Genomic editing of KOZAK sequences for treatment of disease

Abstract

The present invention relates to the medical field of monogenic diseases caused by loss or acquisition of allele function. The innovative method is developed on the basis of editing the human genome at the Kozak sequence level by means of CRISPR-Cas programmable nuclease. In particular, the present invention relates to variant Kozak sequences and related in vitro or in vivo methods of obtaining the variant Kozak sequences for the treatment of monogenic diseases caused by loss or acquisition of monoalleles. These in vitro and in vivo methods include CRISPR-Cas homologous mediated repair, CRISPR-Cas pilot editing, CRISPR-Cas base editing or genome editing with the use of other programmable RNA-directed nuclease, and the introduction of specific nucleotide transformations in the Kozak sequence of the disease virulence gene. These nucleotide transformations enhance or inhibit the translation of the gene produced mRNA, compensating for the loss of function or acquisition of an allele in the disease.

Description

Technical Field

[0001] The present invention relates to the medical field of monogenic diseases caused by loss or gain of allele function. The innovative method is developed based on editing the human genome at the Kozak sequence level by CRISPR-Cas programmable nucleases. In particular, the present invention relates to variant Kozak sequences and related in vitro or in vivo methods for obtaining the variant Kozak sequences for the treatment of monogenic diseases caused by loss or gain of monoallelic genes. These in vitro and in vivo methods include CRISPR-Cas homology-mediated repair, CRISPR-Cas lead editing, CRISPR-Cas base editing, or genome editing with other programmable RNA-guided nucleases, and the introduction of specific nucleotide conversions in the Kozak sequence of disease-causing genes. These nucleotide conversions enhance or inhibit the translation of mRNA produced by the gene, compensating for the loss or gain of function of one allele in the disease. Background Art

[0002] There are hundreds of diseases caused by loss or gain of function of a single allele. In particular, haploinsufficiency (HI) is a genetic disease in which a mutational inactivation of a single allele results in reduced protein levels and is sufficient to produce a disease phenotype. More than 300 human diseases, ranging from cancer susceptibility to developmental and neurological monogenic disorders, are caused by haploinsufficiency. Gene duplications (GDs) are gains of a single allele that result in increased protein levels and produce a disease phenotype. Gene duplications are responsible for dozens of human diseases.

[0003] The Kozak consensus sequence was first defined in the 1980s as the optimal nucleotide context around protein start codons. It is now widely accepted that the Kozak sequence plays an important role in translation regulation by confining specific interactions to the 48S initiation complex.

[0004] The impact of the Kozak sequence on disease is exemplified by hereditary and sporadic diseases, in which translation efficiency is affected by point mutations and allelic variations near the AUG start codon, respectively. For example, a C-to-T mutation (cl.C>T) in the Kozak sequence (located at position 1 of the AUG codon) of the α-tocopherol transfer protein gene reduces protein levels and leads to the monogenic disease AVED (vitamin E deficiency ataxia). As another example, the T / C polymorphism at position 1 of the CD40 gene (-1T>C, rs1883832) is associated with increased CD40 translation, thereby susceptibility to Graves' disease and coronary heart disease.

[0005] Despite the apparently strict pattern of the motif describing the Kozak consensus sequence, there is sequence variability around the AUG start codon in the human genome and other vertebrate genomes, and recent studies aimed at measuring the strength of different AUG start codons as a function of surrounding bases have found substantial differences. These results reveal the possibility that multiple genes are translationally controlled by suboptimal Kozak sequences.

[0006] Given the cited data and their major role in regulating translation, Kozak sequences are also potential targets for regulating gene expression. Summary of the invention

[0007] Applicants have now discovered a genome editing method that aims to rescue diseases caused by loss or gain of function of a single allele. This method can utilize any genome editing platform that can introduce single or multiple base transitions in short DNA fragments. Therefore, CRISPR-CAS homology-mediated repair, CRISPR-CAS lead editing, CRISPR-CAS base editing, or any other programmable RNA-guided nuclease fused to an effector protein that allows base transitions into the genome is suitable.

[0008] In one embodiment of the present invention, applicants focus on modifying the Kozak sequence with reference to the HI gene of interest to increase the translation efficiency of the wild-type allele. In other words, in this embodiment of the present invention, the process upregulates the production of proteins by altering the Kozak sequence for therapeutic purposes.

[0009] In particular, as an example of this approach, the applicant has now discovered specific nucleotide transitions in the Kozak sequence of selected HI genes for the treatment of HI diseases. These diseases are developmental disorders, metabolic syndrome, eye disorders and hematopoietic system diseases.

[0010] In another embodiment of the present invention, the applicant focuses on modifying the Kozak sequence with reference to the GD gene of interest to suppress the translation efficiency of the three alleles. In other words, in this embodiment of the present invention, the process downregulates the production of proteins by altering the Kozak sequence for therapeutic purposes.

[0011] Therefore, a first object of the present invention relates to the nucleotide sequence according to claim 1.

