: SEQ ID NO: 124). CMV stands for Cytomegalovirus Promoter. Figure 5B shows the single V713_dC9 mutant used for transient co-transfer of each cell. The dCas9 fragments correspond to the dCas9N1 and dCas9C1 constructs shown in Figure 4.

[0044] [Figure 5-2]Figure 5 shows dCas9-VPR (SEQ ID NO: 95) and split V713_dC9-mediated transcriptional activation of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells. Figures 5C and 5D show the binding sites (in bp) of single Opn1mw (Figure 5C) or Opn1sw (Figure 5D) specific gRNAs (g1-g3) (target sequences of g1-g3 in Opn1mw (SEQ ID NOs: 79-81) and Opn1sw (SEQ ID NOs: 83-85) containing PAM sequences to transcription start sites (TSSs) within their promoters (black arrows). Promoters and TSSs were obtained from the website http: / / epd.vital-it.ch / mouse / mouse_database.php.

[0045] [Figure 5-3] Figure 5 shows dCas9-VPR (SEQ ID NO: 95) and split V713_dC9-mediated transcriptional activation of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells. Figures 5E to 5G show qRT-PCR results for determining the transcriptional activation efficiency of single genes using full-length dCas9-VPR (SEQ ID NO: 95) (Figures 5E, 5G, and 5I) or V713_dC9 (Figures 5F and 5H), as illustrated. Statistical analysis was performed using unpaired Student's t-tests for comparison between the two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0046] [Figure 5-4]Figure 5 shows dCas9-VPR (SEQ ID NO: 95) and split V713_dC9-mediated transcriptional activation of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells. Figures 5E to 5G show qRT-PCR results for determining the transcriptional activation efficiency of single genes using full-length dCas9-VPR (SEQ ID NO: 95) (Figures 5E, 5G, and 5I) or V713_dC9 (Figures 5F and 5H), as illustrated. Statistical analysis was performed using unpaired Student's t-tests for comparison between the two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0047] [Figure 5-5] Figure 5 shows dCas9-VPR (SEQ ID NO: 95) and split V713_dC9-mediated transcriptional activation of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells. Figures 5E to 5G show qRT-PCR results for determining the transcriptional activation efficiency of single genes using full-length dCas9-VPR (SEQ ID NO: 95) (Figures 5E, 5G, and 5I) or V713_dC9 (Figures 5F and 5H), as illustrated. Statistical analysis was performed using unpaired Student's t-tests for comparison between the two groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0048] [Figure 6-1] Figure 6 shows the in vivo transcriptional activation of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) using V713_dC9. Figure 6A shows a single V713_dC9 AAV vector expression cassette used for co-expression of rod photoreceptors. hRHO stands for human rhodopsin promoter. Wild-type mice were injected subretinally at P14 using the AAV2 / 8 capsid.

[0049] [Figure 6-2] Figure 6 shows the in vivo transcriptional activation of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) using V713_dC9. Figures 6B–6D show immunolabeling of mouse retinas 3 weeks after injection in injected eyes (Figures 6B and 6D) or sham-injected control eyes (Figures 6C and 6E). Peanut agglutinin lectin (PNA) antibodies were used as cone photoreceptor markers. Specific antibodies described elsewhere were used for staining Opn1mw (SEQ ID NO: 44) and Opn1sw (SEQ ID NO: 46) (e.g., Becirovic et al., 2016, Nguyen et al., 2016).

[0050] [Figure 6-3] Figure 6 shows the in vivo transcriptional activation of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) using V713_dC9. Figures 6B–6D show immunolabeling of mouse retinas 3 weeks after injection in injected eyes (Figures 6B and 6D) or sham-injected control eyes (Figures 6C and 6E). Peanut agglutinin lectin (PNA) antibodies were used as cone photoreceptor markers. Specific antibodies described elsewhere were used for staining Opn1mw (SEQ ID NO: 44) and Opn1sw (SEQ ID NO: 46) (e.g., Becirovic et al., 2016, Nguyen et al., 2016).

[0051] [Figure 6-4] Figure 6 shows the in vivo transcriptional activation of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) using V713_dC9. Figures 6F and 6G are images taken 3 weeks after injection of mice injected with viruses expressing V713_dC9 and Opn1mw or Opn1sw gRNA (target sequences g1-g3 in Opn1mw (SEQ ID NOs: 79-81) and Opn1sw (SEQ ID NOs: 83-85) containing PAM sequences, using qRT-PCR with isolated RNA. Sham-injected eyes were used as controls (ctrl).

[0052] [Figure 7-1] Figure 7 shows that M-opsin activation improves the retinal phenotype in heterozygous Rho mice. Heterozygous (hz) Rho mice (n=10) were injected with P14, and electroretinography (ERG, Figure 7A) and optical coherence tomography (OCT, Figure 7B) were performed 12 months after injection. One eye was injected with dCas9-VPR (SEQ ID NO: 95) (hz treatment), and the other eye was sham injected with NaCl (hz sham). Both eyes (OD: right eye and OS: left eye) of 10 untreated wild-type (wt) mice (12 months) were used as controls. Figure 7A, upper panel shows statistical values ​​of single ERG measurements from three groups at different light intensities. ns indicates non-significant. The lower panel plots scotopic b-wave amplitude against light intensity. Figure 7B shows optical coherence tomography performed on the same group of mice used for ERG in Figure 7A. ONL stands for outer nucleus. All statistics were performed using ANOVA with Bonferroni's post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0053] [Figure 7-2] Figure 7 shows that M-opsin activation improves the retinal phenotype in heterozygous Rho mice. Heterozygous (hz) Rho mice (n=10) were injected with P14, and electroretinography (ERG, Figure 7A) and optical coherence tomography (OCT, Figure 7B) were performed 12 months after injection. One eye was injected with dCas9-VPR (SEQ ID NO: 95) (hz treatment), and the other eye was sham injected with NaCl (hz sham). Both eyes (OD: right eye and OS: left eye) of 10 untreated wild-type (wt) mice (12 months) were used as controls. Figure 7A, upper panel shows statistical values ​​of single ERG measurements from three groups at different light intensities. ns indicates non-significant. The lower panel plots scotopic b-wave amplitude against light intensity. Figure 7B shows optical coherence tomography performed on the same group of mice used for ERG in Figure 7A. ONL stands for outer nucleus. All statistics were performed using ANOVA with Bonferroni's post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0054] [Figure 8-1] Figure 8 shows dCas9-VPR-mediated transcriptional activation of USH2A (SEQ ID NO: 49) in human fibroblasts. Figure 8A is a diagram depicting the q41 region of chromosome 1 where the KCDT3 (SEQ ID NO: 109) and USH2A (SEQ ID NO: 49) genes are located on opposite strands (the (+) strand for KCDT3 (SEQ ID NO: 122) and the (-) strand for USH2A). Of note is the overlap of the 3'-UTRs of both genes. The transcriptional activation site is indicated by an arrow. Figure 8B is a non-life-size diagram of the USH2A (SEQ ID NO: 49) transcript, consisting of 72 exons (see box). The 5' and 3'-UTRs are shown at the ends of the diagram. Primers used to amplify a single USH2A (SEQ ID NO: 49) fragment (SEQ ID NOs: 98-121) are shown as double arrows. The corresponding PCR products (a-l) are shown as lines containing the fragment length in base pairs (bp).

[0055] [Figure 8-2] Figure 8 shows dCas9-VPR-mediated transcriptional activation of USH2A (SEQ ID NO: 49) in human fibroblasts. Figure 8C shows RT-PCR from human fibroblasts transfected with dCas9-VPR (SEQ ID NO: 95) using the primer pairs shown in Figure 8B, in combination with USH2A gRNA (left panel) (target sequences of USH2A (SEQ ID NO: 86-88) gRNA containing PAM sequences) or control lacZ gRNA (target sequences of lacZ (SEQ ID NO: 125) containing PAM sequences) (right panel). All PCR products were amplified under the same PCR cycling conditions. Kb represents a kilobase pair. The band in line 1 (right panel) corresponds to the 3'-UTR of KCDT3 (SEQ ID NO: 122). Band identity was evaluated by Sanger sequencing.

[0056] [Figure 8-3]Figure 8 shows dCas9-VPR-mediated transcriptional activation of USH2A (SEQ ID NO: 49) in human fibroblasts. Figure 8D shows qRT-PCR from three independent experiments using human fibroblasts transfected with dCas9-VPR (SEQ ID NO: 95) in combination with lacZ (left) (target sequence in lacZ containing the PAM sequence: SEQ ID NO: 125) or USH2A (right) gRNA (target sequence of gRNA in USH2A (SEQ ID NO: 86-88) containing the PAM sequence). Data are presented as multiplicative changes in mRNA transcription numbers normalized to housekeeping (constantly expressed or functional) aminolevulinate synthase (ALAS). Statistical analysis was performed using unpaired Student's t-tests. **, p = 0.0033. Data are shown as standard error of the mean (dCas9-VPR_lacZ: 0.86 0.04, dCas9-VPR_USH2A: 600 0.70 95.56). The figure on the right shows the binding sites of qRT-PCR primers in the USH2A transcript, with qU2_forward binding to exon 12 and qU2_reverse binding to exon 13.

