Mutation-independent allele-specific CRISPR targeting strategies for treatment of genetic diseases

Abstract

Novel compositions and methods are provided that are useful for treating, preventing and potentially curing genetic diseases, such as familial Alzheimer's disease, by disrupting a genomic sequence comprising one or more SNPs that are highly epidemic in a population but independent of a particular disease, in some embodiments, their genomic positions are within the same gene exon as the disease-related alleles, and upstream of such disease-related alleles present in the genome of a treatment recipient.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 582,523, filed September 14, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Background of the Invention

[0004] Autosomal dominant diseases are genetic disorders that manifest clinical symptoms even when only one of the two alleles located on one of the numbered chromosomes (1 to 23) other than the sex chromosomes (X and Y) contains a mutation or variation that causes or contributes to the disease. Alzheimer's disease (AD), especially familial AD, is such an autosomal dominant disease.

[0005] Gene-based therapeutic strategies for treating autosomal dominant diseases have so far focused on targeting disease-associated gene mutations or alleles for genome editing. This includes disrupting DNA coding sequences through deletions, substitutions, or inactivating mRNA transcripts by creating reading frame shifts or premature termination, thereby silencing the target allele. For example, brain diseases, including neurodegenerative diseases such as Alzheimer's disease (AD), are catastrophic conditions affecting a large portion of the population. Many of these diseases are incurable, highly debilitating, and often lead to progressive deterioration of brain structure and function over time. The prevalence of these diseases is rapidly increasing due to the growing global aging population, as older adults are at high risk of developing these conditions. Currently available treatments for these conditions are ineffective and fail to meet market demand, which is increasing significantly annually due to the aging population. Gene-based therapies are an attractive alternative, especially for individuals with a family history of such neurodegenerative diseases and / or identified through genetic screening as carrying one or more known disease-associated gene variants / alleles. A drawback of these methods is the significant variability in suitable gene-editing therapeutics; however, treatment can be targeted due to the unique genetic background of each individual, for example, in the precise sequence and location of disease-related gene variants. Therefore, there is an urgent need to develop novel and effective gene therapy methods that can provide relatively universal tools that are directly useful to most specified populations. This invention addresses this and other related needs by providing novel gene therapy methods that target one or more non-disease-specific alleles that are highly prevalent in the population and located in close physical proximity to disease-related alleles, thereby providing an effective treatment option with substantially universal applicability in the population.

[0006] Brief Overview of the Invention

[0007] This application provides the first disclosure of a novel therapeutic strategy for treating genetic diseases, which disrupts the genome sequence and thus inactivates disease-related alleles by targeting one or more single nucleotide polymorphisms (SNPs) unrelated to the disease itself, utilizing SNPs located closely adjacent to disease-related alleles. The high prevalence of these SNPs in the general population also provides a significant degree of versatility to this treatment approach.

[0008] Therefore, in a first aspect, the present invention provides a method for treating autosomal dominant diseases in persons in need or for reducing the risk of said diseases in persons in need. The method comprises administering to the person an effective amount of a composition that destroys a genomic sequence containing at least one pair (i.e., two or more) single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5, provided that (1) the person is heterozygous at all said SNPs, i.e., has two different nucleotide sequences at each of the SNP loci on two alleles; and (2) the person has a gene mutation or variant known to be associated with or related to said disease; and (3) the SNP and the disease-associated mutation are located in close proximity within the genomic sequence, for example, within the same exon sequence of the same gene.

[0009] In some embodiments, the claimed method includes sequencing at least a portion of the human genome prior to the application step, such that the nucleotide sequence of at least one SNP, its heterozygous nature, and the presence of known disease-related variants in the human genome sequence, particularly within gene exons. In some embodiments, the autosomal dominant disease is Alzheimer's disease (AD). In some embodiments, the gene is the amyloid precursor protein (APP) gene or the presenilin-1 (PSEN1) gene. In some embodiments, the person has been diagnosed with AD. In some embodiments, the person has not been diagnosed with AD but has known AD risk factors, such as a family history of AD or carrying one or more gene alleles known to increase the risk of AD, such as being a female APOE-ε4 carrier. In some embodiments, at least one pair of SNPs includes any pair of SNPs listed in Table 1, such as rs2830026 and rs6516719. In some embodiments, at least one pair of SNPs includes any pair of SNPs listed in Table 5. In some embodiments, at least one SNP pair includes rs17125457 and rs17408630. In some embodiments, at least one SNP pair includes any two SNP pairs listed in Table 2, such as rs2830026 and rs6516719; rs2248682 and rs8130594. In some embodiments, at least one SNP pair includes any three SNP pairs listed in Table 3, such as rs2830026 and rs6516719; rs10154121 and rs2070655; rs2830046 and rs2070654. In some embodiments, the composition comprises one or more vectors encoding a small guide RNA (sgRNA)-guided endonuclease and at least two sgRNAs targeting at least one, and possibly more, SNP pairs listed in Tables 1, 2, 3, or 5. In some embodiments, at least two sgRNAs include at least two nucleotide sequences listed in Table 4 or Table 6. In some embodiments, the endonuclease is a Cas9 nuclease. In some embodiments, the one or more vectors are one or more viral vectors, such as adenovirus or adeno-associated virus (AAV) vectors. In some embodiments, the composition is administered via subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or intracranial injection, or via oral or nasal administration. In some embodiments, the composition is administered in the form of a solution (including nebulized solution), suspension, powder, paste, tablet, or capsule.

[0010] In a second aspect, the present invention provides a composition comprising: (1) an effective amount of one or more agents that disrupt a genomic sequence containing at least one pair of single nucleotide polymorphisms (SNPs) listed in Table 1, Table 2, Table 3 or Table 5, and (2) one or more physiologically acceptable excipients.

[0011] In some embodiments, the composition comprises one or more vectors encoding a nuclease guided by a small guide RNA (sgRNA) and two sgRNAs targeting at least one pair of SNPs listed in Tables 1, 2, 3, or 5. In some embodiments, the nuclease is a Cas9 nuclease. In some embodiments, each of the one or more vectors is a viral vector. In some embodiments, the composition is formulated for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or for oral or nasal administration. In some embodiments, the composition is in the form of a solution, suspension, powder, paste, tablet, or capsule.

[0012] In a third aspect, the present invention provides a kit for treating autosomal dominant diseases in individuals in need or reducing the risk of such individuals developing the disease later in life. The kit includes a container containing a composition capable of disrupting genomic sequences comprising at least one pair of single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5.

[0013] In some embodiments, the autosomal dominant disease is Alzheimer's disease (AD). In some embodiments, the composition comprises one or more vectors encoding a nuclease guided by a small guide RNA (sgRNA) and two sgRNAs targeting at least one pair of SNPs listed in Tables 1, 2, 3, or 5. In some embodiments, the nuclease is a Cas9 nuclease. In some embodiments, each of the one or more vectors is a viral vector. In some embodiments, the composition is formulated for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial administration, or for oral or nasal administration. In some embodiments, the composition is in the form of a solution, suspension, powder, paste, tablet, or capsule. In some embodiments, the kit product may also include a second container or multiple containers containing one or more reagents useful for sequencing at least a portion of the genome sequence of the human containing at least one pair of single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5. Optionally, the kit may also include an instruction manual for applying the composition.

[0014] In connection with this aspect of the invention, the disclosure herein also provides the use of one or more reagents for disrupting the genomic sequence of at least one, possibly two or three, SNPs listed in Tables 1, 2, 3 or 5 for the preparation of (1) a medicament for treating autosomal dominant genetic diseases (e.g., familial Alzheimer's disease); and / or (2) a package product containing a medicament for treating said disease.

[0015] Brief description of the attached figures

[0016] Figure 1A and Figure 1B A diagram illustrating current allele-specific CRISPR targeting strategies and SNP-directed CRISPR disruption strategies for mutant alleles. Figure 1A Mutation-dependent allele-specific targeting. This method disrupts mutated alleles by directly targeting the mutation site. Different targeting tools are needed to target various pathogenic mutations in different individuals. Figure 1B SNP-pair-targeted CRISPR disruption strategy for mutant alleles. This approach focuses on targeting exons on common SNP pairs or deletion mutant alleles. Exon excision leads to frameshift mutations and premature stop codon generation, ultimately disrupting mutant allele expression.

[0017] Figure 2 : A diagram of targetable exons in the APP gene that can be targeted using mutation-independent targeting methods. The APP gene consists of 18 exons, with most pathogenic mutations located in exons 16 and 17. Deletions of exons 3, 5, 6, 9, and 13 (highlighted in orange) can each individually lead to the formation of premature stop codons (indicated by red arrows).