[0012] Advantageously, the present invention allows the use of Kozak sequences targeted by genome editing methods aimed at regulating translation efficiency, manipulating it to achieve therapeutic goals of upregulating or downregulating protein production. Other advantages of the method on which the present invention is based are as follows. First, genome editing methods can install permanent nucleotide transitions in genomic DNA; second, nucleotide transitions on Kozak sequences allow targeting HI genes by inducing small, controlled increases in translation efficiency, which is crucial because the ultimate goal is to obtain protein levels that are sufficient to rescue the HI phenotype but not enough to cause an imbalance and be incompatible with cell physiology; third, the action on cis sequences that universally control translation produces the desired effect on any cell type in the body, regardless of the variation in gene expression, and reduces noise in the system; fourth, several different Kozak variants can be tested for each gene, ensuring flexibility; finally, any mutant HI gene that causes disease can in principle be rescued in any patient, because the method does not rely on the correction of a specific disease.

[0013] Other objects, features, preferred embodiments and advantages of the present invention are described below.The scope of the present invention is defined in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Enhancement of a suboptimal Kozak sequence by base editing is shown. A. Sanger sequencing chromatograms representing wild-type (EGFP-1C) and mutant (EGFP-1T) EGFP, the mutant having a variation at position 1 of the Kozak sequence. B. Western blot analysis of the expression of EGFP and mCherry in HEK293T cells transiently transfected with EGFP-1C or EGFP-IT plasmids. C. Representative FACS dot plots of HEK293T cells 3 days after transient transfection. D. FACS analysis of HEK293T cells transiently transfected with the corresponding plasmids. Data were normalized to EGFP-1C and presented as mean ± SD of n = 3 biological replicates. Statistically significant differences were calculated by unpaired t-test. E. Representative Sanger sequencing chromatograms of HEK293T cells edited with the ABE7.10 base editor and sg-1, compared with ABE7.10 combined with a scrambled sgRNA (sgCTRL). F. Percentage of correct T-to-C conversions analyzed with EditR software. G. Western blot analysis of EGFP and mCherry expression in HEK293T cells edited with ABE 7.10 or ABEmax in combination with sg-1 or sgCTRL. H. Representative FACS dot plots of cells edited with ABE7.10 and sg-1 at day 3 post-transfection, compared with ABE7.10 combined with scrambled sgRNA (sgCTRL). I. FACS analysis of EGFP expression in cells transfected with base editors (ABE7.10 and ABEmax) and sgCTRL or sg-1. Data are mean ± SD of n = 3 biological replicates. Statistically significant differences were calculated by unpaired t-test (p value = 0.0483).

[0015] Figure 2 High-throughput assays involving protein levels from Kozak sequence variants. A. mCherry expression in transduced cells in the first round of FACS-seq sorting. 6mCherry-positive cells (23.1% of the total) were sorted. B. Second round of FACS-seq sorting. C. mCherry-positive cells from the gate drawn in B. were divided into 4 gates based on EGFP / mCherry expression, defined as each dish containing 25% of the total population of interest. D. Heat map representing the distribution of candidate HI genes and variants through statistical analysis. The upper figure shows the Kozak variant. The WT Kozak sequence of the HI gene is shown in the figure below. Each column corresponds to one of the four gates, while each row represents a Kozak variant. E. Logo representation of the Kozak sequence extracted from each of the four gates. In each figure, the position along the Kozak sequence (A of ATG is at position +1) is represented on the x-axis, and the probability of occurrence of each base is represented on the y-axis. Gate 1 (upper figure) represents the lowest translation efficiency, while Gate 4 (lower figure) represents the Kozak sequence with the highest probability of occurrence. The relevant positions (-3 and +5) are highlighted in yellow. F. Percentage of counts per million reads (CPM) in four gates for wild type (WT) and corresponding variants (Var) of five selected genes.

[0016] Figure 3 Validation of actionable hit variants. A. Wild-type (WT) and variant (Var) Kozak sequences of selected hit genes. B. Translation enhancement was analyzed as EGFP / mCherry expression by high-content image analysis. Violin plots report the data distribution of n=3 biological replicates. The dotted line represents the population median. C. Histograms represent the population mean analyzed by high-content image analysis. The data are the mean ± SD of n=3 biological replicates. The numbers represent the percentage increase of the variant over the WT mean. Statistically significant differences were calculated using an unpaired t-test for each variant versus the corresponding WT.

[0017] Figure 4 Validation of actionable hit variants involving translational inhibition of PMP22. A. Wild-type and variant Kozak sequences of the PMP22 gene. B. Translational inhibition was analyzed as EGFP / mCherry expression by high-content image analysis. Violin plots report data distributions for n=2, 3 biological replicates. Dashed lines represent population medians. C. Histograms represent population means analyzed by high-content image analysis. Data are mean ± SD of n=2, 3 biological replicates. Statistically significant differences were calculated using an unpaired t-test for each variant versus WT.