[0057] [Figure 9] Figure 9 shows that transcriptional activation of Opn1mw in heterozygous Rho mice does not induce any microglial activation or reactive gliosis. Figures 9A and 9B are representative immunostaining of the retina from heterozygous Rho mouse #1 injected with either V713_dC9 and Opn1mw (M-opsin) specific gRNA (Figure 9A, treated) or saline (Figure 9B, pseudo, contralateral eye). Peripherin-2 antibody (PRPH2) was used as an outer segment marker for rods and cones, and peanut agglutinin (PNA) was used as a cone marker. Figures 9C–9F show visualization of microglial cells or reactive gliosis in the treated eye (Figures 9C, 9E) and the contralateral eye injected with saline (Figures 9D, 9F) after immunolabeling of the same retina with Iba1 or GFAP. Figures 9G and 9H show the retinas of P13 Pde6b-deficient (rd1) mice, immunolabeled with Iba1 (Figure 9G) or GFAP (Figure 9H) to serve as positive controls. The scale is 30 μm.

[0058] [Figure 10] Figure 10 shows that transcriptional activation of Opn1mw reduces apoptosis in heterozygous Rho mice. Figure 10A shows representative cross-sections of the immunolabeled retina from heterozygous Rho mouse #1 injected with V713_dC9 and Opn1mw (M-opsin) specific gRNAs, showing the introduced (left panel) or unintroduced (right panel) portion of the same retina one year after injection. Figure 10B shows immunolabeling of P13 rd1 mouse retina as a positive control. Apoptosis was visualized by TUNEL staining (top panel), and PRPH2 was used as a rod and cone outer segment marker (bottom panel). The scale is 30 μm. Figure 10C shows quantification of TUNEL+ cells in the introduced versus unintroduced regions of the retina from eight heterozygous Rho mice (+ / -) injected with V713_dC9 and Opn1mw specific sgRNAs. Paired t-tests (two-sided) were used for statistical analysis.

[0059] [Figure 11-1]Figure 11 shows a multiplexing method using three guide RNAs to simultaneously perform Rho knockdown (i.e., inactivation) and Opn1mw activation. Figure 11A shows that Rho knockdown is achieved using a single guide RNA (sgRNA) with a protospacer (PS) > 16 bp, and Cas9 catalytic activity can be preserved. Opn1mw activation, on the other hand, is achieved in the presence of an sgRNA with a short protospacer sequence (< 16 bp). Under these conditions, Cas9 binds but cannot cleave DNA. Figures 11B and 11C show rAAV cassettes used for protein-level reconstruction of cleaved Cas9 using cleavage inteins (Figure 11B) or RNA-level reconstruction using mRNA trans-splicing (REVeRT) (Figure 11C). ITR stands for inverted terminal repeat, g1-g3 stands for gRNA as described in Figure 11A, and U6 stands for U6 promoter. N-Int and C-Int refer to the N-terminal or C-terminal portion of the cleavage intein. Rho refers to the human rhodopsin promoter, SDS to the splice donor site, SAS to the splice receptor site, pA to the polyadenylation signal, and BD to the binding domain.

[0060] [Figure 11-2] Figure 11 shows a multiplexing technique using three guide RNAs to simultaneously perform Rho knockdown (i.e., inactivation) and Opn1mw activation. Figures 11D to 11F show qRT-PCR analysis from the retinas of wild-type mice injected with dual rAAV expressing the SpCas9-VPR cassette shown in Figure 11B (Intein) or Figure 11C (REVeRT), in the presence of two Opn1mw gRNAs and one Rho gRNA (multiplexing technique) or a single lacZ sgRNA alone. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc tests for multiple comparisons. *; p < 0.05; **, p < 0.01; ***, p < 0.001. [Modes for carrying out the invention]

[0061] The present invention relates to a method for transcriptionally activating a homologous gene of at least one target gene and, optionally, inactivating at least one target gene, wherein the mRNA encoded by the at least one target gene contains mutations compared to a control. The method comprises a conjugation step of a complex comprising an unmodified or recombinant DNA-binding protein, a transcription activator or at least one transcription activation domain of a transcription factor, and at least one guide RNA. The at least one guide RNA binds to the promoter region of the homologous gene of at least one target gene or to other elements that regulate the expression of the mRNA encoded by the homologous gene of at least one target gene, and optionally, further guide RNAs bind to the coding region, promoter region and / or other elements that regulate the expression of the mRNA encoded by the at least one target gene. Furthermore, the at least one target gene is selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes and myosin genes. This method further includes the steps of inducing the expression of mRNA encoded by a homolog of at least one target gene, optionally inactivating the expression of mRNA encoded by at least one target gene, and thereby transcriptionally activating at least one target gene. The optional inactivation of at least one target gene preferably involves the inactivation of at least one target gene whose homologous gene has been transcriptionally activated, but also includes the inactivation of at least one further target gene. The target gene and further target genes are genes whose function is impaired by mutation, in other words, the mRNA encoded by the target gene contains mutations.

[0062] As used in the context of this invention, "transcriptional activation" refers to an increase in gene expression rates induced by either biological processes or artificial means, through the expression of an intermediate transcriptional activation protein such as the complex of this invention. Therefore, the term "transcriptional activation of a target gene" in the context of this invention always refers to a situation that leads to functional compensation of a defective / non-functional target gene, thereby enabling the treatment of a disease.

[0063] As used in the context of this invention, the term "gene" means any nucleic acid sequence or portion thereof that has a functional role in coding or transcribing RNA (rRNA, tRNA, or mRNA, the latter being translatable as a protein) or in regulating the expression of other genes. A gene may consist of all nucleic acids that play a role in coding a functional protein, or only a portion of nucleic acids that plays a role in coding or expressing a protein. A nucleic acid sequence may contain genetic abnormalities within exons, introns, start or end regions, promoter sequences, other regulatory sequences, or gene-specific facies regions.

[0064] As used in the context of this invention, the term "target gene" means a gene whose function is impaired by mutation and is therefore a target whose function is replaced by a homologous gene. As used herein, the term "mRNA encoded by the target gene contains mutations" means mutations in the mRNA sequence (nucleotide deletions, insertions, and / or substitutions, preferably point mutations), but also includes modifications of mRNA such as changes in the splice pattern (also called splice mutations), decreased mRNA stability, and / or decreased expression (compared to a control), indicating that the mRNA modification is due to a mutation in the target gene. Mutations can occur in coding regions or non-coding regions, such as promoters, activation regions, and / or introns (e.g., creation, modification, or removal of splice donor or splice receiver sites). Preferably, the mutation is a mutation in the coding region or a splice mutation. The function of the target gene may also be impaired by chromosome excision or the like.

[0065] As used in the context of this invention, the term "homologous gene" means a gene whose sequence, structure, and / or function are identical or similar to that of the target gene, and which can therefore replace or complement the function of the target gene after transcriptional activation.

[0066] As used in the context of this invention, the term "inactivation" means any manipulation of a gene such that the gene-mediated function is inhibited. This may include, but is not limited to, a reduction in gene activity or complete inactivation. It may also include, but is not limited to, cleaving the target gene.

[0067] In the context of this invention, the term "mRNA" refers to a large family of RNA molecules called messenger RNAs that transmit genetic information to protein translation, which is carried out from DNA by ribosomes. In other words, such RNAs are produced by transcription and carry the code for a specific protein from nuclear DNA to ribosomes in the cytoplasm, functioning as a template for protein formation.

[0068] As used in the context of this invention, the term "mutation" means any (pathogenic) alteration or permanent alteration of the nucleotide sequence of a gene (e.g., by point mutation or frameshift mutation). This includes insertion, deletion, or substitution of nucleotides.

[0069] As used in the context of this invention, the term "complex" means a whole composed of two or more parts. In the particular context of this invention, a complex consists of an undenatured or recombinant DNA-binding protein, a transcription activator or at least one transcription activation domain of a transcription factor, and at least one guide RNA as defined elsewhere herein.

[0070] As used in the context of this invention, the term “undenatured or recombinant DNA-binding protein” means any protein capable of binding to DNA. Such proteins are, in particular in the context of this invention, any Cas enzyme, zinc finger nuclease, or transcription activator-like nuclease (TALEN) from any known organism. Such an undenatured DNA-binding protein may also be the Cas9 enzyme of Streptococcus pyogenes (SEQ ID NO: 92). The term “recombined” in this particular context may include any modification in the coding sequence of a DNA-binding protein that alters protein function, preferably its DNA editing properties, more preferably its DNA editing properties. Such recombinant DNA-binding proteins may also be dCas9 having mutations D10A and H840A according to SEQ ID NO: 96, and dCas9 having mutations D10A, D839A, H840A and N863A according to SEQ ID NO: 97.

[0071] As used in the context of this invention, the term "transcriptional activator or transcriptional activation domain of a transcription factor" generally refers to any protein, domain, or sequence that has the ability to activate the expression of a factor or activator involved in the transcription of another sequence. For example, "transcriptional activation domains" include, but are not limited to, VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), or Rta (SEQ ID NO: 75).

[0072] The terms “transcription factor” and “transcription factor” are used synonymously herein and refer to proteins that bind to a gene promoter and enable gene transcription by recruiting RNA polymerase. Transcription factors act alone or in complex with other proteins, such as one or more transcription activators and / or transcription repressors.

[0073] As used in the context of this invention, the term "guide RNA" may refer to a sequence that targets the CRISPR / Cas9 complex at a specific location in genomic DNA, preferably in the promoter region of a specific gene. For example, guide RNA may mean a sequence containing two RNAs, namely CRISPR RNA (crRNA) and transcription-activating crRNA (tracrRNA), or a single-stranded RNA (sgRNA) produced by the fusion of essential parts of crRNA and tractRNA. sgRNA consists of a protospacer complementary to DNA, a tractRNA that stabilizes the complex, and a linker sequence connecting these two parts. For the CRISPR / Cas9-guide RNA complex to be recruited to a target gene locus (e.g., a promoter), a protospacer adjacency motif (PAM) must be present in the sequence of the corresponding gene locus. Guide RNA is introduced into a cell or organism in the form of RNA or DNA encoding the guide RNA. Guide RNA may be isolated RNA, RNA incorporated into a viral vector, or in a form encoded by a vector. Preferably, the vector may be, but is not limited to, a viral vector, a plasmid vector, or an Agrobacterium vector. The DNA encoding the guide RNA may be a vector containing the nucleotide sequence encoding the guide RNA. For example, the guide RNA can be introduced into a cell or organism by transtransferring isolated guide RNA, or plasmid DNA containing the sequence encoding the guide RNA and a promoter (e.g., the U6 promoter), into the cell or organism.