[0018] Figure 3 Frequency analysis of common heterozygous SNP pairs in the APP gene. The arc plot shows the heterozygous SNP pairs in the APP gene that are targeted for deletion candidate exons. Arc nodes represent their relative positions within the APP genomic loci, while the height of the arc indicates the population frequency of each heterozygous SNP pair. Only SNP pairs with a population frequency >0.2 are included as heterozygous in the plot. The most prevalent heterozygous SNP pairs, rs6516719 and rs2830026, are indicated, with a population frequency of 48.9%. The population data used for this analysis were derived from the gnomAD database, with a specific focus on European populations.

[0019] Figure 4 A diagram illustrating an EGFP alternative reporter assay used to evaluate the efficiency and specificity of sgRNA targeting. sgRNA was validated using a targeting reporter and a control reporter. Double-positive EGFP was calculated. + mCherry +Cells and mCherry + The targeting efficiency of sgRNA was determined by the ratio of the total number of cells to the target. The targeting specificity of the CRISPR / Cas9 system was evaluated by comparing the targeting efficiency of the target template with that of the control template.

[0020] Figure 5 Design and evaluation of a CRISPR toolset for targeting candidate SNP haplotypes in the APP gene. The specific SNPs rs6516719 and rs2830026 have two haplotypes. Haplotype 1 (H1) consists of two major allele sites (H1L and H1R), while haplotype 2 (H2) consists of two minor allele sites (H2L and H2R). Two CRISPR targeting tools were designed and validated to target each haplotype. Targeting efficiency was evaluated using templates from H1 and H2.

[0021] Figures 6A to 6D Evaluation of the targeting efficiency and specificity of CRISPR tools at high-coverage target sites in the APP gene. Figure 6A The performance of CRISPR targeting tools against SNP pairs (rs8130594 and rs2248682) in the APP gene was evaluated using an EGFP alternative reporter assay. Figure 6B The performance of CRISPR targeting tools against SNP pairs (rs2830052 and rs2830046) in the APP gene was evaluated using an EGFP alternative reporter assay. Figure 6C The performance of CRISPR targeting tools against SNP pairs (rs8130594 and rs2830048) in the APP gene was evaluated using an EGFP alternative reporter assay. Figure 6D The performance of CRISPR targeting tools against SNP pairs (rs2070654 and rs2830046) in the APP gene was evaluated using an EGFP reporter substitution assay. For each SNP pair, the most common double heterozygous haplotype was selected for CRISPR tool design. To target each haplotype, two sets of CRISPR targeting tools were designed and validated, with targeting efficiency evaluated using templates H1 and H2.

[0022] Figure 7Evaluation of the targeting efficiency and specificity of CRISPR tools at high-coverage target sites in the PSEN1 gene. We evaluated CRISPR targeting tools for targeting SNP pairs in the PSEN1 gene (with a particular focus on rs165931 and rs362340). Evaluation was performed using EGFP surrogate reporter assays. For this specific SNP pair, the most common diheterozygous haplotypes were selected for CRISPR tool design. To target each haplotype, two sets of CRISPR targeting tools were designed and validated, with targeting efficiency evaluated using templates H1 and H2.

[0023] Figure 8A and Figure 8B In iPSC-derived neurons carrying the APP-V717I mutation, genome editing eliminated the expression of the mutated APP and restored the Aβ42 / Aβ40 ratio. Figure 8A : Allelic expression of APP mRNA. NDC, non-dementia control; CON, non-editing control. Figure 8B The level of the Aβ42 / 40 ratio in conditioned medium from neurons of 28 DIVs. Values ​​are mean ± SEM (n=5 independent biological replicates for NDC, n=12 independent biological replicates for APP-V717I, and n=7 independent biological replicates for edited APP-V717I). p < 0.05, p < 0.01 or p < 0.001 vs. CON-APP V717I neurons (Student's t-test).

[0024] definition

[0025] “Nucleic acid” or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. Unless specifically defined, the term includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected codons (or all codons) is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985) and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The terms nucleic acid, gene, cDNA, and gene-encoded mRNA are used interchangeably.

[0026] The term "gene" refers to a segment of DNA that is involved in the production of a polypeptide chain. It can include regions before and after coding regions (leader and tail) as well as insertion sequences (introns) between individual coding regions (exons).

[0027] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimics" are compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0028] Various known methods in the art allow for the incorporation of non-natural amino acid derivatives or analogs into polypeptide chains in a site-specific manner, see, for example, WO02 / 086075.

[0029] Amino acids may be referred to in this article by either their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0030] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. As used herein, the term includes amino acid chains of any length (including full-length proteins) where amino acid residues are linked by covalent peptide bonds.

[0031] As used herein, “a composition that disrupts a genomic sequence containing [any one or more specific single nucleotide polymorphisms (SNPs)]” means any composition containing one or more such agents capable of inhibiting or eliminating transcription or translation of a genomic sequence. This can be achieved by directly deleting or altering at least a portion of a genomic sequence that results in, for example, a frameshift of a coding sequence or premature termination of translation (e.g., by genome editing techniques, such as the CRISPR system, etc.), or by the action of small repressive DNA or RNA molecules or other enzymes (e.g., antisense oligonucleotides, small repressive RNAs (e.g., siRNA or shRNA), and ribozymes, etc.) by reducing or eliminating mRNA transcribed from the genomic sequence. Terms and phrases used in this disclosure and similarly worded when referring to other loci (e.g., “any one or more pairs of SNPs listed in Table 1, Table 2, or Table 3”) are defined in a functionally identical or similar manner.

[0032] When used to describe a person as "heterozygous" at any particular SNP, the term is intended to indicate that the person has two distinct alleles at that particular locus, which can be distinguished from each other due to their different nucleotide sequences.

[0033] Disease-associated gene mutations or alleles are genomic sequence variants that have been shown to be associated with the risk, presence, or prognosis of disease because they may play a role in causing or contributing to the onset of disease, accelerating disease progression, and / or aggravating disease severity or outcome.

[0034] When used in the context of describing repressive oligonucleotides (e.g., small repressive RNAs or antisense oligonucleotides) or sgRNAs associated with repressive oligonucleotides or gene editing systems for negative regulation of genomic sequences, the term "targeted" refers to sufficient sequence complementarity between at least a portion of the oligonucleotide or sgRNA and the genomic sequence, for example, at least 80%, 85%, 90%, 95%, or higher percentage of nucleotide sequence complementarity based on the Watson-Crick base pairing principle, such that specific hybridization between the sgRNA or oligonucleotide and the genomic sequence or its mRNA transcript is permitted, which subsequently results in the cleavage of the genomic sequence at a predetermined location or the disruption of its mRNA transcript.

[0035] When used in conjunction with, for example, cells or nucleic acids, proteins, or vectors, the term "recombinant" means that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein, or by altering the native nucleic acid or protein, or that the cell is derived from a cell that has been so modified. Thus, for example, recombinant cells express genes that are not present in the natural (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, poorly expressed, or not expressed at all.

[0036] A “promoter” is defined as a set of nucleic acid control sequences that direct the transcription of a polynucleotide sequence. As used herein, a promoter includes an essential polynucleotide sequence near the transcription start site, for example, in the case of a polymerase II promoter, including a TATA element. A promoter may also optionally include distal enhancer or repressor elements, which can be located up to several thousand base pairs from the transcription start site. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional link between a polynucleotide expression control sequence (e.g., a promoter or an array of transcription factor binding sites) and a second polynucleotide sequence, wherein the expression control sequence directs the transcription of a polynucleotide sequence corresponding to the second sequence.

[0037] An "expression cassette" is a recombinant or synthetically produced nucleic acid construct that has a set of designated polynucleotide elements that allow a specific polynucleotide sequence to be transcribed in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette comprises a polynucleotide to be transcribed that is operatively linked to a promoter.

[0038] The term "heterogeneous," as used in the context of describing the relative positions of two elements, refers to two elements that do not naturally exist in the same relative position, such as polynucleotide sequences (e.g., a promoter and a sequence encoding mRNA or a protein / peptide) or polypeptide sequences (e.g., two peptides acting as fusion partners within a fusion protein). Thus, a "heterogeneous promoter" of a coding sequence refers to a promoter that is not naturally operatively linked to that coding sequence. Similarly, a "heterogeneous polypeptide" or "heterogeneous polynucleotide" of a particular protein or its coding sequence is a polypeptide or polynucleotide derived from a different origin than that particular protein, or, if derived from the same origin, a polypeptide or polynucleotide not naturally linked to that particular protein or its coding sequence in the same manner. The fusion of a polypeptide (or its coding sequence) with a heterologous polypeptide (or polynucleotide sequence) does not produce a longer polypeptide or polynucleotide sequence that could exist in nature.