[0018] Figure 5Base editing of NCF1 is involved to replicate the desired variants. A. Schematic diagram of the NCF1 wild-type (WT), variant 2 (Var2), and variant 4 (Var 4) Kozak sequences. The start codon is bold blue; the base changes of the variants are highlighted in pink. B. Editing efficiency at target and bystander (red) guanines in the Raji bulk population was analyzed using EditR software 5 days after electroporation of AncBE4max and sgNCFl or sgCTRL. The corrected G-to-A conversion percentage (y-axis) is shown for each position in the NCF1 Kozak sequence (x-axis, A of ATG is +1 position). Data are mean ± SD of n = 3 independent experiments. C. Editing efficiency at target and bystander (red) guanines of two clones isolated from the bulk population (Var 2 and Var 4 cells). D. Sanger sequencing chromatograms of the NCF1 Kozak sequence in Raji WT, Var 2, and Var 4 cells. E. p47 in Raji cells (WT, Var 2 and Var 4) phox Western blot analysis of proteins. A representative blot result is shown. The arrow indicates the protein corresponding to p47 phox F. Western blot quantification. p47 phox Levels were normalized to housekeeping proteins and fold changes relative to WT levels are shown, n=3 biological replicates. G. qPCR of NCF1 was performed on WT, Var 2 or Var4Raji cells. Data are mean ± SD of n=3 independent experiments. H. Representative western blot of two polysomal markers (RPS6 and RPL26) in fractions separated by sucrose gradient centrifugation. Input is cell cytoplasmic lysate loaded on sucrose gradient. tot = fraction corresponding to total RNA; pol = fraction selected as polysomes and used for I. I. Quantitative analysis of translation efficiency (TE) of NCF1 in Var 2 and Var 4 cells compared with WT cells. TE refers to the ratio of polysomal (fractions 8-9) to total (fractions 4-9) mRNA levels measured by qPCR (polysomal fold change / total fold change). Data are mean ± SD of n=3 independent experiments. Statistically significant differences were calculated by unpaired t-test for each variant versus WT. DETAILED DESCRIPTION

[0019] To the scope of the present invention, some terms and expressions used in this specification and the appended claims are as follows.

[0020] As used herein, the term "CRISPR" refers to a system or locus of regularly interspaced clustered short palindromic repeats, a bacterial adaptive immune system that prevents invasion of dynamic genetic elements. The term "Cas" (CRISPR-associated protein) refers to a programmable endonuclease guided by RNA that recognizes a protospacer adjacent motif (PAM sequence) and cuts invading nucleic acids in a region complementary to the sequence encoded by the spacer, which is encoded in the CRISPR array. The term "Cas9n" refers to a partially inactive Cas9 endonuclease. References: Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, JA, & Charpentier, E. (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science, 337 (6096), 816-821.

[0021] As used herein, "base editor" refers to the fusion of a deaminase to Cas9n in a tool capable of making a single base conversion. There are three types of base editors: cytosine base editors (CBEs) that can convert a CG to a TA base pair and adenine base editors (ABEs) that can make a base pair substitution from AT to GC, and adenine to cytosine base editors that can make a substitution from AT to CG. References: Komor, AC, Kim, YB, Packer, MS, Zuris, JA, & Liu, DR (2016). "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage". Nature, 533(7603), 420-424. Chen L, Hong M, Luan C, Gao H, Ru G, Guo X, Zhang D, Zhang S, Li C,Wu J,Randolph PB,Sousa AA,Qu C,Zhu Y,Guan Y,Wang L,Liu M,Feng B,Song G,Liu DR,Li D.,Nat Biotechnol.2023Jun15.

[0022] As used herein, the term "gRNA" (single guide RNA) refers to an RNA molecule that acts as a guide to direct the Cas9 protein or base editor or prime editor to the locus of interest, complementary to its spacer sequence, and adjacent to the PAM motif located within 30 nucleotides upstream or downstream of the ATG start codon.

[0023] As used herein, the term "haploinsufficiency" refers to a molecular mechanism whereby mutational inactivation of one allele of a gene is sufficient to produce a disease phenotype. "Haploinsufficiency disease" refers to a disease caused by this process, characterized by insufficient amounts of a particular protein. The term "gene duplication disease" refers to a molecular mechanism whereby duplication of a gene generally results in an increase in the amount of a particular protein, leading to disease.

[0024] As described herein, the term "homologous directed repair" (HDR) refers to a repair pathway induced by double-strand breaks in DNA. In contrast to non-homologous end joining repair, HDR allows precise editing to be inserted into DNA by providing a donor DNA molecule encoding the desired edit, which will be used as a template for DNA repair. In genome editing applications, HDR is used to insert genetic information encoded by donor DNA after Cas9 cuts the target locus. References: Doudna, JA, & Charpentier, E. (2014). "The new frontier of genome engineering with CRISPR-Cas9". Science, 346 (6213), 1258096.

[0025] As used herein, the expression "Kozak consensus sequence" or "Kozak sequence" refers to a DNA sequence motif that serves as a translation start site in the mRNA of most eukaryotic organisms. It ensures that translation starts from the correct position in the mRNA, mediating the reading of the AUG start codon in the ribosome.

[0026] As used herein, the expression "Kozak variant sequence" or "Kozak variant" refers to an alternative Kozak sequence designed by replacing some of the 4 nucleotides on both sides of the ATG codon of the wild-type Kozak sequence, with the ATG codon remaining unchanged (ie, NNNN ATG NNNN). A variant may contain multiple transitions of the same type.

[0027] As described herein, the expression "lead editor" refers to a complex characterized by the fusion of Cas9n and reverse transcriptase. With programmable pegRNA (lead editing guide RNA), the complex is able to write new genetic information at the target locus, which encodes the target site and the template to be inserted and edited. Reference: Anzalone, AV, Randolph, PB, Davis, JR, Sousa, AA, Koblan, LW, Levy, JM, ... & Liu, DR (2019). "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature, 576 (7785), 149-157.

[0028] As used herein, the expression "translation regulator", "translation enhancer" or "translation repressor" refers to cis sequences and trans factors that are respectively endowed with the ability to regulate, enhance or inhibit the translation efficiency of a specific mRNA. More specifically, in the context of this article, the expression refers to variant Kozak sequences with the same ability.