[0074] As used in the context of this invention, the term "promoter region" refers to a region of DNA that triggers the initiation of transcription of a particular gene. The promoter is located near the transcription start site of a gene, upstream of the DNA (towards region 5 of the sense strand). A promoter typically consists of 100 to 1000 base pairs.

[0075] As used in the context of this invention, the term "other elements that regulate mRNA expression" may refer to transcription promoters, silencers, and / or boundary regions, or insulators, relating to the expression of each RNA or mRNA.

[0076] As used in the context of this invention, the term "opsin gene" means any gene for various colorless proteins that, in combination with retinal or associated prosthetic sites, form visual pigments (such as rhodopsin) in a reversible photocatalytic reaction. Such genes include, for example, the M-opsin gene (OPN1MW) (SEQ ID NO: 43), the L-opsin gene (OPN1LW) (SEQ ID NO: 41), or the S-opsin gene (OPN1SW) (SEQ ID NO: 45).

[0077] The term "cyclic nucleotide-sensitive channel (CNG) gene" refers to any member of the CNG channel gene family, which consists of six members in vertebrates. These genes are classified into two subspecies, CNGA and CNGB, based on sequence similarity. Genes encoding CNG channels have also been cloned from Caenorhabditis elegans and Drosophila melanogaster. The CNG channel subunit CNGA1, formerly called the rod-α subunit, is expressed in rod photoreceptor cells and, when exogenously expressed in either Xenopus oocytes or human embryonic kidney cell lines (HEK293), generates a functional channel regulated by cGMP. In humans, mutations in the CNGA1 gene cause retinitis pigmentosa, an autosomal recessive degenerative blindness. CNGB1, formerly called the rod-β subunit, is the second subunit of the rod channel. Unlike CNGA1, expressing the CNGB1a subunit alone does not produce a functional CNG channel. However, co-expression of CNGA1 and the CNGB1a subunit has been shown to generate a heteromeric channel with regulatory ability, permeability, pharmacological activity, and cyclic nucleotide specificity equivalent to the undenatured channel. CNG channels form tetramers, and recent studies have shown that the undenatured rod channel consists of three CNGA1 subunits and one CNGB1a subunit. The CNGA3 subunit (formerly called the cone α subunit) forms a functional channel in heterologous expression systems. On the other hand, CNGB3 (formerly called the cone β subunit) cannot form a functional channel on its own. Mutations in human CNGA3 and CNGB3 are associated with complete color blindness, a rare autosomal recessive congenital disorder characterized by color blindness, decreased visual acuity, and photophobia. The cone CNG channel, like the rod subunit, consists of three CNGA3 subunits and one CNGB3 subunit. CNGA2, formerly known as the olfactory α subunit, CNGA4, formerly known as the olfactory β subunit, and CNGB1b are involved in the transmission of odor signals in olfactory neurons. The olfactory CNG channel consists of two CNGA2, one CNGA4, and one CNGB1b subunit.

[0078] The term "retina-specific ATP-binding cassette transporter (ABC transporter) gene," as used in the context of this invention, refers to any gene encoding a member of the ABC transporter family. These are a group of specific membrane proteins that utilize ATP hydrolysis to power the translocation of a wide variety of substrates across the cell membrane. ABC transporters consist of a minimum of two conserved regions: a highly conserved nucleotide-binding domain (NBD) and a less conserved transmembrane domain (TMD). Eukaryotic ABC proteins are typically organized as complete transporters (containing two NBDs and two TMDs) or semi-transporters (containing one NBD and one TMD), and require the formation of homodimers or heterodimers to constitute a functional protein. The retina-specific ATP-binding cassette transporter ABCA4 (also known as the lim protein, ABCR) is a eukaryotic protein belonging to the ABC-A subfamily of the ABC transporter family. In humans, ABCA4 is localized with opsin photopigments in the outer segment disc membrane of rod and cone photoreceptor cells. It functions as an uptake transporter for N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine. Mutations in the ABCA4 gene cause Stargardt disease (STGD1), a recessive disorder characterized by decreased central vision, progressive bilateral atrophy of photoreceptor cells and retinal pigment epithelial (RPE) cells, accumulation of fluorescent deposits in the macula, and delayed dark adaptation.

[0079] As used in the context of this invention, the term "myosin gene" refers to a gene that codes for a related protein called myosin. Myosin is often called a molecular motor because it consumes energy to move. Myosin can interact with actin. Actin proteins are organized into filaments, forming the reticular structure (cytoskeleton) that gives structure to cells, and can function as pathways for myosin to move. Some myosin proteins bind to other proteins and move within and between cells along actin orbitals. Some myosins are involved in muscle contraction. These myosins interact with other myosin proteins to form thick filaments. In muscle cells, thick filaments made of myosin and thin filaments made of actin constitute a structure called a sarcomere, which is the basic unit of muscle contraction. The overlapping thick and thin filaments bind to and separate from each other, causing the filaments to move relative to each other and allowing the muscle to contract. Mutations in genes encoding muscle myosin can lead to serious abnormalities in the muscles used for exercise (skeletal muscle) and the muscle of the heart (cardiac muscle). Abnormalities in the cardiac muscle can lead to heart failure and sudden death. Myosin proteins are involved in many cellular functions. They are important in the process of cell division because they can transport substances and generate force through contraction. Myosin is also involved in cell movement. Some myosin is present in special structures called stereotactic cilia in the inner ear. These myosin are thought to help organize the stereotactic cilia properly. Abnormalities in these myosin can lead to hearing loss. Examples of genes included in this group of genes are MYH3, MYH6, MYH7, MYH9, MYH11, MYO5A, MYO5B, and MYO7A. Mutations in the MYO7A gene cause Usher syndrome, the leading cause of hereditary hearing loss worldwide. Patients suffer from severe retinitis pigmentosa, congenital hearing loss, and vestibular dysfunction (balance disorder).

[0080] As used in the context of this invention, the term "control" refers to a target gene that does not contain any mutations leading to the respective disease, and whose presence is investigated by any of the methods according to the present invention. Since genomes naturally differ between different subjects, the "wild-type" sequence of the same gene will have a certain degree of deviation between different subjects (same species). These differences usually do not alter the function of the gene. Therefore, even if there are some differences in sequence, the function of the expression product of the target gene is not impaired. However, these differences do not include mutations that could cause disease. Such disease-associated mutations may include not only single nucleotide deletions or changes, but also deletions or changes of longer portions within the affected gene.

[0081] In one embodiment of the transcriptional activation method according to the present invention, the method further includes inducing the expression of a protein encoded by the mRNA of a homologous gene of at least one target gene, and analyzing the sequence, expression level, localization, or function of the at least one mRNA-encoded protein.

[0082] When used in the context of this invention, the term "expression level" means any degree of expression of a particular sequence.

[0083] As used in the context of this invention, the term "protein localization" means any method that enables the detection of a specific protein. Such a method may include the use of a localization signal. However, in most cases, a specific antibody (homemade, commercially available, or imported from elsewhere) is used to detect protein localization. This antibody recognizes an epitope in the undenatured protein. Recombinant proteins can be tagged for easier detection. The tag may be recognized by standard commercially available antibodies (e.g., flag tag, His tag, myc tag). Finally, a small fluorescent tag can be attached to the protein to be examined, which can then be easily detected by microscopic measurement. Since the methods according to the present invention activate genes or proteins present in the undenatured state, antibody-based methods for detecting protein localization are preferred.

[0084] As used in the context of this invention, the term "protein function" or "protein function" means any function mediated by a protein. Several methods exist for classifying protein functions. Among them, gene ontology (GO) and function catalog (FunCat) are two commonly used methods based on general biological phenomena occurring in a wide variety of organisms and eukaryotes (Riley, 1998; Rison et al., 2000; Ouzounis et al., 2003).

[0085] Homologous genes can have the same or similar function as the target gene and, therefore, can replace or complement the function of the target gene after transcriptional activation. Examples of such homologous genes can be found below. Accordingly, in one embodiment of the transcriptional activation method, at least one homologous gene of the target gene is selected from the group consisting of ABCA1 (SEQ ID NO: 1), ABCA2 (SEQ ID NO: 3), ABCA7 (SEQ ID NO: 7), ABCA12 (SEQ ID NO: 9), ABCA13 (SEQ ID NO: 11), CNGA1 (SEQ ID NO: 13), CNGA2 (SEQ ID NO: 15), CNGA3 (SEQ ID NO: 17), CNGA4 (SEQ ID NO: 19), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), MYO7B (SEQ ID NO: 33), MYO5A (SEQ ID NO: 25), MYO5B (SEQ ID NO: 27), MYO5C (SEQ ID NO: 29), MYO10 (SEQ ID NO: 35), MYO15B (SEQ ID NO: 39), MYO15A (SEQ ID NO: 37), OPN1LW (SEQ ID NO: 41), OPN1MW (SEQ ID NO: 43), and OPN1SW (SEQ ID NO: 45).