[0039] The phrase "specific hybridization with..." refers to the binding, duplexing, or hybridization of a polynucleotide sequence with another polynucleotide sequence under stringent hybridization conditions, based on Watson-Crick nucleotide base pairing, when the polynucleotide sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). The phrase "stringent hybridization conditions" refers to conditions, typically in a complex mixture of nucleic acids, under which a nucleic acid (e.g., a polynucleotide probe) will hybridize with its target nucleotide sequence but not with other sequences. Stringent conditions are sequence-dependent and will vary under different conditions. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization exist. Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993) In general, stringent conditions are chosen to be at a specified ionic strength pH than the thermal melting point (T0) of a particular sequence. m Temperatures will be approximately 5°C to 10°C lower. m At a temperature (within defined ionic strength, pH, and nucleic acid concentration), 50% of the probes complementary to the target hybridize with the target sequence at equilibrium (because of the excess of the target sequence, at T...). m(At this point, 50% of the probe is occupied at equilibrium). Tight conditions would be those where the salt concentration is less than about 1.0 M sodium ions, typically about 0.01 M to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Tight conditions can also be achieved by adding a destabilizing agent such as formamide. For high-tight hybridization, the positive signal is at least twice the background hybridization, preferably ten times the background hybridization. Exemplary high-tight hybridization conditions include: 50% formamide, incubation at 42°C with 5×SSC and 1% SDS, or incubation at 65°C with 5×SSC and 1% SDS, followed by washing at 65°C in 0.2×SSC and 0.1% SDS.

[0040] The term "host cell" refers to a cell that contains an expression vector and supports its replication or expression. Host cells can be prokaryotic cells, such as E. coli; or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells, such as CHO, HeLa, etc., including cultured cells, explants, and in vivo cells.

[0041] As used herein, the term "inhibiting" or "inhibition" refers to any detectable negative effect of an inhibitor on a target biological process, such as the expression of a predetermined target protein (e.g., APP or PSEN1), the formation of amyloid-β (Aβ) plaques in the brains of AD patients, cognitive decline in AD patients, protein phosphorylation, cell signal transduction, protein synthesis, cell proliferation, tumorigenicity, and metastatic potential. Typically, inhibition is reflected by a reduction of at least 10%, 20%, 30%, 40%, or 50% in the target process (e.g., target protein expression or Aβ plaque accumulation) or any of the downstream parameters described above, compared to a control unexposed to the inhibitor. In a similar manner, the terms "increasing" or "increase" are used to describe any detectable positive effect of the enhancer on the target biological process, such as a positive change of at least 25%, 50%, 75%, 100%, or up to 2, 3, 4, 5, or up to 10 or 20 times compared to a control without the enhancer. Conversely, the term "substantially unchanged" describes a state where the positive or negative change is less than 10%, 5%, 2%, 1%, or lower.

[0042] As used herein, the term "effective amount" refers to an amount sufficient to produce the intended effect of the substance administration. Effects can include desired changes in biological processes (e.g., a detectable reduction in the expression of target proteins such as APP or PSEN1, a reduction in Aβ plaque formation, or a slowing of cognitive decline in AD patients), and prevention, correction, or inhibition of the progression of symptoms and associated complications of the disease / condition to any detectable extent. The exact amount that is "effective" in achieving the desired effect will depend on the nature of the therapeutic agent, the route of administration, and the purpose of treatment, and will be determined by a person skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999) ).

[0043] As used herein, the term "treatment" or "treating" includes therapeutic and preventative measures taken to address the presence of a disease or condition or the risk of its subsequent development. This includes therapeutic or preventative measures used to relieve ongoing symptoms, inhibit or slow disease progression, delay the onset of symptoms, or eliminate or reduce side effects caused by the disease or condition. Preventative measures and their variations in this context do not require the complete elimination of the event; rather, they refer to suppressing or reducing the likelihood or severity of such occurrence or delaying its occurrence.

[0044] A "pharmaceuticalally acceptable" or "pharmacologically acceptable" excipient is a substance that is not biologically harmful or otherwise undesirable; that is, the excipient can be administered to an individual together with a bioactive agent without causing any undesirable biological effects. The excipient also will not interact in a harmful manner with any component of the composition in which it is contained.

[0045] The term "excipient" refers to any substantially auxiliary substance that may be present in the final dosage form of the compositions of the present invention. For example, the term "excipient" includes media, binders, disintegrants, fillers (diluents), lubricants, flow aids (flow enhancers), compression aids, colorants, sweeteners, preservatives, suspending / dispersing agents, film-forming agents / coatings, flavoring agents, and printing inks.

[0046] When used in the context of describing a composition containing one or more active ingredients, the term "substantially composed of" means that the composition does not contain any other ingredient having similar or related biological activity to the active ingredient or capable of enhancing or inhibiting that activity, while one or more inactive ingredients, such as physiologically or pharmaceutically acceptable excipients, may be present in the composition. For example, a composition substantially composed of an active agent that effectively disrupts a genomic sequence containing one or more pairs of specified SNPs or inhibits the transcription of mRNA from a subject's genomic sequence is such a composition that does not contain any other agent that may have any detectable positive or negative effect on the same target process or that may increase or decrease the occurrence or symptoms of disease in the receiving subject to any measurable extent.

[0047] The term “about” indicates a range of + / - 10% of a predetermined value. For example, “about 10” indicates a range of 90% to 110% of 10, that is, 9 to 11. Detailed Implementation

[0048] I. Introduction

[0049] This invention relates to therapeutic genome editing strategies for autosomal dominant disorders. Specifically, it relates to the development of a general mutation-independent, allele-specific CRISPR targeting strategy for familial Alzheimer's disease (AD), applicable to other genetic disorders, particularly autosomal dominant disorders. This contrasts with previous therapeutic strategies that directly target disease-associated gene variants, see, for example, WO 2023 / 005959 and WO 2023 / 072225. Using this novel strategy, the inventors of this application target one or more non-pathogenic SNP pairs that are cis-positive to a pathogenic mutation, deleting a specific exon. These SNP pairs are naturally occurring variants present in the population at heterozygous and high frequencies, making them available for identifying any allele containing the mutation site in most individuals. The expression of the mutated gene can then be disrupted by excision of a specific exon. This invention relates to identifying candidate targetable SNP pairs in familial AD and validating the targeting efficiency and specificity of corresponding CRISPR targeting tools.

[0050] II. General Recombination Techniques

[0051] Basic textbooks on general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0052] For nucleic acids, sizes are given in kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, sequencing of nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or the number of amino acid residues. Protein sizes are estimated from gel electrophoresis, sequencing of proteins, derived amino acid sequences, or published protein sequences.

[0053] Oligonucleotides that are not commercially available can, for example, be obtained from... Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981) The solid-phase phosphoramidite trimer method, described for the first time, uses, as Van Devanter et al. al., Nucleic Acids Res. 12: 6159-6168 (1984) The automated synthesizer described herein is used for chemical synthesis. Purification of the oligonucleotides is performed using any field-recognized strategy, such as natural acrylamide gel electrophoresis or anion exchange HPLC, as described above. Pearson & Reanier, J. Chrom. 255: 137-149 (1983) As described in [the text].

[0054] The sequences of the target gene, the polynucleotide encoding the target polypeptide, and the synthetic oligonucleotides can be used after cloning or subcloning, for example... Wallace et al., Gene 16: 21-26 (1981) Validation of the chain termination method for sequencing double-stranded templates.

[0055] II. Compositions that disrupt genome sequences

[0056] The inventors' earlier work in this application demonstrated that various genes and the proteins they encode are involved in the development of Alzheimer's disease. Their latest findings indicate that disrupting genomic sequences containing certain loci can reduce the expression of related genes and the accumulation of Aβ plaques in the brain. This discovery leads to compositions that disrupt such genomic sequences for the treatment of Alzheimer's disease in patients already diagnosed with the disease, and for the prevention / reduction of Alzheimer's disease in undiagnosed individuals at high risk (e.g., due to family history or known genetic background (e.g., one or two APOEs)). Therapeutic and preventative uses of point mutations in the genome sequence encoding amyloid precursor protein (APP) on chromosome 21, point mutations in the genome sequence encoding presenilin 1 (PSEN1) on chromosome 14, and point mutations in the genome sequence encoding presenilin 2 (PSEN2) on chromosome 1) to reduce the risk of later Alzheimer's disease in individuals. Various classes of possible agents acting through different mechanisms (e.g., through genome editing or mRNA repression) are useful in formulating such compositions for disrupting the genome sequence containing rs1921622, and are disclosed, for example, in WO 2023 / 005959 and WO 2023 / 072225.