[0029] As used herein, the term "vector" refers to a nucleic acid that is capable of entering a host cell, mutating and replicating within the host cell, and then transferring the replicated form of the vector to another host cell.

[0030] Advantageously, in one embodiment of the invention, a base editor is used to mutate the Kozak sequence. In addition, in one embodiment of the invention, the disease gene targeted is an HI disease gene. In this case, the change of one or several nucleotides in the Kozak sequence must be compatible with the action of the base editor and must be able to increase the amount of encoded protein, thereby compensating for the deleterious effects of loss of function of one allele. To this end, the present invention appropriately selects gRNA to induce the base editor to modify one of several nucleotides, thereby causing translation enhancement relative to the wild-type Kozak sequence. Therefore, the present invention relates to a Kozak variant nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 58, which is used to treat HI disease.

[0031] According to a preferred embodiment, the HI disease is selected from the following disease areas: developmental disorders, metabolic syndrome, eye disorders and hematopoietic system diseases.

[0032] For each of the above HI disease areas, the corresponding specific sequences and further relevant details are listed below.

[0033] According to a preferred embodiment, when the HI disease belongs to the category of developmental disorders, the nucleotide sequence is selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 28. The HI disease gene that causes the developmental disorder after mutation is selected from the group consisting of: DYRK1A, GRIN2B, NRXN1 and STXBP1. The developmental disorder is selected from the group consisting of: intellectual development disorder, autosomal dominant 7 (OMIM#614104), caused by loss of function of the allele of the DYRK1A gene; intellectual development disorder, autosomal dominant 6, with or without epileptic seizures (OMIM#613970), caused by loss of function of the allele of the GRIN2B gene; chromosome 2pl6.3 deletion syndrome (OMIM#614332), caused by loss of function of the allele of the NRXN1 gene; developmental epileptic encephalopathy 4 (OMIM#612164), caused by loss of function of the allele of the STXBP1 gene.

[0034] When the developmental disorder is intellectual developmental disorder, autosomal dominant 7, the nucleotide sequence is selected from SEQ ID NO:1 to SEQ ID NO:3; when the developmental disorder is intellectual developmental disorder, autosomal dominant 6, with or without epileptic seizures, the nucleotide sequence is selected from SEQ ID NO:4 to SEQ ID NO:8; when the developmental disorder is chromosome 2pl6.3 deletion syndrome, the nucleotide sequence is selected from SEQ ID NO:9 to SEQ ID NO:14; when the developmental disorder is developmental epileptic encephalopathy 4, the nucleotide sequence is selected from SEQ ID NO:15 to SEQ ID NO:28.

[0035] According to another preferred embodiment, when the HI disease belongs to the category of metabolic syndrome, the nucleotide sequence is selected from the group consisting of: SEQ ID NO: 29 to SEQ ID NONE. The HI disease genes causing metabolic syndrome are selected from the group consisting of: HNF1A, GHRL and PROXI. The metabolic syndrome is selected from the group consisting of: maturity-onset diabetes mellitus type 3 in the young (OMIM#600496), caused by loss of function of one allele of the HNF1A gene; susceptibility to obesity (OMIM#601665), caused by loss of function of one allele of the GHRL gene; adult-onset obesity and lymphatic disease, caused by loss of function of one allele of the PROXI gene.

[0036] When the metabolic syndrome is maturity-onset diabetes type 3 in adolescents, the nucleotide sequence is selected from SEQ ID NO:29 to SEQ ID NO:33; when the metabolic syndrome is susceptibility to obesity, the nucleotide sequence is selected from SEQ ID NO:34 to SEQ ID NO:38; when the metabolic syndrome is adult-onset obesity and lymphatic disease, the nucleotide sequence is selected from SEQ ID NO:39 to SEQ ID NO:41.

[0037] According to another preferred embodiment, when the HI disease belongs to the category of eye disorders, the nucleotide sequence is selected from the group consisting of: SEQ ID NO: 42 to SEQ ID NO: 53. The HI disease gene causing eye disorders is selected from the group consisting of: EYA1, OPA1, COL2A1. The eye disorder is selected from the group consisting of: branchio-oto-renal syndrome 1 (OMIM#113650), caused by loss of function of one allele of the EYA1 gene; optic atrophy 1 (OMIM#165500), caused by loss of function of one allele of the OPA1 gene; Stickler syndrome type 1, non-syndromic eye type (OMIM#609508), caused by loss of function of one allele of the COL2A1 gene.

[0038] When the eye disorder is branchio-oto-renal syndrome 1, the nucleotide sequence is selected from SEQ ID NO:42 or SEQ ID NO:43; when the eye disorder is optic atrophy 1, the nucleotide sequence is selected from SEQ ID NO:44 to SEQ ID NO:50; when the eye disorder is Stickler syndrome type 1, non-syndromic eye type, the nucleotide sequence is selected from SEQ ID NO:51 to SEQ ID NO:53.

[0039] According to another preferred embodiment, when the HI disease is a hematopoietic disease, the nucleotide sequence is selected from: SEQ ID NO: 54 to SEQ ID NO: 58. The HI gene involved in the hematopoietic disease is NCF1. The hematopoietic disease is chronic granulomatous disease (OMIM#233700).