[0086] In this invention, the target gene is a gene whose function is impaired due to a mutation, and therefore is a gene that is targeted for functional substitution by a homologous gene. As outlined herein, the target gene and homologous gene have the same or similar functions, but do not necessarily have the same sequence or structure. In one embodiment of the transcriptional activation method of this invention, at least one target gene is selected from the group consisting of rhodopsin gene (RHO) (SEQ ID NO: 47), M-opsin gene (OPN1MW) (SEQ ID NO: 43), L-opsin gene (OPN1LW) (SEQ ID NO: 41), or S-opsin gene (OPN1SW) (SEQ ID NO: 45), ABCA4 (SEQ ID NO: 5), CNGA1 (SEQ ID NO: 13), CNGA3 (SEQ ID NO: 17), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), and MYO7A (SEQ ID NO: 31).

[0087] In one embodiment of the transcriptional activation method according to the present invention, at least one target gene is selected from the group consisting of the M-opsin gene (OPN1MW) (SEQ ID NO: 43), the L-opsin gene (OPN1LW) (SEQ ID NO: 41), and the S-opsin gene (OPN1SW) (SEQ ID NO: 45).

[0088] Therefore, some typical examples of related homologous gene pairs include ABCA4 / ABCA1, CNGA1 / CNGA3, CNGB1 / CNGB3, GUCY2E / GUCY2F, GUCA1A / GUCA1B, and MYO7A / MYO7B. Given the functional and / or structural similarities of each homologous gene pair, the deficiency of the mutant gene is functionally compensated for by switching on each homologous gene through transcriptional activation in the affected cell type (pyramid, rod, or RPE cells).

[0089] As outlined herein, the basic principle of the present invention is a combination of a DNA-binding protein and a transcriptional activation domain. The DNA-binding protein may be undenatured or genetically modified. The DNA-binding protein may be selected from the group consisting of Cas enzymes, zinc finger nucleases, and transcriptional activator-like nucleases (TALENs). Since these undenatured DNA-binding molecules may have endonuclease function, they may be genetically modified to lose their endonuclease function. Furthermore, undenatured Cas enzymes may lose their endonuclease function if the gRNA target sequence (protospacer) is shortened. In the present invention, the "targeting sequence" refers to the portion of the guide RNA that directly binds to the target DNA. In combinations of Cas9 and guide RNA with a targeting sequence of less than 16 base pairs, Cas9 cannot cleave DNA and therefore cannot function as an endonuclease.

[0090] Various transcriptional activation domains are known to those skilled in the art. These transcriptional activation domains include, but are not limited to, VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), or Rta (SEQ ID NO: 75). These transcriptional activation domains may be fused to DNA-binding proteins. Thus, DNA-binding proteins induce transcriptional activation domains in homologous genes, thereby enabling transcription of homologous genes. Therefore, in one embodiment of the transcription activation method, the undenatured or recombinant DNA-binding protein is selected from the group consisting of Cas-enzymes (preferably Cas9 (SEQ ID NO: 92), dCas9-enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), or Cas12b (SEQ ID NO: 94)), zinc finger nucleases, and transcription activator-like nucleases, and / or the transcription activator or transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75), and combinations thereof, and preferably the nucleotide sequences of the undenatured or recombinant DNA-binding protein and the transcription activator or transcription factor are separated into two split fragments. The use of split fragments allows the DNA-binding protein-transcription activator fusion protein to be dispersed into separate vectors. Each of these separate vectors is smaller and is thereby incorporated into smaller viral particles that can be administered to a target. Thus, in one embodiment of the transcriptional activation method according to the present invention, the transcription activator or at least one transcriptional activation domain of the transcription factor is the transcriptional activation domains VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), and Rta (SEQ ID NO: 75), and preferably the transcriptional activation domain of the transcription activator or transcription factor comprises or consists of the nucleotide sequences described in SEQ ID NOs: 73, 74, and 75.

[0091] Cas9 (SEQ ID NO: 92) may be cleaved at the E573 or V713 position for cleavage intein-mediated protein transsplicing. However, any other position for cleavage may also be considered in the context of any method of the present invention. Thus, in one embodiment of the transcriptional activation method, the undenatured or recombinant DNA-binding protein is Cas9 (SEQ ID NO: 92), and the cleavage nucleotide sequence, consisting of the nucleic acid sequence of at least one transcriptional activation domain of a transcription activator or transcription factor and the nucleic acid sequence of Cas9, is cleaved into one of the dCas9-enzymes at the E573 or V713 position of dCas9, preferably by SEQ ID NO: 96 or SEQ ID NO: 97.

[0092] In one embodiment of the transcriptional activation method according to the present invention, the undenatured or recombinant DNA-binding protein is a Cas enzyme, preferably Cas9 (SEQ ID NO: 92), dCas9-enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), or Cas12b (SEQ ID NO: 94), and the complex further comprises at least one guide RNA, which can bind to the promoter region of a homologous gene of at least one target gene or to other elements that regulate the expression of a homologous gene of at least one target gene. More preferably, the undenatured DNA-binding protein is the Cas9 enzyme of Streptococcus pyogenes (SEQ ID NO: 92). More preferably, the recombinant DNA-binding protein is selected from the group consisting of dCas9 having mutations D10A and H840A according to SEQ ID NO: 96, and dCas9 having mutations D10A, D839A, H840A, and N863A according to SEQ ID NO: 97. However, in principle, all Cas enzymes from any known organism can be used in this method of the present invention.

[0093] In one embodiment of the transcriptional activation method according to the present invention, the guide RNA includes or consists of the nucleotide sequences described in SEQ ID NOs. 76 to 88. In a further embodiment of the transcriptional activation method according to the present invention, at least one guide RNA has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more guide RNAs.

[0094] In one embodiment of the transcriptional activation method, the DNA-binding protein is C-terminally or N-terminally fused to at least one transcriptional activation domain of a transcription activator or transcription factor. In one embodiment of the transcriptional activation method of the present invention, the DNA-binding protein is N-terminally fused to at least one transcriptional activation domain of a transcription activator or transcription factor. In one embodiment of the transcriptional activation method, the DNA-binding protein is C-terminally fused to at least one transcriptional activation domain of a transcription activator or transcription factor.

[0095] In one embodiment of the transcriptional activation method according to the present invention, the transcription activator or at least one transcriptional activation domain of the transcription factor includes or consists of VPR (SEQ ID NO: 89), preferably at least one transcriptional activation domain of the transcription activator is the transcriptional activation domains VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), and Rta (SEQ ID NO: 75), and more preferably at least one transcriptional activation domain of the transcription activator includes or consists of the amino acid sequences described in SEQ ID NOs: 73, 74, and 75.

[0096] In one embodiment of the transcriptional activation method according to the present invention, the nucleotide sequences of an undenatured or recombinant DNA-binding protein and at least one transcriptional activation domain of a transcription activator or transcription factor are present in two separate plasmids and / or vectors.

[0097] In one embodiment of the transcriptional activation method according to the present invention, the coding sequence of at least one target gene has a size of at least 0.5 kb, preferably at least 5 kb.

[0098] In one embodiment of the transcriptional activation method according to the present invention, the method further comprises using a recombinant AAV vector of natural or artificial origin, preferably an AAV vector variant having retinal cell-specific targeting and enhanced retinal transduction efficiency. Compared to classical rAAV-mediated gene supplementation, the dCas9-VPR-mediated gene transcriptional activation method offers several significant advantages: i) Transcriptional activation can activate homologous genes regardless of size, enabling the development of therapies for diseases caused by mutations in very large genes (violating the genome size limitations of AAV); ii) Activation of endogenous gene promoters enables near physiological gene expression, eliminating the excessively strong and harmful overexpression that can occur with commonly used rAAV vectors equipped with strong promoters and intron-less cDNA; iii) Multiple genes can be efficiently activated simultaneously, potentially useful in treating bigenic and polygenic diseases; and iv) It enables the development of mutation-independent therapies with broader applicability (in contrast to time-consuming and sophisticated mutation-dependent gene editing techniques (personalized medicine)).

[0099] The method described herein for transcriptionally activating homologous genes of at least one target gene and optionally inactivating at least one target gene (in which the mRNA encoded by the target gene contains mutations compared to a control) can be performed in vitro as well as in vivo in cell culture, and is preferably preferred for therapeutic use in vivo. Therefore, in certain embodiments, the method relates to a method for treating a patient in need, comprising transcriptionally activating homologous genes of at least one target gene and optionally inactivating at least one target gene (for example, in which the mRNA encoded by at least one target gene contains mutations compared to a control). Furthermore, the method includes a step of binding a complex comprising an undenatured or recombinant DNA-binding protein, a transcription activator or at least one transcriptional activation domain of a transcription factor, and at least one guide RNA, wherein at least one guide RNA binds to the promoter region of a homologous gene of at least one target gene, or to other elements that regulate the expression of mRNA encoded by a homologous gene of at least one target gene, and optionally, further guide RNAs bind to coding regions, promoter regions, and / or other elements that regulate the expression of mRNA encoded by at least one target gene, and the at least one target gene is selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes. Furthermore, the method includes a step of inducing the expression of mRNA encoded by a homologous gene of at least one target gene (a step of transcriptionally activating at least one target gene), and optionally, a step of inactivating the expression of mRNA encoded by at least one target gene. Patients requiring this treatment may be those with hereditary retinal dystrophy (IRD), preferably those in which the IRD is caused by a mutation in at least one target gene selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes.The complex used in the therapeutic method of the present invention may be defined as described herein in the context of the method of the present invention.