[0057] Compared to previously discussed therapeutic approaches, this latest disclosure involves disrupting the genome sequence by using gene-editing tools such as the CRISPR system to target gene alleles (SNPs) that are unrelated to the disease or unrelated to it (except for their proximity in the genome sequence to disease-related alleles), rather than targeting specific alleles directly related to the disease being treated (e.g., autosomal dominant diseases such as familial AD). For example, the target SNPs used in the methods of this invention are typically located within the same gene as the disease-related allele, particularly within the same exon of the same gene. In some cases, the target SNP is located upstream of the disease-related allele. For example, there may be at least about 50, 100, 200, 250, 500, 1000, 2000, 3000, 5000, 10,000, or 20,000 or more nucleotides upstream of the disease-related allele, such that gene editing at the SNP site will result in deletion or substitution of the genome sequence, causing the encoded mRNA transcript to be completely unable to be generated or to suffer reading frame shifts and / or premature termination.

[0058] Furthermore, the target SNPs were carefully selected so that their presence in the general population reached a relatively high percentage, for example, at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, or higher. The high prevalence of these target SNPs (as named in Tables 1, 2, or 3) endows them with substantial universality, thus providing invaluable universal availability for gene-editing tools and the therapeutic methods of this invention.

[0059] A. antisense oligonucleotides

[0060] In some implementations, the agent is an antisense oligonucleotide. Antisense oligonucleotides are relatively short nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of RNA transcribed from a genomic sequence containing specific SNPs (e.g., those named in Tables 1, 2, or 3). Although antisense oligonucleotides are typically RNA-based, they can also be DNA-based. Furthermore, antisense oligonucleotides are often modified to improve their stability.

[0061] Unbound by theory, the binding of these relatively short oligonucleotides to mRNA is thought to induce the stretching of double-stranded RNA, thereby triggering the degradation of mRNA by endogenous RNases. Furthermore, sometimes oligonucleotides are specifically engineered to bind near the promoter of the coding sequence, and in these cases, antisense oligonucleotides may additionally interfere with mRNA translation. Regardless of the specific mechanism by which antisense oligonucleotides function, their administration to cells or tissues allows for the degradation of RNA transcribed from genomic sequences containing specific SNPs (e.g., those in Tables 1, 2, or 3). Therefore, antisense oligonucleotides reduce the expression and / or activity of the coding products derived from genomic sequences.

[0062] Oligonucleotides can be single-stranded or double-stranded DNA or RNA, or chimeric mixtures, derivatives, or modified versions thereof. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization, etc. Oligonucleotides may include other additional groups, such as peptides (e.g., for targeting host cell receptors), or those that facilitate transmembrane transport (see, for example, ...). Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553- 6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; WO 88 / 09810 Drugs transported via the blood-brain barrier (see, e.g., WO 89 / 10134), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976 ), or an inserter (see, for example, Zon, 1988, Pharm. Res. 5:539-549 For this purpose, oligonucleotides can conjugate with another molecule.

[0063] The oligonucleotides of the present invention can be synthesized using standard methods known in the art, for example, by using an automated DNA synthesizer (e.g., commercially available from Biosearch, Applied Biosystems, etc.). As an example, phosphate thioester oligonucleotides can be synthesized using Stein et al. (… 1988, Nucl. Acids Res. 16:3209 Methylphosphonate oligonucleotides can be synthesized using a controlled-pore glass polymer support. Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451 ),etc.

[0064] Various methods have been developed for delivering antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into the target anatomical site, or modified antisense molecules designed to target desired cells can be systematically administered (e.g., antisense molecules linked to peptides or antibodies that specifically bind to receptors or antigens expressed on the surface of target cells).

[0065] In some cases, it may be difficult to achieve intracellular concentrations of antisense molecules sufficient to inhibit the translation of endogenous mRNA. Therefore, another approach utilizes recombinant DNA constructs in which antisense oligonucleotides are placed under the control of a strong pol III or pol II promoter. For example, a vector can be introduced in vivo, allowing it to be taken up by the cell and direct the transcription of antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed using recombinant DNA techniques standard in the art. The vector can be a plasmid, virus, or other vector known in the art for replication and expression in mammalian cells. Expression of the sequence encoding antisense RNA can be achieved using any promoter known in the art that functions in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (…). Bernoist and Chambon, 1981, Nature 290:304-310 The promoter contained in the 3' long terminal repeat sequence of Rous sarcoma virus ( Yamamoto et al., 1980, Cell 22:787-797 ), herpes thymidine kinase promoter ( Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 ), regulatory sequence of metallothionein gene ( Brinster et al., 1982, Nature 296:39-42 Any type of plasmid, granule, YAC, or viral vector can be used to prepare recombinant DNA constructs that can be directly introduced into target tissue sites. Alternatively, viral vectors that selectively infect desired tissues can be used, in which case administration can be accomplished via another route (e.g., systematically).

[0066] B. Small interfering RNA

[0067] In some implementations, the agent is a small interfering RNA (siRNA or RNAi) molecule. RNAi constructs comprise double-stranded RNA that can specifically block the expression of a target gene. "RNA interference" or "RNAi" is the term originally applied to such a phenomenon, where double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. RNAi provides a useful method for suppressing gene expression in vitro or in vivo. RNAi constructs can include small interfering RNA (siRNA), short hairpin RNA (shRNA), and other RNA species that can be cleaved in vivo to form siRNA. The RNAi constructs described herein also include expression vectors ("RNAi expression vectors") capable of producing transcripts that form dsRNA or hairpin RNA in cells and / or transcripts capable of producing siRNA in vivo.

[0068] RNAi expression vectors express (transcribe) RNA, which produces siRNA portions in the cells of the expression construct. Such vectors include a transcription unit containing the following components (1), (2), and (3): (1) a gene element (e.g., a promoter, operon, or enhancer) that plays a regulatory role in gene expression, operatively linked to (2) a “coding” sequence that is transcribed to produce double-stranded RNA (two RNA portions that anneal in the cell to form siRNA, or a single hairpin RNA that can be processed into siRNA), and (3) appropriate transcription initiation and termination sequences. The choice of promoters and other regulatory elements typically varies depending on the intended host cell.

[0069] The RNAi construct contains a nucleotide sequence that hybridizes under physiological conditions with at least a portion of the mRNA transcript of the gene to be repressed (i.e., RNA transcribed from a genomic sequence containing one or more pairs of SNPs listed in Tables 1, 2, or 3). The double-stranded RNA only needs to be sufficiently similar to the native RNA to enable it to mediate RNAi. Therefore, this invention has the advantage of being resistant to sequence variations that may be expected due to genetic mutations, strain polymorphism, or evolutionary differentiation. The number of resistant nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than one per 5 base pairs, or no more than one per 10 base pairs, or no more than one per 20 base pairs, or no more than one per 50 base pairs. Mismatches at the center of the siRNA double strand are most critical and can potentially eliminate target RNA cleavage. In contrast, nucleotides at the 3' end of the siRNA strand complementary to the target RNA do not significantly contribute to the specificity of target identification.

[0070] RNAi constructs can be generated through chemical synthesis or recombinant nucleic acid technology. Endogenous RNA polymerases from treated cells can mediate intracellular transcription, or cloned RNA polymerases can be used for in vitro transcription. RNAi constructs may include modifications to the phosphate-sugar backbone or nucleosides, for example, to reduce sensitivity to cellular nucleases, improve bioavailability, enhance formulation properties, and / or alter other pharmacokinetic properties. For example, the phosphodiester bonds of native RNA can be modified to include at least one of nitrogen or sulfur heteroatoms. Modifications in the RNA structure can be tailored to allow for specific gene repression while avoiding general responses to dsRNA. Similarly, bases can be modified to block adenosine deaminase activity. RNAi constructs can be enzymatically generated or produced through partial / complete organic synthesis, and any modified ribonucleotides can be introduced via in vitro enzymatic or organic synthesis.

[0071] In some embodiments, the subject RNAi construct is a “small interfering RNA” or “siRNA.” These nucleic acids are approximately 19 to 30 nucleotides in length, and even more preferably 21 to 23 nucleotides, for example, fragments corresponding in length to those produced by nuclease “cleavage” of longer double-stranded RNA. siRNA is understood to recruit nuclease complexes by pairing with specific sequences and directing those complexes to target mRNA. As a result, the target mRNA is degraded by nucleases within the protein complex. In a particular embodiment, the 21 to 23 nucleotide siRNA molecule contains a 3' hydroxyl group.

[0072] In some implementations, the RNAi construct is in the form of a short hairpin structure (named shRNA). shRNA can be synthesized exogenously or formed in vivo via transcription from the RNA polymerase III promoter. For example, Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature, 2002, 418:38-9; Yu et al., Proc Natl Acad Sci USA, 2002, 99:6047-52 Examples of preparing and using such hairpin RNAs for gene silencing in mammalian cells are described. Typically, such shRNAs are engineered in cells or animals to ensure sustained and stable suppression of the desired gene. It is known in the art that siRNAs can be produced by processing hairpin RNA in cells.