[0040] In another embodiment of the present invention, the target gene of the disease is a GD disease gene. In this case, the change of one or several nucleotides in the Kozak sequence must be compatible with the action of the base editor, and must allow the amount of encoded protein to be reduced, thereby compensating for the deleterious effects of the gain of function of one allele. To this end, the present invention appropriately selects gRNA to induce the base editor to modify one of several nucleotides and obtain translational inhibition relative to the wild-type Kozak sequence. Therefore, the present invention relates to a Kozak variant nucleotide sequence selected from SEQ ID NO:61 to SEQ ID NO:65, which is used to treat HI disease.

[0041] According to a preferred embodiment, the HI disease is selected from the following disease areas: developmental disorders, metabolic syndrome, eye disorders and hematopoietic system diseases.

[0042] For each of the above HI disease areas, the corresponding specific sequences and further relevant details are listed below.

[0043] According to a preferred embodiment, when the HI disease belongs to the category of developmental disorders, the nucleotide sequence is selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 28. The HI disease gene that causes the developmental disorder after mutation is selected from the group consisting of: DYRK1A, GRIN2B, NRXN1 and STXBP1. The developmental disorder is selected from the group consisting of: intellectual development disorder, autosomal dominant 7 (OMIM#614104), caused by loss of function of the allele of the DYRK1A gene; intellectual development disorder, autosomal dominant 6, with or without epileptic seizures (OMIM#613970), caused by loss of function of the allele of the GRIN2B gene; chromosome 2pl6.3 deletion syndrome (OMIM#614332), caused by loss of function of the allele of the NRXN1 gene; developmental epileptic encephalopathy 4 (OMIM#612164), caused by loss of function of the allele of the STXBP1 gene.

[0044] In addition, the present invention also relates to a vector suitable for genome editing, comprising any gRNA designed to edit a wild-type Kozak sequence to obtain any variant Kozak nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 58. The targeting sequences of these gRNAs correspond to a target domain adjacent to a PAM sequence located within 30 nucleotides upstream or downstream of the ATG start codon embedded in the Kozak sequence.

[0045] According to a preferred embodiment, when the disease is chronic granulomatous disease, the gRNA for editing the NCF1 wild-type Kozak sequence is encoded by SEQ ID NO:59.

[0046] According to a preferred embodiment, when the disease is chronic optic atrophy, the gRNA for editing the OPA1 wild-type Kozak sequence is encoded by SEQ ID NO:60.

[0047] In addition, the present invention also relates to a pharmaceutical composition comprising at least one gRNA designed to obtain any one of the variant Kozak sequences selected from SEQ ID NO: 1 to SEQ ID NO: 58, a suitable vector for genome editing and a genome editing complex, the genome editing complex selected from CRISPR-Cas homology-directed repair, CRISPR-Cas lead editing, CRISPR-Cas base editing or any other method based on fusion to a programmable RNA-guided nuclease fused to an effector protein that allows the introduction of one or more nucleotide conversions, and at least one pharmaceutically acceptable excipient. According to a preferred embodiment, the pharmaceutical composition is intravenously injectable.

[0048] Therefore, the object of the present invention is a variant Kozak nucleotide sequence obtained by a genome editing method, which is used as a translation regulator of protein-coding genes in the treatment of diseases that alter the expression of said protein, wherein the variant Kozak nucleotide sequence replaces the wild-type Kozak sequence in vitro or in vivo.

[0049] Preferably, an object of the present invention is a variant Kozak nucleotide sequence according to claim 1, wherein the genome editing method is selected from the group consisting of CRISPR-Cas homology-directed repair, CRISPR-Cas lead editing, CRISPR-Cas base editing or any other method based on a programmable RNA-guided nuclease fused to an effector protein that allows the introduction of one or more nucleotide transitions.

[0050] According to one embodiment, a variant Kozak nucleotide sequence as described above is preferred, wherein the translational regulatory factor is a translational enhancer or a translational repressor.

[0051] According to one embodiment, the above-mentioned variant Kozak nucleotide sequence is preferred, which is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 58 as a translation enhancer, wherein the variant Kozak nucleotide sequence replaces the wild-type Kozak sequence in vitro or in vivo.

[0052] Another object is the use of the above variant Kozak nucleotide sequence for increasing the translation efficiency of protein coding genes in the treatment of haploinsufficiency diseases.

[0053] According to a preferred embodiment, the use of the above-mentioned variant Kozak nucleotide sequence is preferred, wherein the haploinsufficiency disease is selected from the following disease classes: developmental disorders, metabolic syndrome, eye disorders and hematopoietic system diseases.

[0054] According to one embodiment, the use of the above-mentioned variant Kozak nucleotide sequence is preferred, wherein the developmental disorder is selected from: intellectual disability, autosomal dominant 7; intellectual disability 6, with or without epileptic seizures; 2pl6.3 deletion syndrome; developmental epileptic encephalopathy 4.

[0055] According to one embodiment, the use of a variant Kozak nucleotide sequence according to any one of claims 6 to 7 is preferred, wherein:

[0056] When the developmental disorder is intellectual developmental disorder, autosomal dominant 7, the nucleotide sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 3;

[0057] When the developmental disorder is intellectual developmental disorder, autosomal dominant 6, with or without epileptic seizures, the nucleotide sequence is selected from SEQ ID NO: 4 to SEQ ID NO: 8;

[0058] When the developmental disorder is chromosome 2pl6.3 deletion syndrome, the nucleotide sequence is selected from SEQ ID NO: 9 to SEQ ID NO: 14;

[0059] When the developmental disorder is developmental epileptic encephalopathy 4, the nucleotide sequence is selected from SEQ ID NO:15 to SEQ ID NO:28.