[0100] The present invention further provides a complex comprising an unmodified or recombinant DNA-binding protein, at least one transcriptional activation domain of a transcription activator or transcription factor, and at least one guide RNA, for use in a method for treating hereditary retinal dystrophy (IRD) caused by a mutation in at least one target gene selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes. The method comprises transcriptional activation of a homolog of at least one target gene, optionally deactivation of at least one target gene (e.g., the mRNA encoded by at least one target gene contains a mutation in comparison to a control), at least one guide RNA binding to the promoter region of the homolog of at least one target gene or other elements that modulate the expression of the mRNA encoded by the homolog of at least one target gene, and optionally further guide RNA binding to the coding region, promoter region and / or other elements that modulate the expression of the mRNA encoded by at least one target gene. Expression of mRNA encoded by at least one homologous gene of the target gene is induced, and, if necessary, expression of mRNA encoded by at least one target gene is inactivated. The complex used is defined herein as in the context of the method of the present invention.

[0101] Specifically, in certain embodiments, the undenatured or recombinant DNA-binding protein is selected from the group consisting of Cas-enzymes (preferably Cas9 (SEQ ID NO: 92), dCas9-enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), or Cas12b (SEQ ID NO: 94)), zinc finger nucleases, and transcription activator-like nucleases, and / or at least one transcriptional activation domain of the transcription activator or transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75), and combinations thereof. Preferably, the undenatured or recombinant DNA-binding protein and at least one transcriptional activation domain and at least one guide RNA of the transcription activator or transcription factor are provided as nucleotide sequences, more preferably the undenatured or recombinant DNA-binding protein and at least one transcriptional activation domain of the transcription activator or transcription factor are separated into two split fragments. In certain embodiments, the nucleotide sequences of an undenatured or recombinant DNA-binding protein and at least one transcriptional activation domain of a transcription activator or transcription factor are located in two separate plasmids and / or vectors. In certain embodiments, the complex used in the present invention involves the use of a recombinant AAV vector. The AAV vector may be of natural or genetically engineered origin, and preferably, the AAV vector is an AAV vector variant having retinal cell-type targeting and / or enhanced retinal delivery efficiency. Thus, provided in certain embodiments are nucleotide sequences of an undenatured or recombinant DNA-binding protein, at least one transcriptional activation domain of a transcription activator or transcription factor, and at least one guide RNA, used in a method for treating hereditary retinal dystrophy (IRD) resulting from a mutation in at least one target gene selected from the group consisting of opsin genes, cyclic nucleotide-sensitive channel (CNG) genes, retina-specific ATP-binding cassette transporter (ABC transporter) genes, and myosin genes according to the present invention.Preferably, the nucleotide sequences of an undenatured or recombinant DNA-binding protein and a transcription activator or transcription factor, or at least one transcription activation domain of the transcription factor, are located in two separate plasmids and / or vectors. In certain embodiments, the two separate vectors are recombinant AAV vectors. The AAV vectors may be of natural or genetically engineered origin and are preferably AAV vector variants having retinal cell-type targeting and / or enhanced retinal transduction efficiency.

[0102] The present invention further relates to a method for diagnosing diseases in vitro. Here, rather than homologous genes, genes that may cause a disease, or genes that may be associated with a disease, are transcriptionally activated. The usefulness of this method becomes apparent when, although gene sequencing is theoretically possible, it can be substituted with less expensive methods such as PCR or Western blotting to search for mutations at the mRNA or protein level rather than the genome level. This is particularly effective when analyzing mRNA and proteins expressed in cells that are not routinely accessible, for example, when sample collection from retinal or brain tissue is required. By applying the method described herein, mRNA and / or proteins of genes expressed in cells and tissues that are rarely obtainable from patients can be analyzed without invasively extracting tissue samples such as retina or brain tissue.

[0103] Accordingly, the present invention further relates to a method for diagnosing a disease in vitro, the method comprising: a) inducing the expression of mRNA encoded by at least one target gene in a cell or tissue sample obtained from a subject; b) isolating the mRNA expressed in step a); c) analyzing the sequence of the mRNA isolated in step b); and d) detecting mRNA mutations that suggest the presence of a disease compared to a control. The term “mRNA mutation” as used herein includes not only mutations in the mRNA sequence (nucleotide deletions, insertions, and / or substitutions), but also modifications of mRNA such as changes in the splice pattern (also called splice mutations), decreased mRNA stability, and / or decreased expression (compared to a control). Typically, mRNA modifications result from mutations in the target gene, and the mutations may be in coding regions or non-coding regions such as promoters, activation regions, and / or introns (e.g., generating, modifying, or removing splice donor or splice receiver sites). Preferably, the mutations are coding region mutations or splice mutations. In certain embodiments, the mutations and / or modifications are caused by disease-causing mutations.

[0104] In one embodiment of the method for diagnosing a disease in vitro according to the present invention, the method further includes inducing the expression of an mRNA-encoded protein and analyzing the sequence, expression level, localization, or function of at least one mRNA-encoded protein in a cell or tissue sample.

[0105] In one embodiment of the method for diagnosing a disease in vitro according to the present invention, step a) includes specifically binding a complex comprising an undenatured or genetically modified DNA-binding protein and a transcription activator or at least one transcription activation domain of a transcription factor to the promoter region of at least one target gene or other element that modulates the expression of at least one target gene.

[0106] In one embodiment of the method for diagnosing diseases in vitro according to the present invention, the undenatured or recombinant DNA-binding protein is selected from the group consisting of Cas enzymes (preferably Cas9 (SEQ ID NO: 92), dCas9 enzymes (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93) or Cas12b (SEQ ID NO: 94), zinc finger nucleases (ZFNs), and transcription activator-like nucleases (TALENs). More preferably, the undenatured DNA-binding protein is the Cas9 enzyme of Streptococcus pyogenes (SEQ ID NO: 92). More preferably, the recombinant DNA-binding protein is selected from the group consisting of dCas9 having mutations D10A and H840A according to SEQ ID NO: 96, and dCas9 having mutations D10A, D839A, H840A and N863A according to SEQ ID NO: 97. However, in principle, all Cas enzymes from any known organism can be used in this method of the present invention.

[0107] In one embodiment of the method for diagnosing a disease in vitro according to the present invention, the undenatured or recombinant DNA-binding protein is a Cas enzyme, preferably Cas9 (SEQ ID NO: 92), dCas9-enzyme (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), or Cas12b (SEQ ID NO: 94), and the complex further comprises at least one guide RNA, which can bind to the promoter region of at least one target gene or to other elements that regulate the expression of at least one target gene.

[0108] In one embodiment of the method for diagnosing a disease in vitro according to the present invention, the DNA-binding protein is fused at its C-terminus or N-terminus to at least one transcriptional activation domain of a transcription activator or transcription factor, preferably the at least one transcriptional activation domain of the transcription activator or transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75), and combinations thereof.

[0109] In one embodiment of the method for diagnosing diseases in vitro according to the present invention, diseases suitable for this diagnostic method include neurodegenerative diseases, epilepsy, mental disorders (especially depression, mania, bipolar disorder, schizophrenia, or autism), or retinal diseases, particularly hereditary retinal dystrophy, among which hereditary retinal dystrophy is selected from the group consisting of age-related macular degeneration (AMD), hereditary age-related macular degeneration (AMD), autosomal dominant, autosomal recessive, X-linked or bihereditary retinitis pigmentosa, color vision deficiency, Stargart disease, retinal dystofy, Best disease, Leber congenital amaurosis, retinal aneurysm, congenital stationary night blindness, choroidemia, early-onset retinal dystrophy, cone-rod-cone dystrophy, pattern dystofy, syndromic ciliary protozoa such as Usher syndrome, particularly Valde-Viddl syndrome, Hubert syndrome, Sr.-Loken syndrome, or Alström syndrome.

[0110] To perform a method for diagnosing a disease in vitro, cells or tissue samples obtained from a subject can be transfected or transtransported with undenatured or recombinant DNA-binding proteins, transcription activators, or at least one transcriptional activation domain of a transcription factor, and at least one guide RNA. Therefore, in one embodiment, the method further includes transfecting or transducing cells or tissue samples obtained from a subject.

[0111] The in vitro diagnostic method for disease according to the present invention can also be used to analyze the splice patterns of genes and / or proteins involved in the disease. Therefore, the diagnostic method for disease according to the present invention may further include a step of analyzing the differences between the splice pattern of at least one target gene and a control splice pattern to detect changes in the splice pattern of at least one target gene, where the altered splice pattern suggests the presence of disease. As used in the context of this invention, the term "splice pattern" means the complete result of the splicing process. Intron splicing occurs in all eukaryotes, but the splicing method employed and the frequency of splicing differ among organisms. Bacteria and archaea lack the spliceosome pathway, and splicing by self-splicing introns is rare. In single-celled eukaryotes, there is considerable variation in the frequency of splicing. The number of introns and the recognized splice sites may differ among mRNA transcripts of a single gene, leading to splice diversity and the phenomenon of alternative splicing. The latter leads to different splice patterns.

[0112] In one embodiment of the method for diagnosing a disease in vitro according to the present invention, the cell sample from the subject is a blood sample, saliva sample, urine sample, skin sample, or mucous membrane sample.

[0113] The present invention is also directed toward nucleic acid sequences comprising or consisting of any of the sequences specified by SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 123, which are used for the treatment or prevention of diseases.

[0114] Furthermore, the present invention is also directed to nucleic acids comprising, or consisting of, one nucleic acid sequence described in SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 123, used in any of the methods described herein.