[0073] Plasmids can be used to deliver, for example, double-stranded RNA as a transcription product. In such embodiments, the plasmid is designed to contain a “coding sequence” for each of the sense and antisense strands of the RNAi construct. The coding sequences can be, for example, the same sequence flanked by inverted promoters, or they can be two separate sequences, each under the transcriptional control of an independent promoter. After transcription of the coding sequences, the complementary RNA transcripts pair bases to form double-stranded RNA.

[0074] C. Ribozyme

[0075] In some implementations, the agent is a ribozyme. Ribozyme molecules designed to catalyze the cleavage of mRNA transcripts are also used to disrupt and prevent downstream effects of mRNA (see, for example, WO 90 / 11364; Sarver et al., 1990, Science 247:1222-1225 (and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy specific mRNAs, hammerhead ribozymes are preferred. Hammerhead ribozymes cleave mRNA at a location determined by a flanking region that forms a complementary base pair with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5'-UG-3'. The construction and production of hammerhead ribozymes are well known in the art, and... Haseloff and Gerlach, 1988, Nature, 334:585-591 A more comprehensive description is provided.

[0076] The ribozymes used in this invention may also include endoRNA ribonucleases (hereinafter referred to as "Cech-type ribozymes"), for example, in Tetrahymena thermophila ( Tetrahymena thermophila The naturally occurring RNA endonuclease (called IVS or L-19 IVS RNA) in ) has been developed by Thomas Cech and collaborators ( Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; WO 88 / 04300; Been and Cech, 1986, Cell, 47:207-216 This has been extensively described. Chech-type ribozymes possess an 8-base-pair active site that hybridizes to a target RNA sequence, subsequently leading to the cleavage of the target RNA. This invention includes those Chech-type ribozymes that target the 8-base-pair active site sequence.

[0077] Similar to antisense methods, ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and can be delivered to cells in vitro or in vivo. Preferred delivery methods involve using DNA constructs that “encode” the ribozyme under the control of strong constitutive polIII or pol II promoters, such that transfected cells will produce sufficient amounts of the ribozyme to disrupt the target mRNA and inhibit its function. Because ribozymes are catalytic, unlike antisense molecules, lower intracellular concentrations are required for efficiency.

[0078] There are currently two basic types of DNases, both of which were identified by Santoro and Joyce (see, for example, U.S. Patent No. 6,110,462). DNases contain a loop structure connecting two arms. These two arms provide specificity by recognizing specific target nucleic acid sequences, while the loop structure provides catalytic function under physiological conditions.

[0079] In short, in order to design an ideal DNase that specifically recognizes and cleaves target nucleic acids, those skilled in the art must first identify a unique target sequence. This can be accomplished using the same methods outlined for antisense oligonucleotides. Preferably, the unique or substantially unique sequence is approximately 18 to 22 nucleotides rich in G / C. A high G / C content helps ensure a stronger interaction between the DNase and the target sequence.

[0080] During the synthesis of DNase, the specific antisense recognition sequence that targets the enzyme to the mRNA is separated, so that it contains the two arms of the DNase, and the DNase loop is placed between these two specific arms.

[0081] For example, methods for preparing and administering DNases can be found in U.S. Patent No. 6,110,462. Similarly, methods for delivering DNA ribozymes in vitro or in vivo include methods for delivering RNA ribozymes, as detailed above. Furthermore, those skilled in the art will recognize that, like antisense oligonucleotides, DNases can optionally be modified to improve stability and resistance to degradation.

[0082] D. Genome editing

[0083] Inhibition of target protein (e.g., APP or PSEN1) expression can be achieved by disrupting gene sequences containing one or more pairs of SNPs named in Tables 1, 2, or 3. An effective method for targeted gene cleavage is the CRISPR system.

[0084] The term CRISPR (an abbreviation for "clustered regularly spaced short palindromic repeats") was originally coined to refer to segments of prokaryotic DNA containing short, repetitive base sequences first discovered in bacteria and archaea. In palindromic repeats, the nucleotide sequence is identical in both directions. Each repeat is followed by a short segment of spacer DNA derived from previously exposed foreign DNA (e.g., viral DNA). Small clusters of Cas (CRISPR-associated) genes are located next to the CRISPR sequence. It was later recognized that the CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements, particularly viral-derived ones, thus providing a form of acquired immunity. RNA containing spacer sequences helps Cas (CRISPR-associated) proteins recognize and cleave foreign DNA. Other RNA-guided Cas proteins cleave the foreign RNA. CRISPR is present in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea, and recently the CRISPR / Cas system has been engineered for targeted gene editing in eukaryotic cells. (The text then abruptly shifts to a different topic:) Ledford (2016), Nature 531 (7593):156–9 .

[0085] A simplified version of the CRISPR / Cas system, CRISPR / Cas9, has been modified to edit the genome. By delivering a Cas9 nuclease, complexed with one or more synthetic guide RNAs (gRNAs), into cells (typically by transfecting cells with one or more expression vectors encoding Cas9 nucleases and gRNAs), the cell's genome can be cut at one or more pre-selected locations, allowing target genes (e.g., genome sequences containing one or more pairs of SNPs listed in Tables 1, 2, or 3) to be removed and / or replaced with new sequences.

[0086] In this context, an expression vector (e.g., a viral vector) carrying a coding sequence of one or more gRNAs specific to genomic sequences containing one or more pairs of SNPs listed in Tables 1, 2, or 3 can be introduced into cells, where the endogenous genomic sequence containing the specified SNP will be knocked out (e.g., endothelial cells or endothelial progenitor cells, or neuronal cells). The same expression vector may optionally carry a coding sequence for a CRISPR / Cas9 nuclease or its equivalent. Alternatively, a separate expression vector can be used to introduce the CRISPR / Cas9 nuclease coding sequence to express the CRISPR / Cas9 nuclease in target cells. In some cases, more than one (e.g., two) different gRNAs are used to ensure the removal and / or replacement of the target genomic sequence (e.g., a target genomic sequence containing one or more pairs of SNPs listed in Tables 1, 2, or 3).

[0087] Other gene editing systems that can be used to implement the present invention include TALEN (transcription activator-like effector nuclease), ZFN (zinc finger nuclease), and base editing, as well as newly developed technologies such as homing endonucleases and megNs (which target and cut DNA sequences) and prime editing (which generate RNA templates for gene alteration).

[0088] III. Pharmaceutical Composition and Administration

[0089] The present invention also provides pharmaceutical or physiological compositions comprising an effective amount of one or more agents for use in preventative and therapeutic applications in the methods of the present invention. Such pharmaceutical or physiological compositions further comprise one or more pharmaceutically or physiologically acceptable excipients or carriers. For example, one exemplary composition of the present invention comprises one or more expression vectors encoding a CRISPR system (e.g., a Cas9 nuclease or equivalent and one or more sgRNAs) and one or more physiologically acceptable excipients or carriers, or substantially composed of the above. Another exemplary composition of the present invention comprises one or more expression vectors encoding one or more repressive oligonucleotides (e.g., small repressive RNA molecules or antisense DNA or RNA oligonucleotides) and one or more physiologically acceptable excipients or carriers, or substantially composed of the above. The pharmaceutical compositions of the present invention are applicable to various drug delivery systems. Suitable formulations for use in the present invention are found in […]. Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) For a brief overview of drug delivery methods, see [link to relevant documentation]. Langer, Science 249: 1527-1533 (1990) .

[0090] The pharmaceutical compositions of the present invention can be administered via various routes, such as oral, nasal, subcutaneous, percutaneous, intramuscular, intravenous, or intracranial. A preferred route of administration is local delivery of the pharmaceutical composition to the recipient's target disease-related organ or tissue at a predetermined daily dose. Appropriate doses can be administered as a single daily dose or in fractions provided at appropriate intervals, such as sub-dose administration twice, three, four, or more times daily.

[0091] To prepare pharmaceutical compositions containing one or more active agents of the present invention, an inert and pharmaceutically acceptable carrier is also used. Typically, the pharmaceutical carrier can be solid or liquid. Preparations in solid form include, for example, powders, tablets, dispersible granules, capsules, suppositories, and sachets. The solid carrier can be one or more substances that can also be used as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, or tablet disintegrant; it can also be an encapsulation material.

[0092] In powders, the carrier is typically a finely ground solid mixed with the finely ground active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in a suitable proportion and compacted to the desired shape and size.

[0093] To prepare a pharmaceutical composition in suppository form, a mixture of low-melting-point waxes, such as fatty acid glycerides and cocoa butter, is first melted, and the active ingredient is dispersed therein by, for example, stirring. The molten, homogeneous mixture is then poured into a suitably sized mold and allowed to cool and solidify.

[0094] Powders and tablets preferably contain about 5% to about 70% of the active ingredient by weight. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, low-melting-point wax, cocoa butter, etc.