[0060] According to one embodiment, the use of the above variant Kozak nucleotide sequence is preferred, wherein the metabolic syndrome is selected from: maturity-onset diabetes of the adolescent, type 3; susceptibility to obesity; lymphatic vessel defects and / or adult-onset obesity.

[0061] According to one embodiment, the use of the above variant Kozak nucleotide sequence is preferred, wherein:

[0062] When the metabolic syndrome is maturity-onset diabetes mellitus type 3 in adolescents, the nucleotide sequence is selected from SEQ ID NO: 29 to SEQ ID NO: 33;

[0063] When the metabolic syndrome is susceptibility to obesity, the nucleotide sequence is selected from SEQ ID NO: 34 to SEQ ID NO: 38;

[0064] When the metabolic syndrome is lymphatic vessel defect and / or adult-onset obesity, the nucleotide sequence is selected from SEQ ID NO: 39 to SEQ ID NO: 41.

[0065] According to one embodiment, the use of the above variant Kozak nucleotide sequence is preferred, wherein the eye disorder is selected from the group consisting of branchio-oto-renal syndrome, optic atrophy, Stickler syndrome type 1, non-syndromic ocular type.

[0066] Use of the above variant Kozak nucleotide sequence, wherein:

[0067] When the eye disorder is branchio-oto-renal syndrome, the nucleotide sequence is selected from SEQ ID NO:42 or SEQ ID NO:43;

[0068] When the eye disorder is optic atrophy, the nucleotide sequence is selected from SEQ ID NO:44 to SEQ ID NO:50;

[0069] When the eye disorder is Stickler syndrome type 1, non-syndromic eye type, the nucleotide sequence is selected from SEQ ID NO:51 to SEQ ID NO:53.

[0070] According to one embodiment, the use of the above variant Kozak nucleotide sequence is preferred, wherein when the hematopoietic disease is chronic granulomatous disease, the nucleotide sequence is selected from SEQ ID NO: 54 to SEQ ID NO: 58.

[0071] Another object is a gRNA designed to edit a wild-type Kozak sequence to obtain any one variant Kozak nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 58, characterized in that its targeting sequence corresponds to a target domain adjacent to a PAM sequence located within 30 nucleotides upstream or downstream of the ATG start codon.

[0072] According to one embodiment, the above-mentioned gRNA is preferred, which edits the wild-type Kozak sequence of NCF1, wherein the gRNA has the nucleotide sequence SEQ ID NO:59.

[0073] According to another embodiment, the above-mentioned gRNA is preferred, which edits the wild-type Kozak sequence of OPA1, wherein the gRNA has the nucleotide sequence SEQ ID NO:60.

[0074] Another object is a vector for genome editing, comprising any of the above-mentioned gRNAs.

[0075] Another object is a pharmaceutical composition comprising the above-mentioned vector and other suitable components for in vivo genome editing.

[0076] According to one embodiment, it is preferred that the above pharmaceutical composition is intravenously injectable.

[0077] Example

[0078] Experimental Section

[0079] Base editing-mediated Kozak optimization enhances translation in a reporter system

[0080] To demonstrate the feasibility of the proposed approach, an experiment was performed that focused on enhancing EGFP translation from a reporter vector. We created two versions of the bicistronic reporter vector pWPT-EGFP-IRESmCherry: EGFP with a wild-type Kozak sequence (C at position 1, EGFP-1C) or a suboptimal motif with a T at position 1 (EGFP-1T) ( Figure 1 A). This single base change reduced EGFP translation by 4-5 fold ( Figure 1 B, C, D). Next, we corrected this nucleotide variation using base editing and observed a significant increase in EGFP expression (Figure IE-1). These data confirm that base editing can be used to selectively mutate single nucleotides in the Kozak sequence.

[0081] Design and generation of an actionable Kozak variant library

[0082] We screened wild-type (WT) Kozak sequences of annotated HI genes and compared them with their respective variants to identify specific sets of actionable changes that upregulate translation efficiency of each WT Kozak sequence. We started with 230 haploinsufficient genes, from which we created an unbiased library of Kozak mutants. We obtained 5539 variants, of which 4838 were unique. As destination vector, we used pWPT-EGFP-IRESmCherry. After assembly, the library of wild-type and variant Kozak sequences will replace the EGFP·Kozak sequence, directing EGFP expression. The reporter gene obtained with this library was used to transform HEK293T cells.

[0083] Evaluation of protein levels of wild-type and variant Kozak sequences

[0084] To quantify the translation efficiency of wild-type and variant Kozak sequences of the HI gene, we sorted HEK293T cells transduced with the reporter gene with the library in four gates according to the normalized EGFP translation efficiency (EGFP / mCherry). 6 mCherry-positive cells were sorted to ensure 1000× library coverage ( Figure 2A). In the second round, the obtained mCherry positive cells were sorted according to their EGFP / mCherry ratio in 4 culture dishes with different fluorescence intensity ratios ( Figure 2 B, C). The Kozak sequence regions obtained from cells collected from each culture dish were PCR-amplified. By deep sequencing all fragments, we can compare the intensity of each HI wild-type Kozak with its variants. 89 wild-type sequences and 403 variant sequences passed the statistical analysis ( Figure 2 D). We then generated a motif for each of the four gates, representing the nucleotide frequency at each position of the Kozak sequence ( Figure 2 E). To select Kozak variants that up-regulated the corresponding WT, we selected only the variants with the largest distance to the corresponding WT. We obtained 47 wild-type and 149 variant sequences. From this list, we selected 5 HI genes and their corresponding variants for validation: PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1 ( Figure 2 F).