[0115] The present invention also comprises any of the above nucleic acid sequences used for the treatment or prevention of a disease, the disease being a neurodegenerative disease, epilepsy, a mental disorder (preferably depression, mania, bipolar disorder, schizophrenia, or autism), or a retinal disease, preferably a hereditary retinal dystrophy, more preferably the hereditary retinal dystrophy being selected from the group consisting of age-related macular degeneration (AMD), genetically caused age-related macular degeneration (AMD), autosomal dominant, autosomal recessive, X-linked, or bihereditary retinitis pigmentosa, color blindness, Stargart disease, Best disease, Leber congenital amaurosis, retinopathy, congenital static night blindness, choroidopathy, early-onset retinal dystrophy, cone-rod-cone dystrophy, pattern dystofy, symptomatic cilioid disorders such as Usher syndrome, and even more preferably Valde-Biedl syndrome, Hubert syndrome, retinitis pigmentosa (Sennis-Loken syndrome), or Alström syndrome.

[0116] As a result, the method of the present invention offers several major advantages, including: i) its simplicity makes it suitable for routine diagnostics; ii) it can be used to detect novel nucleotide variants in known genes; iii) it can be used to reclassify known disease variants in pathogenic genes; iv) it can be used to verify (or dispute) the proposed pathogenicity of detected mutations; and v) it is applicable to any genetic disease.

[0117] Diseases include, for example, neurodegenerative diseases, epilepsy, mental disorders (especially depression, mania, bipolar disorder, schizophrenia, or autism), or retinal diseases (especially hereditary retinal dystrophy), among which hereditary retinal dystrophy is selected from the group consisting of age-related macular degeneration (AMD), genetically caused age-related macular degeneration (AMD), autosomal dominant, autosomal recessive, X-linked or bihereditary retinitis pigmentosa, color blindness, Stargart disease, Best disease, Leber congenital amaurosis, retinal aneurysm, congenital stationary night blindness, choroidemia, early-onset retinal dystrophy, cone-rod-cone dystrophy, pattern dystofy, syndromic ciliosis such as Usher syndrome, and more specifically, Valde-Biedl syndrome, Hubert syndrome, Sr.-Loken syndrome, or Alström syndrome.

[0118] Various sequence-based sequence comparison methods well known to those skilled in the art can be used to determine the identity between sequences. These include, but are not limited to, the use of the local identity / homology algorithm of Smith, F. and Waterman, MS (1981) Adv. Appl.Math. 2: 482-89, the homology sequence comparison algorithm of Peason, WR and Lipman, DJ (1988) Proc. Natl.Acad. Sci. USA 85: 2444-48, the Basic Local Alignment Search Tool (BLAST) described in Altschul, SF et al. (1990) J. Mol. Biol. 215 403-10, or the Best Fit program described in Devereau, J. et al. (1984) Nucleic Acids. Res. 12: 387-95, and the FastA and TFASTA sequence comparison programs, preferably using initial value settings or by examination. Alternatively, amino acid sequences can be compared manually / visually as follows: The percentage of identity between the amino acid sequence in question (inquiry sequence) and the amino acid sequence of the present invention disclosed in the sequence list (reference sequence), as defined herein, is determined by pairwise sequence comparison to obtain the greatest possible identity between both amino acid sequences. The percentage of identity is obtained by counting the identical amino acid residues between the two amino acid sequences and dividing by the total number of residues in the reference sequence (including positions that do not contain amino acid residues, e.g., one or more gaps).

[0119] Where used herein, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. Therefore, for example, "reagent" includes one or more such different reagents, and "method" also includes equivalent steps and methods known to those skilled in the art that may modify or substitute the methods described herein.

[0120] Unless otherwise specified, the term “at least” preceding a set of elements refers to all elements in that set. The term “at least one” refers to one or more such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, unless otherwise specifically defined. Those skilled in the art will recognize, or confirm in the course of routine experimentation, many equivalents to the particular embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0121] As used herein, the terms "and / or" include "and," "or," and "all or any combination of the elements connected by the term."

[0122] The terms "less than" and, conversely, "greater than" do not include specific numerical values.

[0123] For example, "less than 20" means less than the number shown. Similarly, "greater than" or "greater than" means greater than the number shown; for example, "greater than 80%" means greater than 80% of the number shown.

[0124] Throughout this specification and the subsequent claims, unless otherwise required by context, variations of the words “comprising” and “including” shall mean including the integer or process or group of integers or processes described, but not excluding other integers or processes or other groups of integers or processes. Where used herein, the term “comprising” may be replaced with “containing” or “including,” and may be replaced with the term “having.” Where used herein, “consisting of” shall exclude any element, process, or component not specified.

[0125] The term "including" means "to include but without limitation." The terms "including" and "to include but without limitation" are used interchangeably.

[0126] The term "approximately" means ±10%, preferably ±5%, more preferably ±2%, and most preferably ±1%.

[0127] Throughout this specification and in its claims, the singular form includes the plural form unless otherwise required by context. In particular, where the indefinite article is used, this specification intends the plural form as well as the singular form unless otherwise required by context.

[0128] The present invention is not limited to the specific methods, procedures, materials, reagents, substances, etc. described herein, and is naturally subject to change. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0129] All publications cited throughout this Specified Publication (including all patents, patent applications, scientific publications, and manuals), whether as stated above or below, are incorporated herein by reference in their entirety. Nothing in this Specified Publication shall be construed as admitting that the present invention does not have prior rights to such disclosure by prior art. To the extent that any material incorporated by reference conflicts with or is inconsistent with this Specified Publication, the provisions of this Specified Publication shall prevail over such material.

[0130] The contents of all documents and patent documents cited herein are incorporated in their entirety by reference.

[0131] A deeper understanding of the present invention and its advantages can be obtained from the following examples, which are provided for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way. [Examples]

[0132] The following examples illustrate the present invention, but are not intended to limit the scope of the invention.

[0133] Example 1: dCas9-VPR-mediated transcriptional activation for ophthalmic gene therapy

[0134] Transcriptional activation of Cnga1 in 661w cells expressing inducible full-length dCas9-VPR cassettes

[0135] The transcriptional activation efficiency of the mouse Cnga1 (SEQ ID NO: 13) gene was tested using dCas9-VPR in combination with three different gRNAs that bind to the promoter region of this gene. For Cnga1 (SEQ ID NO: 13) activation, 661w cells (al-Ubaidi et al., 1992) derived from immortalized mouse retinoblasts lacking Cnga1 (SEQ ID NO: 13) expression were used, expressing several cone-specific markers. In 661w cells stably expressing a doxycycline-inducible dCas9-VPR cassette (SEQ ID NO: 123) combined with Cnga1 gRNA (target sequences of Cnga1 gRNAs (SEQ ID NOs: 76-78) containing PAM sequences), Cnga1 signaling could be detected at both mRNA and protein levels. This was not observed at all in 661w control cells stably expressing the dCas9-VPR lacZ gRNA cassette (SEQ ID NO: 124) (Figure 3A-F). Furthermore, using patch-clamp recordings, 661w cells possessing the dCas9-VPR Cnga1 gRNA cassette (SEQ ID NO: 123) exhibited two important functional characteristics of Cnga1-specific currents: cGMP-dependent activation and Ca 2+ / Mg 2+ We were able to demonstrate that it inhibits dependence (Figure 3G-L).

[0136] Example 2: Reconstruction efficiency via Cas9 cleavage intein

[0137] As mentioned above, the dCas9-VPR cassette (SEQ ID NO: 123) exceeds the packaging capabilities of AAV vectors. To broaden the in vivo application range of the dCas9-VPR system, we tested the efficiency of cleavage-intine techniques to split dCas9-VPR into two different parts, reconstitute them, and deliver them to two separate plasmids. The reconstitution efficiency of cleavage-intine is known to depend on the corresponding splitting site within the protein. In recent studies, two independent research groups used cleavage-intine techniques to examine the nuclease activity of Cas9 split at either the aa position E573 (Truong et al., 2015) or V713 (Chew et al., 2016). Both groups showed that the nuclease activity of the split and reconstituted Cas9 remained unchanged in principle. However, there are no absolute or relative data on the reconstitution efficiency at the protein level of Cas9 split at these two positions. Therefore, in initial experiments using transiently transfected HEK293 cells, we quantified the reconstruction efficiency of Cas9 split intein fragments that crossed these two positions. As shown in Figure 4, the reconstruction efficiency of the Cas9 mutant split at the V713 position (56.9% ± 2.1%) was found to be considerably higher than that of the split at the E573 position (33.3% ± 2.1%).

[0138] Example 3: Transcriptional activation of Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 45) genes in transiently transfected 661w or MEF cells by dCas9-VPR and split dCas9-VPR.

[0139] The inventors also analyzed the transcriptional activation efficiency of the Cnga1 (SEQ ID NO: 13), Opn1mw (SEQ ID NO: 43), and Opn1sw (SEQ ID NO: 13) genes in cells transiently transfected with either full-length dCas9-VPR (SEQ ID NO: 95) or dCas9-VPR split at position V713 (referred to here as V713_dC9) in combination with their respective gRNAs (Figure 5). Mouse embryonic fibroblast (MEF) cells that did not express these genes in substantial amounts (in contrast to 661w cells) were used for the transcriptional activation of pyramidal opsins. Using full-length dCas9-VPR (SEQ ID NO: 95), the inventors observed efficient transcriptional activation of all three genes. Similarly, V713_dC9 and Cnga1 (target sequence of gRNA in Cnga1 containing the PAM sequence; SEQ ID NOs. 76-78) or Opn1mw (target sequence of gRNA containing the PAM sequence; SEQ ID NOs. 79-81) gRNAs were found to be able to transcribe and activate both genes, albeit less efficiently, compared to full-length dCas9 mutants. To date, the inventors have not included the combination of V713_dC9 and Opn1sw gRNA (target sequence of gRNA containing the PAM sequence; SEQ ID NOs. 83-85) in this in vitro setting. In all cases, transcriptional activation of each gene was not detected in cells expressing lacZ control gRNA (target sequence of lacZ gRNA containing the PAM sequence; SEQ ID NOs. 125).