[0095] Pharmaceutical compositions may include an active agent and a formulation providing an encapsulating material as a carrier, wherein one or more pharmaceutical agents (with or without other carriers) are surrounded by the carrier, such that the carrier is thus bound to the pharmaceutical agent. In a similar manner, capsules may also be included. Tablets, powders, capsules, and tablets can be used as solid dosage forms suitable for oral administration.

[0096] Liquid pharmaceutical compositions include, for example, solutions, suspensions, and emulsions suitable for oral or parenteral administration. Examples of liquid compositions suitable for parenteral administration include sterile aqueous solutions of the active ingredient or sterile solutions of the active ingredient in solvents including water, buffered water, saline, PBS, ethanol, or propylene glycol. The composition may contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, tonic modifiers, wetting agents, detergents, etc.

[0097] Sterile solutions can be prepared by dissolving the active component in a desired solvent system and then sterilizing the resulting solution by passing it through a membrane filter, or alternatively, by dissolving the sterile component in a pre-sterilized solvent under aseptic conditions. The resulting aqueous solution can be used as is or lyophilized, with the lyophilized preparation combined with a sterile aqueous carrier prior to application. The pH of the preparation is typically from 3 to 11, more preferably from 5 to 9, and most preferably from 7 to 8.

[0098] Pharmaceutical compositions containing one or more active agents may be administered for preventative and / or therapeutic treatment. In therapeutic applications, a composition is administered to a patient already diagnosed with Alzheimer's disease in an amount sufficient to prevent, cure, reverse, or at least partially alleviate or stop the symptoms of the disease and its complications, such as the onset, progression, duration, and severity of the disease. An amount sufficient to achieve this purpose is defined as a "therapeuticly effective dose." The effective dose for this purpose will depend on the severity of the disease, the patient's weight and general condition, and the nature of the active agent.

[0099] In prophylactic applications, a pharmaceutical composition containing one or more active agents is administered to a patient susceptible to or at risk of developing Alzheimer's disease in an amount sufficient to delay or prevent the onset of symptoms. Such an amount is defined as the "preventative effective dose." In this application, the precise amount of the active agent again depends on the patient's health status and weight, as well as the nature of the active agent.

[0100] The composition can be administered single or multiple times, with the dosage level and mode chosen by the treating physician. In any case, the pharmaceutical formulation should provide a quantity of the drug that is therapeutically or preventively effective in inhibiting the expression levels of the target protein and Aβ plaque formation in the patient.

[0101] IV. Therapeutic applications using nucleic acids

[0102] Various conditions can be treated through therapeutic methods involving the introduction of nucleic acids into cells that encode one or more agents that disrupt a genomic sequence containing one or more pairs of SNPs listed in Tables 1, 2, or 3, or that inhibit mRNA encoded by such genomic sequences (e.g., antisense or miRNA, or Cas9 nuclease and sgRNA), causing transcription of the coding sequence and the production of polypeptide or oligonucleotide agents within the cells. For a discussion of the application of gene therapy in the treatment of hereditary and acquired diseases, see Miller Nature 357:455-460 (1992); and Mulligan Science 260:926-932 (1993).

[0103] A. Vectors used for gene delivery

[0104] For delivery to cells or organisms, polynucleotides encoding one or more active agents can be incorporated into a vector. Examples of vectors for such purposes include expression plasmids capable of directing the expression of nucleic acids in target cells. In other cases, the vector is a viral vector system in which the polynucleotide is incorporated into the genome of a virus capable of transfecting target cells. In one embodiment, the encoding polynucleotide can be operatively linked to expression and control sequences that can direct the expression of peptides or oligonucleotides in desired target host cells. Thus, under appropriate conditions, expression of peptide or oligonucleotide inhibitors in target cells can be achieved.

[0105] B. Gene delivery system

[0106] Viral vector systems useful in the expression of polypeptides or oligonucleotides that disrupt genomic sequences containing one or more pairs of SNPs listed in Tables 1, 2, or 3 include, for example, naturally occurring or recombinant viral vector systems. Depending on the specific application, suitable viral vectors include replicative viral vectors, replication-defective viral vectors, and conditionally replicative viral vectors. For example, viral vectors can be derived from the genomes of human or bovine adenovirus, vaccinia virus, herpesvirus, adeno-associated virus (AAV), mouse parvovirus (MVM), HIV, Sindbis virus, and retroviruses (including but not limited to Rous sarcoma virus and lentiviruses), as well as MoMLV. Typically, the target coding sequence (e.g., the coding sequence encoding the polypeptide or oligonucleotide active agent of the present invention) is inserted into such a vector to allow the gene construct to be packaged, typically along with viral DNA, and then used to infect susceptible host cells and express the target coding sequence.

[0107] As used herein, "gene delivery system" refers to any means for delivering a target polynucleotide sequence to target cells. In some embodiments of the invention, nucleic acids are delivered via appropriate linkers (e.g., DNA linkers). Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92 / 06180 Or via an ultrasonic microbubble delivery system ( Lan HY et al., J. Am Soc. Nephrol. 14:1535-1548 Nucleic acids can conjugate with cell receptor ligands to facilitate uptake (e.g., invagination of the fovea and internalization of endosomes). For example, nucleic acids can be linked to asialo-oromucocid via the polylysine moiety; asialo-oromucocid is a ligand for the desialyl glycoprotein receptor on hepatocytes.

[0108] Similarly, the viral envelope used to package gene constructs containing target nucleic acids can be modified by adding receptor ligands or antibodies specific to the receptor to allow receptor-mediated endocytosis into specific cells (see, for example, WO 93 / 20221, WO 93 / 14188, and WO 94 / 06923). In some embodiments of the invention, the DNA constructs of the invention are linked to viral proteins such as adenovirus particles to facilitate endocytosis. Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991) In other embodiments, the active agent of the present invention may include a microtubule inhibitor (WO / 9406922), a synthetic peptide mimicking influenza virus hemagglutinin (WO / 9406922), etc. Plank et al., J. Biol. Chem. 269:12918-12924 (1994) ) and nuclear localization signals such as SV40 T antigen (WO 93 / 19768).

[0109] Retroviral vectors may also be useful for introducing the coding sequences of the polypeptide or oligonucleotide active agents of the present invention into target cells or tissues. Retroviral vectors are generated by genetically manipulating retroviruses. The viral genome of a retrovirus is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy, which integrates with high stability and efficiency into the chromosomal DNA of the transfected cell. The integrated DNA copy is called a provirus and is inherited by daughter cells like any other gene. The wild-type retroviral genome and proviral DNA have three genes: the gag gene, the pol gene, and the env gene, flanked by two long terminal repeats (LTRs). The gag gene encodes an internal structural (nucleocapsid) protein; the pol gene encodes an RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes a viral envelope glycoprotein. The 5' LTR and 3' LTR are used to promote transcription and polyadenylation of viral particle RNA. Adjacent to the 5' LTR are the sequences required for genomic reverse transcription (tRNA primer binding site) and efficient encapsulation of viral RNA into particles (Psi site) (see [link to relevant documentation]). Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984) ).

[0110] The design of retroviral vectors is well known to those skilled in the art. In short, if the viral genome lacks the sequences required for capsidation (or packaging retroviral RNA into infectious viral particles), the result is a cis-acting defect that prevents capsidation of the genomic RNA. However, the resulting mutants are still able to direct the synthesis of all viral particle proteins. Retroviral genomes that have already lacked these sequences, as well as cell lines containing mutant genomes stably integrated into the chromosome, are well known in the art and used to construct retroviral vectors. The preparation and uses of retroviral vectors are described in numerous publications, including, for example, European Patent Application EPA 0 178 220; and U.S. Patent 4,405,712. Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al. Biotechniques 7:981-990 (1989); Miller (1992), ibid.; Mulligan (1993), ibid. ; and WO 92 / 07943.

[0111] Retroviral vector particles are prepared by inserting a desired nucleotide sequence into a retroviral vector and packaging the vector with a retroviral capsid protein using a packaging cell line. The resulting retroviral vector particles cannot replicate in host cells but can be integrated into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, patients are able to produce, for example, peptides or polynucleotide activators useful in the method of this invention, thereby restoring target cells (e.g., brain endothelial cells) to a normal phenotype.

[0112] Packaging cell lines used to prepare retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins for packaging but not infectious viral particles. On the other hand, the defective retroviral vectors used lack these structural genes but encode the remaining proteins required for packaging. To prepare packaging cell lines, an infectious clone of the desired retrovirus can be constructed, in which the packaging site has been deleted. Cells containing such a construct will express all the structural viral proteins, but the introduced DNA will not be packaged. Alternatively, packaging cell lines can be generated by transforming cell lines with expression plasmids encoding appropriate nuclear and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.

[0113] Many packaging cell lines suitable for this invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317, and PG13 (see [link to previous section]). Miller et al., J. Virol. 65:2220-2224 (1991) Examples of other packaging cell lines are in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988), ibid.; and Miller (1990), ibid. As described in the text.