[0085] Validation of protein upregulation by selected hit Kozak sequence variants

[0086] To validate the selected hits, we cloned each Kozak sequence (wild-type and hit variants of the five selected genes) to replace the plasmid EGFP Kozak sequence in our reporter vector, creating a new plasmid for each sequence. We transiently transfected HEK293T cells with the respective wild-type and hit Kozak variants and measured fluorescence by high-content image analysis three days after transfection ( Figure 3 These analyses confirmed that 10 of the 11 Kozak variants tested had increased translation efficiency compared to their respective wild-type sequences ( Figure 3 B, C).

[0087] Enhancing NCF1 translation by base editing of its Kozak sequence

[0088] We then replicated two variants that emerged from the screen (Var 2: SEQ ID NO: 55; Var 4: SEQ ID NO: 57) by base editing at the endogenous locus in Raji cells, a B lymphocyte line derived from Burkitt's lymphoma that constitutively expresses the gene of interest. We performed base editing by electroporating AncBE4max and the guide RNA sgNCF1 (SEQ ID NO: 59). Figure 4To improve the readout of editing, we then decided to isolate cell clones, and we found and expanded clones with the same desired base editing-mediated nucleotide changes as Kozak NCF1 variants 2 and 4 (SEQ ID NO:55 and SEQ ID NO:57). Western blot analysis showed that the NCF1-encoded protein p47 was upregulated in both variants compared to wild-type. phox The expression of Figure 4 E, F). We also analyzed NCF1 mRNA levels in wild-type cells and clones and found that they were unchanged. These results strongly support the view that the increase in gene expression is the result of enhanced translation of NCF1 due to Kozak sequence editing ( Figure 4 G). Sucrose gradient fractionation in WT and edited cells shows that increased protein levels correspond to increased mRNA loading on polysomes ( Figure 4 H, I). Taken together, these results indicate that this is a new gene editing method targeting the Kozak sequence of a gene. It introduces suitable variants that trigger upregulation of target gene translation through base editing.

[0089] Table 1 below shows variant Kozak SEQ ID NOs: 1-58 of the present invention.

[0090] Table 2 below shows gRNA SEQ ID NOs: 59 and 60 of the present invention.

[0091] Table 3 below shows variant Kozak SEQ ID NOs: 61-65 of the present invention.

[0092] Table 1

[0093]

[0094]

[0095]

[0096] Table 2

[0097]

[0098] Table 3

[0099]

Claims

1. A variant Kozak nucleotide sequence obtained by a genome editing method, which serves as a translational regulator of a protein-coding gene in the treatment of a disease in which the expression of the protein is altered, wherein the variant Kozak nucleotide sequence replaces the wild-type Kozak sequence in vitro or in vivo.

2. The variant Kozak nucleotide sequence of claim 1, wherein the genome editing method is selected from the group consisting of CRISPR-Cas homology-directed repair, CRISPR-Cas lead editing, CRISPR-Cas base editing, or any other method based on a programmable RNA-guided nuclease fused to an effector protein that allows for the introduction of one or more nucleotide transitions.

3. The variant Kozak nucleotide sequence according to any one of claims 1-2, wherein the translational regulatory factor is a translational enhancer or a translational repressor.

4. The variant Kozak nucleotide sequence according to claim 3, which is selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 58 as a translation enhancer, or selected from the group consisting of SEQ ID NO: 61-SEQ ID NO: 65 as a translation repressor, wherein the variant Kozak nucleotide sequence replaces the wild-type Kozak sequence in vitro or in vivo.

5. Use of the variant Kozak nucleotide sequence according to claim 4 for increasing the translation efficiency of protein-coding genes in the treatment of haploinsufficiency diseases.

6. Use of a variant Kozak nucleotide sequence according to claim 5, wherein the haploinsufficiency disease is selected from the following disease categories: developmental disorders, metabolic syndrome, eye disorders and hematopoietic system diseases.

7. Use of a variant Kozak nucleotide sequence according to claim 6, wherein the developmental disorder is selected from: intellectual disability, autosomal dominant 7; intellectual disability 6, with or without epileptic seizures; 2pl6.3 deletion syndrome; developmental epileptic encephalopathy 4.

8. Use of a variant Kozak nucleotide sequence according to any one of claims 6-7, wherein: - When the developmental disorder is intellectual disability, autosomal dominant 7, the nucleotide sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 3; - When the developmental disorder is intellectual disability, autosomal dominant 6, with or without epileptic seizures, the nucleotide sequence is selected from SEQ ID NO: 4 to SEQ ID NO: 8; - When the developmental disorder is chromosome 2pl6.3 deletion syndrome, the nucleotide sequence is selected from SEQ ID NO: 9 to SEQ ID NO: 14; - When the developmental disorder is Developmental Epileptic Encephalopathy 4, the nucleotide sequence is selected from SEQ ID NO: 15 to SEQ ID NO:

28.

9. Use of a variant Kozak nucleotide sequence according to claim 6, wherein the metabolic syndrome is selected from: maturity-onset diabetes of the adolescent, type 3; susceptibility to obesity; lymphatic vessel defects and / or adult-onset obesity.