[0140] Example 4: V713_dC9-mediated transcriptional activation of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) in rod photoreceptors

[0141] The inventors also analyzed whether V713_dC9 can transcribe and activate the Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) genes in rod photoreceptor cells of wild-type mice. For this purpose, the inventors injected mice with an AAV2 / 8 virus serotype containing a human rhodopsin promoter (Figure 6A) for specific expression in rods. Three weeks after injection, the retinas of the injected animals were used for immunolabeling or for qRT-PCR studies following RNA isolation.

[0142] Compared to cone photoreceptors, rod photoreceptors are present at a much higher density throughout the mouse retina. Furthermore, the outer segments of mouse rods are longer than those of cone photoreceptors. These characteristics allow for easy differentiation of the outer casings of rod and cone photoreceptors. We were able to detect a stable increase in signaling for Opn1mw (SEQ ID NO: 44) and Opn1sw (SEQ ID NO: 46) in more than 50% of injected retinas immunolabeled with specific antibodies. This signaling was spread throughout the photoreceptor outer segments surrounding the injection site and was characteristic of rod outer segment-specific proteins. Therefore, we concluded that the increase in Opn1mw (SEQ ID NO: 44) and Opn1sw (SEQ ID NO: 46) signaling is very likely to originate from V713_dC9-mediated transcriptional activation of the corresponding genes (Figure 6B-E).

[0143] Corresponding qRT-PCR experiments showed elevated mRNA levels of Opn1mw (SEQ ID NO: 43) and Opn1sw (SEQ ID NO: 45) in 50% (Opn1sw, Figure 6G) to 100% (Opn1mw, Figure 6F) of the injected retinas. This increase was lower compared to the corresponding experiment in MEF cells shown in Figure 5G. Nevertheless, this finding is rather expected, as both genes are endogenously highly expressed in the cones of injected mice (in contrast to MEF cells).

[0144] Example 5: Transcriptional activation of Opn1mw slows retinal degeneration and improves retinal function in heterozygous Rho mice.

[0145] The inventors also investigated whether Opn1mw transcriptional activation was sufficient to improve the retinitis pigmentosa phenotype in a heterozygous rhodopsin-deficient RP mouse model (Humphries et al., 1997). For this purpose, heterozygous (hz) Rho mice were subretinally injected with a titer-matched double rAAV vector expressing split dCas9-VPR and Opn1mw sgRNA (hz treatment). The other control eye was injected with NaCl (hz sham) solution (Figure 7).

[0146] Heterozygous Rho mice exhibit a slow progression of retinal degeneration (Humphries et al., 1997), so the effect of the treatment was evaluated one year after injection, and age-matched untreated WT mice were used as additional controls. Retinal degeneration is accompanied by a decrease in photoreceptor cells, a condition that can be addressed non-invasively by measuring the thickness of the outer nucleus (ONL) using optical coherence tomography (OCT). OCT recordings from eyes expressing fragmented dCas9-VPR and Opn1mw sgRNA showed increased ONL thickness compared to the other NaCl-injected eye, suggesting that this treatment can slow degeneration (Figure 7B).

[0147] To evaluate the beneficial effects of the method on rod-mediated (dark-field) retinal function, the inventors performed electroretinography (ERG) in dark-adapted heterozygous Rho mice (Figure 7A). A significant improvement in dark-field b-wave was observed when the treated eyes were compared to the corresponding eyes injected with NaCl. These data conclusively suggest that transcriptional activation of Opn1mw improves retinal degeneration and retinal function in heterozygous Rho RP mice.

[0148] Example 6: dCas9-VPR-mediated transcriptional activation for the diagnosis of genetic disorders

[0149] We focused on USH2A (SEQ ID NO: 49) for several reasons to provide proof of principle for CRISPR / Cas9-mediated transcriptional activation of a frequently occurring IRD-linked gene. Firstly, USH2A (SEQ ID NO: 49) is the most common autosomal recessive retinitis pigmentosa (arRP) and Usher syndrome (USH) gene (accounting for 10–15% of arRP cases and 30–40% of USH cases (Huang et al., 2018)). Secondly, the collaborating LMU Ophthalmology Hospital in Munich has a large cohort of USH2A (SEQ ID NO: 49) patients. In some of these patients, only one USH2A (SEQ ID NO: 49) mutation was identified, suggesting the presence of a second variant in a region not covered by routine genetic diagnostics. Thirdly, USH2A (SEQ ID NO: 49) is not expressed in tissues and / or cell types routinely obtained from patients (https: / / www.proteinatlas.org / ENSG00000042781-USH2A / tissue), hindering USH2A (SEQ ID NO: 49) mRNA analysis in cells from untreated patients. Fourthly, USH2A (SEQ ID NO: 49) belongs to the largest gene in the human genome, particularly hindering the identification of potential pathogenic mutations located in non-coding regions.

[0150] For experiments aimed at transcriptional activation of USH2A (SEQ ID NO: 49), human fibroblasts were isolated from a skin biopsy of one of the inventors. These cells were cultured according to a previously described standard procedure (Chen et al., 2014), and transient translocation was performed using dCas9-VPR (SEQ ID NO: 95) combined with three different USH2A gRNAs (targeting the undenatured USH2A promoter in human fibroblasts (target sequences of USH2A gRNA including PAM sequences (SEQ ID NO: 86-88))). A combination of dCas9 (SEQ ID NO: 96) targeting the undenatured USH2A promoter and lacZ-specific gRNA (target sequence of lacZ gRNA including PAM sequences: SEQ ID NO: 125) was used as a control.

[0151] USH2A (SEQ ID NO: 49) is located on the (-) strand of chromosome 1, q41. Another gene (KCTD3) (SEQ ID NO: 122) is located adjacent to USH2A (SEQ ID NO: 49) on the opposite (+) strand, and the two genes overlap in the distal portion of the 3' untranslated region (UTR) (Figure 8A). Transcriptional activation of USH2A (SEQ ID NO: 49) was analyzed at the RT-PCR and qRT-PCR levels using USH2A-specific primers (SEQ ID NOs: 98-121; see Figures 8B-D). For RT-PCR experiments, 12 primer pairs (SEQ ID NOs: 98-121) covering the entire USH2A (SEQ ID NO: 49) transcript were designed. The size of individual PCR products was between 1.5 and 1.8 kb, allowing for convenient mRNA-level analysis and detection of potential splice mutations from patient cells. In cells transfected with dCas9-VPR (SEQ ID NO: 95) combined with USH2A gRNA (target sequences of USH2A gRNA including the PAM sequence; SEQ ID NOs: 86-88), all primer pairs (SEQ ID NOs: 98-121) led to specific bands of the expected size. The identity of each band was confirmed by Sanger sequencing. Except for the last primer pair covering the distal 3'UTR region, no bands were detected in fibroblasts transfected with lacZ control gRNA (target sequences of lacZ gRNA including the PAM sequence; SEQ ID NO: 125). As expected, Sanger sequencing of the 3'UTR band in lacZ control cells confirmed its origin to the KCDT3 gene (SEQ ID NO: 122), which overlaps with USH2A (SEQ ID NO: 49) in the distal 3'UTR.

[0152] Example 7: Transcriptional activation of Opn1mw reduces apoptosis in heterozygous Rho mice without inducing gliosis or infiltration of immune-responsive cells.

[0153] To evaluate the translational capacity of this method, we investigated whether our treatment induced persistent gliosis or an immune response, accompanied by proliferation of glial fibrillation acid protein (GFAP)-positive Müller glial cells or ionized calcium-binding adapter molecule 1 (Iba-1)-positive microglia or mononuclear cells in the retina. Importantly, when the retina was immunolabeled with these markers, we found no significant increase in the number of glial cells, microglia, or mononuclear cells between the different groups, in contrast to the retina of rd1 (retinal degeneration 1) mice, which showed rapid retinal degeneration peaking at P13 (J. Sancho-Pelluz et al., Mol Neurobiol 38, 253-269 (2008)) (Figure 9C-H). In the heterozygous Rho mouse model, we performed the TUNEL test on retinal sections of treated heterozygous Rho mice to investigate whether photoreceptor degeneration is caused by apoptosis (Figure 10A, B). To detect apoptosis, we performed a dUTP-biotin nickel-end labeling (TUNEL) assay via terminal deoxyribonucleotide transferase using the In Situ Cell Death Detection Kit, Fluorescein (11684795910; Roche), following the manufacturer's instructions. This assay detected a low but significant number of TUNEL-positive cells, indicating that apoptosis is the underlying mechanism of photoreceptor loss in this mouse model. Furthermore, by comparing the number of TUNEL-positive cells per unit area in the treated and untreated retina, we demonstrated that transcriptional activation of Opn1mw reduces apoptosis (Figure 10C). These data further highlight the beneficial effects of our treatment on photoreceptor survival.