[0114] Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins can be used. Alternatively, diphilic or heterophilic envelope proteins, such as those produced by PA317 and GPX packaging cell lines, can be used to package retroviral vectors.

[0115] C. Pharmaceutical preparations

[0116] When used for pharmaceutical purposes, nucleic acids encoding peptides or oligonucleotide active agents are typically prepared in a suitable buffer. A suitable buffer can be any pharmaceutically acceptable buffer, such as phosphate-buffered saline or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to a person skilled in the art, for example... Good et al. Biochemistry 5:467 (1966) Those described.

[0117] The composition may additionally include stabilizers, enhancers, or other pharmaceutically acceptable carriers or mediators. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds, such as those that stabilize the nucleic acids and any related carriers of the present invention. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are known and include, for example, phenols and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in... Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) Furthermore, nucleic acids encoding the polypeptide or oligonucleotide active agents of the present invention can be packaged in lipid nanoparticles to facilitate their crossing of the blood-brain barrier.

[0118] D. Administration of the preparation

[0119] Formulations containing a polynucleotide sequence encoding a polypeptide or oligonucleotide active agent can be delivered to a target tissue or organ using any delivery method known to those skilled in the art. In some embodiments of the invention, the polynucleotide sequence is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or for oral ingestion / nasal inhalation or for topical application.

[0120] Preparations containing the target nucleic acid are typically administered directly to cells. Cells can be provided as part of a tissue, such as red blood cells as part of the circulatory system, or as isolated cells, such as in tissue culture. Cells can be provided in vivo, in vitro, or ex vivo.

[0121] The formulation can be introduced into the target tissue in vivo or in vitro by various methods. In some embodiments of the invention, the target nucleic acid is introduced into cells by methods such as microinjection, calcium phosphate precipitation, liposome fusion, ultrasound, electroporation, or biolistics. In further embodiments, the nucleic acid is directly taken up by the target tissue or organ associated with the disease or condition being treated; for example, intracranial injection is appropriate when the targeted cells are brain endothelial cells.

[0122] In some embodiments of the present invention, the target nucleic acid is administered ex vivo to cells or tissues explanted from a patient and then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23(1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc.Natl. Acad. Sci. USA 93(1):402-6 (1996).

[0123] The effective dose of a formulation will vary depending on many different factors, including the route of administration, target site, patient physiological status, and other medications administered. Therefore, titration of the therapeutic dose will be necessary to optimize safety and efficacy. In determining the effective amount of a vector to be administered, the physician should evaluate the specific nucleic acid used, the diagnosed disease state, the patient's age, weight and overall condition, circulating plasma levels, vector toxicity, disease progression, and the production of anti-vector antibodies. The dose will also be determined by the presence, nature, and extent of any adverse side effects accompanying the administration of the specific vector. For example, antisense oligonucleotides in doses of 1 mg to 1000 mg, 10 mg to 200 mg, or 20 mg to 100 mg can be delivered to the patient via intravenous injection at a frequency of weekly, bi-weekly, or monthly over a period of at least one month to three months or longer. For CRISPR editing targeting genomic regions, as another example, every 5 × 10 5 Transfection of cells (e.g., hCMEC / D3 cells) with a vector carrying a gene encoding Cas9 and at least one pair of sgRNAs at doses ranging from 0.5 µg to 50 µg; 1 µg to 20 µg; or 2 µg to 10 µg. For CRISPR editing targeting genomic regions in human patients, lipid nanoparticles carrying sgRNA and mRNA encoding Cas9 are delivered via intravenous injection in doses ranging from 0.01 mg / kg body weight to 2 mg / kg body weight; 0.02 mg / kg body weight to 1.0 mg / kg body weight; 0.05 mg / kg body weight to 0.5 mg / kg body weight; or 0.10 mg / kg body weight to 0.30 mg / kg body weight over a period of 1 to 4 weeks, once to three times.

[0124] V. Package Products

[0125] The present invention also provides kits for treating autosomal dominant genetic disorders (e.g., Alzheimer's disease (AD)) in people in need or reducing the risk of such disease in people in need using the methods according to the invention. Kits typically include a container containing (1) a pharmaceutical composition having an effective amount of one or more active agents capable of disrupting a genomic sequence containing at least one pair (possibly multiple pairs) of SNPs listed in Tables 1, 2, or 3, or inhibiting / eliminating translation from its RNA transcript; and (2) informative material containing instructions on how to dispense the pharmaceutical composition, including a description of the type of patient who may be treated (e.g., a human patient with Alzheimer's disease, particularly familial AD, or a human patient at increased risk of the disease), the schedule of administration (e.g., dosage and frequency), and the route of administration, etc. In some cases, the kit includes two or more containers to provide multiple pharmaceutical compositions, each containing an effective amount of at least one active agent, such as a component encoding a CRISPR system (e.g., a Cas9 nuclease or equivalent and one or more sgRNAs) or a vector encoding siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotides targeting a genomic sequence containing a specified SNP. Optionally, the kit may also include one or more additional containers, each containing at least one agent useful for sequencing at least a portion of the human genome (especially a genomic sequence containing the relevant SNPs listed in Tables 1, 2, or 3, and a disease-related allele located in the same genomic region, such as the coding sequence of the same gene, or the same exon of a gene (e.g., APP or PSEN1).

[0126] Example

[0127] The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same or similar results.

[0128] introduction

[0129] Alzheimer's disease (AD) accounts for 60% to 80% of dementia, affecting more than 55 million people worldwide, and currently there is no effective treatment. AD is a complex neurodegenerative disease attributed to a combination of genetic, environmental, and lifestyle factors. Familial AD (FAD) is primarily caused by single-gene mutations in APP, PSEN1, and PSEN2; to date, more than 400 FAD-related mutations have been reported. These mutations contribute to amyloidosis by increasing the production of amyloid-β (Aβ) or by Aβ peptides that tend to aggregate. Therefore, silencing these three FAD-related genes (which would lead to reduced Aβ production) is a potential therapeutic approach to alleviate amyloid-related symptoms. However, it has been shown that complete deletion of any of these genes leads to serious side effects related to neuronal function, while the deletion of a single copy of APP or PSEN1 due to nonsense mutations or frameshifts does not result in abnormal neurological symptoms. This supports the view that FAD is not caused by haploid deficiency and that a single functional copy of APP or PSEN1 is sufficient to maintain the integrity of cellular function. Therefore, eliminating alleles containing pathogenic mutations using CRISPR technology while maintaining normal alleles is a feasible treatment for FAD.

[0130] Allele-specific targeting can eliminate mutated alleles without damaging normal alleles, making it ideal for treating autosomal dominant diseases such as FAD. The CRISPR-Cas9 system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. After the sgRNA binds to a target DNA sequence in the genomic DNA adjacent to a protospacer neighboring motif (PAM), the Cas9 nuclease then induces a double-strand break at the target sequence, disrupting gene expression by inducing indel or frameshift mutations. The seed sequence is a 5- to 12-bp sequence within the sgRNA that covers a short DNA region in the target sequence adjacent to the PAM site; the seed sequence and PAM indicate low mismatch tolerance. Once a mutation forms a new PAM located within the seed sequence, the Cas9 nuclease can then target and cleave the sequence carrying the mutation, ultimately leading to silencing of the mutated gene.

[0131] This promising FAD treatment strategy involves targeting FAD mutations and disrupting the mutated alleles. To date, the effectiveness of this approach has been validated in the ablation of two FAD-causing mutations, both showing reduced AD-related symptoms. However, over 400 mutations have been reported in FAD, of which 161 are identified as pathogenic according to the ACMG (American College of Medical Genetics and Genomics) standard classification. To cover all FAD cases by targeting all mutations site-by-site, over 400 targeting tools would be required, making clinical translation difficult. Figure 1A ).

[0132] Conversely, SNP pairs that target pathogenic mutations in cis can also specifically disrupt mutated alleles, independent of the specific mutation involved. Figure 1B These SNP pairs are naturally occurring variants that exist at a heterozygous and high frequency in the population, making them useful for identifying any allele containing the mutation site in most people. Disruption of gene expression can then be achieved through exon excision, which leads to frameshifts and the formation of premature stop codons. Figure 2 This mutation-independent genome editing approach can achieve increased coverage with fewer CRISPR targeting tools.