10. Use of a variant Kozak nucleotide sequence according to any one of claims 6 and 9, wherein: - When the metabolic syndrome is maturity-onset diabetes mellitus type 3 in adolescents, the nucleotide sequence is selected from SEQ ID NO: 29 to SEQ ID NO: 33; - When the metabolic syndrome is susceptibility to obesity, the nucleotide sequence is selected from SEQ ID NO: 34 to SEQ ID NO: 38; - When the metabolic syndrome is lymphatic vessel defect and / or adult-onset obesity, the nucleotide sequence is selected from SEQ ID NO: 39 to SEQ ID NO:

41.

11. Use of a variant Kozak nucleotide sequence according to claim 6, wherein the eye disorder is selected from branchio-oto-renal syndrome, optic atrophy, Stickler syndrome type 1, non-syndromic eye type.

12. Use of a variant Kozak nucleotide sequence according to any one of claims 6 and 11, wherein: - When the eye disorder is branchio-oto-renal syndrome, the nucleotide sequence is selected from SEQ ID NO: 42 or SEQ ID NO: 43; - When the eye disorder is optic atrophy, the nucleotide sequence is selected from SEQ ID NO: 44 to SEQ ID NO: 50; - When the eye disorder is Stickler syndrome type 1, non-syndromic eye type, the nucleotide sequence is selected from SEQ ID NO:51 to SEQ ID NO:

53.

13. Use of the variant Kozak nucleotide sequence according to claim 6, wherein when the hematopoietic disease is chronic granulomatous disease, the nucleotide sequence is selected from SEQ ID NO: 54 to SEQ ID NO:

58.

14. A gRNA designed to edit a wild-type Kozak sequence to obtain any variant Kozak nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 58, characterized in that: Its targeting sequence corresponds to the target domain adjacent to the PAM sequence located within 30 nucleotides upstream or downstream of the ATG start codon. 15 . The gRNA according to claim 14 , which edits the wild-type Kozak sequence of NCF1, wherein the gRNA has the nucleotide sequence SEQ ID NO:

59.

16. The gRNA according to claim 14, which edits the wild-type Kozak sequence of OPA1, wherein the gRNA has the nucleotide sequence SEQ ID NO:

60.

17. A vector for genome editing, comprising any one of the gRNAs according to any one of claims 14-16.

18. A pharmaceutical composition comprising the vector according to claim 17 and other suitable components for in vivo genome editing.

19. The pharmaceutical composition according to claim 18, which is intravenously injectable.

20. The variant Kozak nucleotide sequence according to any one of claims 1 to 4 for use in treating a haploinsufficiency disease or a gene duplication disease.

21. The variant Kozak nucleotide sequence for use according to claim 20, wherein the haploinsufficiency disease is selected from the group consisting of developmental disorders, metabolic syndrome, eye disorders and hematopoietic disorders.

22. A variant Kozak nucleotide sequence for use according to claim 21, wherein: - When the developmental disorder is intellectual disability, autosomal dominant 7, the nucleotide sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 3; - When the developmental disorder is intellectual disability, autosomal dominant 6, with or without epileptic seizures, the nucleotide sequence is selected from SEQ ID NO: 4 to SEQ ID NO: 8; - When the developmental disorder is chromosome 2pl6.3 deletion syndrome, the nucleotide sequence is selected from SEQ ID NO: 9 to SEQ ID NO: 14; - When the developmental disorder is Developmental Epileptic Encephalopathy 4, the nucleotide sequence is selected from SEQ ID NO: 15 to SEQ ID NO:

28.

23. A variant Kozak nucleotide sequence for use according to claim 21, wherein: - When the metabolic syndrome is maturity-onset diabetes of the adolescent, type 3, the nucleotide sequence is selected from SEQ ID NO: 29 to SEQ ID NO: 33; - When the metabolic syndrome is susceptibility to obesity, the nucleotide sequence is selected from SEQ ID NO: 34 to SEQ ID NO: 38; - When the metabolic syndrome is lymphatic vessel defect and / or adult-onset obesity, the nucleotide sequence is selected from SEQ ID NO: 39 to SEQ ID NO:

41.

24. A variant Kozak nucleotide sequence for use according to claim 21, wherein: - When the eye disorder is branchio-oto-renal syndrome, the nucleotide sequence is selected from SEQ ID NO: 42 or SEQ ID NO: 43; - When the eye disorder is optic atrophy, the nucleotide sequence is selected from SEQ ID NO: 44 to SEQ ID NO: 50; - When the eye disorder is Stickler syndrome type 1, non-syndromic eye type, the nucleotide sequence is selected from SEQ ID NO:51 to SEQ ID NO:

53.

25. The variant Kozak nucleotide sequence for use according to claim 21, wherein when the hematopoietic disease is chronic granulomatous disease, the nucleotide sequence is selected from SEQ ID NO: 54 to SEQ ID NO:

58.

26. The variant Kozak nucleotide sequence for use according to claim 20, wherein the gene duplication disease is selected from the group consisting of motor and sensory neuropathy.

27. The variant Kozak nucleotide sequence for use according to claim 26, wherein the motor and sensory neuropathy is Charcot-Marie-Tooth Disease Type 1A, and the nucleotide sequence is selected from the group consisting of SEQ ID NO: 61 to SEQ ID NO: 65.

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