[0154] Example 8: gRNA multiplexing method for simultaneous Rho knockdown and Opn1mw activation

[0155] Transcriptional activation of homologous genes via dCas9-VPR makes it possible to treat loss-of-function mutations in which the deficiency of the protein encoded by the target gene causes disease. However, many genetic diseases develop when the target gene produces harmful proteins due to gain-of-function mutations or dominant inhibitory mutations. To successfully treat such mutations, it is necessary not only to compensate for the deficient functional protein but also to simultaneously remove the mutated harmful protein. To verify the applicability of the above method to this purpose, the inventors used a combination of a catalytically active Cas9-VPR and a gRNA containing a protospacer (PS) of 16 bp or more that retains Cas9 catalytic activity to knock down the mouse rhodopsin gene (RHO) (sgRho target sequence including PAM sequence: SEQ ID NO: 82). Furthermore, by employing two or more gRNAs containing short protospacer sequences (<16 bp) that suppress the catalytic activity of the Cas9 protein, we activated this rhodopsin homolog by targeting the promoter of the mouse M opsin gene (OPN1MW) (target sequence of sgOpn1mw_1_short chain: ggggcctttaaggtaagg, SEQ ID NO: 126 (including PAM sequence) and sgOpn1mw_2_short chain: gccacccctgtggattgg, SEQ ID NO: 127 (including PAM sequence)) (Fig. 11A).

[0156] To test this method in vivo, the Cas9-VPR encoding sequence needs to be split into two parts, delivered via two separate rAAV vectors, and reconstituted in target cells, i.e., photoreceptor cells. However, efficient reconstitution of Cas9-VPR is a crucial factor for effective therapy. Therefore, in this experiment, we compared two reconstitution methods: the cleavage intein method (Figure 11B), which enables reconstitution at the protein level, and the mRNA trans-splicing (REVeRT) method (Figure 11C), which enables reconstitution at the RNA level.

[0157] For this experiment, 2-month-old C57BL / 6J wild-type mice were injected with AAV containing a split Cas9-VPR construct in combination with two Opn1mw-targeted gRNAs and one Rho-targeted gRNA (multiplexing method), or in combination with one single lacZ-targeted control gRNA (Figures 11B and 11C). Four weeks after injection, RNA was extracted from the retina and analyzed by qRT-PCR. The results showed that Cas9-VPR was successfully reconstituted at high levels via REVeRT (Figure 11D). Reconstitution via cleavage intein could not be evaluated because it occurs after translation into protein. Furthermore, we were able to demonstrate efficient Rho knockdown and Opn1mw activation regardless of the reconstitution method employed (Figures 11E and 11F). These results highlight the broad applicability of the described invention and demonstrate its suitability for treating diseases resulting from gain-of-function and dominant inhibition.

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Claims

1. A pharmaceutical composition for use in a method of transcriptionally activating a homologous gene of at least one target gene, wherein the mRNA encoded by at least one target gene comprises mutations compared to a control, wherein the pharmaceutical composition comprises an unmodified or recombinant DNA-binding protein, a transcription activator or at least one transcriptional activation domain of a transcription factor, and at least one guide RNA, and wherein the method is -below: Unmodified or recombinant DNA-binding protein, where said unmodified or recombinant DNA-binding protein is a Cas-enzyme. The transcription activator or at least one transcription activation domain of the transcription factor fused with the unmodified or recombinant DNA-binding protein, and At least one guide RNA, A step of forming a complex comprising, where the at least one guide RNA binds to the promoter region of the homologous gene of the at least one target gene, or to another element that regulates the expression of mRNA encoded by the homologous gene of the at least one target gene, and The at least one target gene is selected from the group consisting of an opsin gene, a cyclic nucleotide-sensitive channel (CNG) gene, a retina-specific ATP-binding cassette transporter (ABC transporter) gene, and a myosin gene, and The homologous gene of at least one target gene is selected from the group consisting of ABCA1 (SEQ ID NO: 1), ABCA2 (SEQ ID NO: 3), ABCA7 (SEQ ID NO: 7), ABCA12 (SEQ ID NO: 9), ABCA13 (SEQ ID NO: 11), CNGA1 (SEQ ID NO: 13), CNGA2 (SEQ ID NO: 15), CNGA3 (SEQ ID NO: 17), CNGA4 (SEQ ID NO: 19), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), MYO7B (SEQ ID NO: 33), MYO5A (SEQ ID NO: 25), MYO5B (SEQ ID NO: 27), MYO5C (SEQ ID NO: 29), MYO10 (SEQ ID NO: 35), MYO15B (SEQ ID NO: 39), MYO15A (SEQ ID NO: 37), OPN1LW (SEQ ID NO: 41), OPN1MW (SEQ ID NO: 43), and OPN1SW (SEQ ID NO: 45); - A step of inducing the expression of the mRNA encoded by a homologous gene of at least one target gene; and - The step of thereby activating the transcription of at least one target gene; A pharmaceutical composition containing the above.

2. The pharmaceutical composition according to claim 1, wherein the method further comprises the steps of inducing the expression of the protein encoded by the mRNA of a homologous gene of the at least one target gene, and analyzing the sequence, expression level, localization, or function of the at least one protein encoded by the mRNA.

3. The pharmaceutical composition according to claim 1 or 2, wherein the homologous gene of the at least one target gene is selected from the group consisting of ABCA1 (SEQ ID NO: 1), CNGA1 (SEQ ID NO: 13), CNGA3 (SEQ ID NO: 17), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), MYO7B (SEQ ID NO: 33), OPN1LW (SEQ ID NO: 41), OPN1MW (SEQ ID NO: 43), and OPN1SW (SEQ ID NO: 45).

4. The transcription activator or at least one transcription activation domain of the transcription factor is selected from the group consisting of VPR (SEQ ID NO: 89), SAM (SEQ ID NO: 90), SunTag (SEQ ID NO: 91), VP64 (SEQ ID NO: 73), p65 (SEQ ID NO: 74), Rta (SEQ ID NO: 75), and combinations thereof. A pharmaceutical composition according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, wherein the nucleotide sequences of the unmodified or recombinant DNA-binding protein and the transcription activator or at least one transcription activation domain of the transcription factor are located in two separate plasmids and / or vectors.

6. A pharmaceutical composition according to any one of claims 1 to 5, further comprising the use of a recombinant AAV vector of natural or artificial origin.

7. A complex for use in a method of treating hereditary retinal dystrophy (IRD) caused by a mutation in at least one target gene selected from the group consisting of an opsin gene, a cyclic nucleotide-sensitive channel (CNG) gene, a retina-specific ATP-binding cassette transporter (ABC transporter) gene, and a myosin gene, wherein the complex is Cas-enzymes as unmodified or recombinant DNA-binding proteins fused with a transcription activator or at least one transcription activation domain of a transcription factor, and At least one guide RNA, Includes, The aforementioned method, A step of transcriptionally activating a homologous gene of at least one target gene, Includes, The at least one guide RNA binds to the promoter region of the homologous gene of the at least one target gene or to other elements that regulate the expression of the mRNA encoded by the homologous gene of the at least one target gene. Furthermore, the expression of the mRNA encoded by the homologous gene of at least one target gene is induced. The homologous gene of at least one target gene is selected from the group consisting of ABCA1 (SEQ ID NO: 1), ABCA2 (SEQ ID NO: 3), ABCA7 (SEQ ID NO: 7), ABCA12 (SEQ ID NO: 9), ABCA13 (SEQ ID NO: 11), CNGA1 (SEQ ID NO: 13), CNGA2 (SEQ ID NO: 15), CNGA3 (SEQ ID NO: 17), CNGA4 (SEQ ID NO: 19), CNGB1 (SEQ ID NO: 21), CNGB3 (SEQ ID NO: 23), MYO7B (SEQ ID NO: 33), MYO5A (SEQ ID NO: 25), MYO5B (SEQ ID NO: 27), MYO5C (SEQ ID NO: 29), MYO10 (SEQ ID NO: 35), MYO15B (SEQ ID NO: 39), MYO15A (SEQ ID NO: 37), OPN1LW (SEQ ID NO: 41), OPN1MW (SEQ ID NO: 43), and OPN1SW (SEQ ID NO: 45). A complex.

8. The aforementioned composite is The aforementioned unmodified or genetically modified DNA-binding proteins, A transcription activator or the at least one transcription activation domain of a transcription factor, and The at least one guide RNA The complex according to claim 7, provided as a nucleotide sequence.

9. The complex according to claim 7 or 8, wherein the nucleotide sequences of the unmodified or recombinant DNA-binding protein and the transcription activator or at least one transcription activation domain of the transcription factor are located in two separate plasmids and / or vectors.

10. The complex according to claim 9, wherein the two separate vectors are recombinant AAV vectors.

11. The pharmaceutical composition according to claim 1, wherein the Cas-enzyme is selected from the group consisting of Cas9 (SEQ ID NO: 92), dCas9-enzyme (SEQ ID NO: 96, SEQ ID NO: 97), Cas12a (SEQ ID NO: 93), and Cas12b (SEQ ID NO: 94).

12. The pharmaceutical composition according to claim 1, wherein the nucleotide sequences of the undenatured or recombinant DNA-binding protein and the transcription activator or the at least one transcription activation domain of the transcription factor are separated into two fragments.

13. The pharmaceutical composition according to claim 6, wherein the method comprises the use of an AAV vector variant having retinal cell-type directivity and enhanced retinal delivery efficiency.

14. The method comprises the following steps: The step of inactivating the at least one target gene, wherein the complex includes a further guide RNA that binds to the coding region, the promoter region, and / or other elements that regulate the expression of the mRNA encoded by the at least one target gene; and The process of thereby inactivating the expression of mRNA encoded by at least one target gene; The pharmaceutical composition according to claim 1, further comprising:

15. The method comprises the following steps: The step of inactivating the at least one target gene, wherein the complex includes a further guide RNA that binds to the coding region, the promoter region, and / or other elements that regulate the expression of the mRNA encoded by the at least one target gene; and The process of thereby inactivating the expression of mRNA encoded by at least one target gene; The composite according to claim 7, further comprising:

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