[0133] This invention relates to the development of a mutation-independent, allele-specific CRISPR targeting strategy for the treatment of febrile autosomal dominant disease (FAD). First, an analytical pipeline was developed to analyze a wide variety of rare autosomal dominant disorders involving multiple pathogenic mutations, including the identification of common targetable SNPs and the frequency of SNP pair combinations in the population, as well as the design of sgRNAs for these SNPs. This analytical pipeline was applied to study FAD, and then targeting strategies were developed using the identified combinations of target sites and their population frequencies (Tables 1-3, Table 5). Simultaneously, a list of targeting sgRNAs for APP and PSEN1 gene disruption was generated (Tables 4, Table 6). Analysis showed that a single targeting tool was sufficient to cover 48% of individuals with any APP mutation. Figure 2 , Figure 3 Furthermore, targeting the optimal SNP combination requires only 3 pairs of CRISPR targeting tools to cover 76% of FAD patients carrying any type of APP mutation (Table 3). The inventors of this application systematically validated the targeting specificity and efficiency of all sgRNAs targeting the optimal SNP combination sites using an alternative reporter subsystem. Figure 4 , Figure 5 , Figures 6A to 6D and Figure 7To verify the efficacy of gene editing methods for APP, induced pluripotent stem cells (iPSCs) derived from FAD patients carrying the APP mutation were used as an experimental model. By using top-ranked sgRNA candidates targeting SNPs rs6516719 and rs2830026 to edit the APP genome, the inventors successfully eliminated the expression of the mutated alleles, resulting in reduced Aβ42 production in iPSC-induced neurons. This validation further supports the potential of this method as an effective therapeutic strategy for FAD. Figure 8A and Figure 8B ).

[0134] Table 1: Candidate target SNP sites in the APP gene and their respective population frequencies

[0135] Table 2: Combinations of two SNP sites in the APP gene and their respective population frequencies

[0136] Table 3: Combinations of three SNP loci in the APP gene and their population frequencies

[0137] Table 4: Sequences of sgRNAs used to target top-ranking candidate SNP sites in the APP gene

[0138] Table 5: Candidate target SNP sites and population frequencies in the PSEN1 gene

[0139] Table 6: Sequences of sgRNAs used to target top-ranking candidate SNP sites in the PSEN1 gene

[0140] method

[0141] Molecular cloning

[0142] The sgRNA-Cas9 plasmid was modified from the pX458--AAV-EFS::NLS-SpCas9-NLS-3xHA-SPA;U6::BsaI-sgRNA and pX601-AAV-EFS::NLS-SaCas9-NLS-3xHA-SPA;U6::BsaI-sgRNA constructs. An mCherry reporter was added to check transfection efficiency. The mCherry fragment was obtained from another plasmid using polymerase chain reaction (PCR), while the original plasmid was cut by double restriction digestion. The mCherry fragment was inserted into the new plasmid via Gibson assembly. Finally, the new plasmid was digested with the BsaI restriction enzyme to generate the sgRNA-Cas9 vector with the mCherry reporter. The template plasmid used was a modified version of pCAG-EGxxFP (Addgene plasmid 50716). The template vector was prepared by double restriction digestion. Dephosphorylated DNA oligonucleotides used for the template and sgRNA spacer were synthesized by Invitrogen (Thermo Fisher Scientific). First, the forward and reverse primers were phosphorylated using T4 polynucleotide kinase and annealed together in a thermal cycler. Then, the products were inserted into their corresponding vectors using T7 ligase. To amplify the plasmid to a sufficient quantity for transfection, the plasmid was transformed into NEB-Stable competent *E. coli* cells according to the manufacturer's protocol (New England Biolabs). The bacteria were allowed to grow overnight in a shaking incubator. Finally, the plasmid was prepared using DNA extraction.

[0143] transfection

[0144] One to two days before transfection, HEK293 cells were seeded on 100 mm culture dishes. Prior to transfection, cells at 50% to 70% confluence were digested with trypsin and suspended in Dulbecco's modified Eagle's medium (DMEM). Then, following the manufacturer's (Thermo Fisher Scientific) protocol, 1.5 × 10⁶ cells were transfected. 5Cells were added to a DNA-Lipofectamine 3000 complex in Opti-MEM (OMEM), which consisted of 1 μg template plasmid and / or 0.5 μg Cas9-sgRNA construct, 2 μl Lipofectamine 3000 reagent, and 2.5 μl p3000 reagent. The mixture was then transferred to 12-well plates and topped with DMEM to 1 ml. Cells were collected 24 h post-transfection.

[0145] Alternative Report Subtest

[0146] The EGFP coding sequence is divided into two segments separated by a stop codon and a template sequence. These two segments contain the same 200 bp homologous region. Following a CRISPR-Cas9 nuclease-induced double-strand break, the cell may undergo homology-directed repair mediated by either single-strand annealing or other methods to repair the DNA. The two EGFP segments will then combine to form the complete EGFP coding sequence. Figure 2 As a result, EGFP was expressed, and a green fluorescent signal could be detected. Compared to other strategies such as the T7EI assay, the EGFP assay provides a highly efficient and rapid detection of cleavage activity. Cells were fixed with 4% paraformaldehyde after collection and stained with DAPI (0.3% DPBST, 1:5000) to visualize the nuclei. Imaging was performed using a Leica TCS SP8 confocal system. The acquired images were analyzed using ImageJ, and the number of cells with fluorescent signals was measured by ImageJ. The signal threshold for each site was adjusted to minimize background signal compared to a control group that only received template plasmid. Editing efficiency was calculated based on the percentage of cells with EGFP signals to the number of cells with mCherry signals.

[0147] All patents, patent applications and other publications (including GenBank accessions or equivalents) cited in this application are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for treating or reducing the risk of said disease in a person in need, comprising administering to the person an effective amount of a composition that destroys a genomic sequence containing at least one pair of single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5, wherein the person is heterozygous at all said SNPs and has a gene mutation associated with said disease, said gene mutation being located within a gene exon of all said SNPs.

2. The method of claim 1, further comprising sequencing at least a portion of the human genome containing the exons of the gene prior to the administration step.

3. The method according to claim 1, wherein the autosomal dominant disease is Alzheimer's disease (AD).

4. The method according to claim 3, wherein the gene is an amyloid precursor protein (APP) gene or a presenilin-1 (PSEN1) gene.

5. The method of claim 3, wherein the person has been diagnosed with AD, or the person has not been diagnosed with AD but has known risk factors for AD.

6. The method of claim 1, wherein the at least one pair of SNPs includes any pair of SNPs listed in Table 1.

7. The method of claim 6, wherein the at least one pair of SNPs comprises rs2830026 and rs6516719.

8. The method of claim 1, wherein the at least one pair of SNPs includes any pair of SNPs listed in Table 5.

9. The method of claim 8, wherein the at least one pair of SNPs comprises rs17125457 and rs17408630.

10. The method of claim 1, wherein the at least one pair of SNPs comprises any two pairs of SNPs listed in Table 2.

11. The method of claim 10, wherein the at least one pair of SNPs comprises rs2830026 and rs6516719; rs2248682 and rs8130594.

12. The method of claim 1, wherein the at least one pair of SNPs comprises any three pairs of SNPs listed in Table 3.

13. The method of claim 12, wherein the at least one pair of SNPs comprises rs2830026 and rs6516719; rs10154121 and rs2070655; rs2830046 and rs2070654.

14. The method of claim 1, wherein the composition comprises one or more vectors encoding a nuclease guided by a small guide RNA (sgRNA) and at least two sgRNAs targeting at least one pair of SNPs listed in Table 1, Table 2, Table 3 or Table 5.

15. The method of claim 14, wherein at least two sgRNAs comprise at least two nucleotide sequences listed in Table 4 or Table 6.

16. The method according to claim 14, wherein the endonuclease is a Cas9 nuclease.

17. The method according to any one of claims 14 to 16, wherein each of the one or more vectors is a viral vector.

18. The method according to any one of claims 14 to 17, wherein the composition is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or intracranial injection, or by oral or nasal administration.

19. The method of claim 18, wherein the composition is administered in the form of a solution, suspension, powder, paste, tablet or capsule.

20. A kit product for treating autosomal dominant diseases in people in need or reducing the risk of said diseases in people in need, comprising a container containing a composition that disrupts a genomic sequence containing at least one pair of single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5.

21. The kit product of claim 20, wherein the autosomal dominant disease is Alzheimer's disease (AD).

22. The kit product of claim 20, wherein the composition comprises one or more vectors encoding a nuclease guided by a small guide RNA (sgRNA) and two sgRNAs targeting at least one pair of SNPs listed in Tables 1, 2, 3 or 5.

23. The kit product according to claim 20, wherein the endonuclease is a Cas9 nuclease.

24. The kit product according to claim 20, wherein each of the one or more carriers is a viral carrier.

25. The kit product of claim 20, wherein the composition is formulated for subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or intracranial injection, or is formulated for oral or nasal administration.

26. The kit product according to claim 20, wherein the composition is in the form of a solution, suspension, powder, paste, tablet or capsule.

27. The kit of claim 20, further comprising a second container containing one or more reagents for sequencing at least a portion of the human genome sequence containing at least one pair of single nucleotide polymorphisms (SNPs) listed in Tables 1, 2, 3, or 5.

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