Differential knockout of heterozygous ELANE alleles

Abstract

1. A method for inactivating a mutant allele of a neutrophil elastase gene (ELANE gene) gene having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) in a cell, the method comprising: inactivating a mutant allele of a neutrophil elastase gene (ELANE gene) gene having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) in a cell; rs8l07095, rs10424470, and rs78302854, and the cell is heterozygous at one or more polymorphic sites selected from the group consisting of rs474, rs3761007, rs172l6649, rs10469327, rs8l07095, rs10424470, and rs78302854, and the method includes introducing into the cell a composition comprising a CRISPR nuclease or a sequence encoding a CRISPR nuclease and a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, wherein the complex of the CRISPR nuclease and the first RNA molecule affects a double-stranded break in the mutant allele of the ELANE gene. [Selection diagram] None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 743,309, filed October 9, 2018, U.S. Provisional Application No. 62 / 723,941, filed August 28, 2018, and U.S. Provisional Application No. 62 / 667,536, filed May 6, 2018, the contents of each of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including by parenthetical references. The disclosures of all publications mentioned in this application are incorporated by reference in their entirety into this application to provide further description of the art to which this invention pertains and features of such art that may be utilized in conjunction with the present invention.

[0003] A reference to an array list This application incorporates by reference the nucleotide sequence present in the file entitled "190506_90522-A-PCTjSequenceJListing_ADR.txt", which is contained in a text file having a size of 249 kilobytes, created on May 3, 2019 in IBM-PC machine format having an operating system compatible with MS-Windows, and submitted as part of this application on May 6, 2019. [Background technology]

[0004] There are several types of DNA variations in the human genome, including insertions and deletions, differences in copy numbers of repetitive sequences, and single nucleotide polymorphisms (SNPs). SNPs are DNA sequence variations that occur when a single base (adenine (A), thymine (T), cytosine (C), or guanine (G)) in the genome differs between human subjects or between a pair of chromosomes in an individual. Over the years, different types of DNA variations have been central to the research community, either as markers in studies to pinpoint inherited traits or disease causation, or as potential causes of genetic disorders. SNPs are usually considered to be benign and not disease-causing.

[0005] Genetic disorders are caused by one or more abnormalities in the genome. Genetic disorders may be considered to be either "dominant" or "recessive". Recessive genetic disorders are disorders that require two copies (i.e., two alleles) of an abnormal / missing gene to be present. In contrast, dominant genetic disorders involve a gene or group of genes that exhibit dominance over a normal (functional / healthy) gene or group of genes. Thus, in dominant genetic disorders, only one copy (i.e., allele) of the abnormal missing gene is required to cause or contribute to the symptoms of a particular genetic disorder. Such mutations include, for example, gain-of-function mutations, where the altered gene product has a new molecular function or a new pattern of gene expression. Gain-of-function mutations are generally dominant-negative mutations. An example of a dominant-negative mutation is haploinsufficiency, where one allele mutates and loses function, leaving a single wild-type allele that does not make enough of a required protein for a particular cellular function. Other examples include dominant-negative mutations that have a gene product that acts antagonistically to the wild-type allele.

[0006] Neutropenia Neutropenia is defined as a reduction in the absolute number of circulating neutrophils and is usually diagnosed by measuring the absolute neutrophil count (ANC) in peripheral blood. The severity of neutropenia is considered mild if the ANC is 1000-1500 / μL, moderate if the ANC is 500-1000 / μL, or severe if the ANC is less than 500 / μL (Boxer 2012).

[0007] Neutropenia can be classified as congenital (genetic) or acquired. The two main types of congenital conditions, usually autosomal dominant inheritance, are cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). Cyclic neutropenia is characterized by neutrophil counts that fluctuate from normal levels to zero, whereas severe congenital neutropenia (SCN) is characterized by a very low ANC (500 / mT) at birth, arrest of myelopoiesis maturation in the bone marrow at the promyelocytic / myelocytic stage, and early onset of bacterial infections (Carlsson et al. 2012; Horwitz et al. 2013).

[0008] SCN may be diagnosed by measuring very low ANC in the blood and examining bone marrow aspirates to identify arrest of bone marrow maturation (Dale 2017). SCN is usually diagnosed before 6 months of age, whereas the diagnosis of CyN is generally made during the first 2 years of life or later, with the primary clinical finding being recurrent acute oral medical illness. When SCN is suspected, a bone marrow examination is often required, with cytogenetic bone marrow studies now being definitive, to rule out malignant hematological disorders, determine cellularity, assess bone marrow maturation, and detect signs of the exact cause. Antineutrophil antibody assays, immunoglobulin assays (Ig GAM), lymphocyte immunophenotyping, pancreatic markers (serum trypsinogen and fecal elastase), and fat-soluble vitamin levels (vitamins A, E, and D) are also of interest when evaluating SCN and CyN (see Donadieu 2011).

[0009] SCN can be inherited in an autosomal-recessive (HAX1, G6PC3), autosomal-dominant (ELANE, GFI1) or X-linked (WAS) manner, or can occur sporadically (Carlsson et al. 2012; Boxer 2012).

[0010] Cyclic and congenital neutropenia are most often caused by mutations in the "neutrophil elastase gene" (ELANE gene) - the gene for neutrophil elastase. ELANE gene mutations are identified in 40-55% of SCN patients, with men and women equally affected (Donadieu et al., 2011; Dale 2017). Mutations in the ELANE gene have been associated with autosomal and sporadic cases of SCN (Carlsson et al., 2012). To date, more than 200 different ELANE mutations have been identified, randomly distributed across all exons and introns 3 and 4 (Skokowa et al., 2017). More than 120 distinct ELANE gene mutations associated with CyN and SCN are now known, e.g., C151Y and G214R, which are particularly associated with poor prognosis (for a comprehensive list of ELANE genes associated with CyN and SCN, see Makaryan et al., 2012; see also Germeshausen et al., 2013).

[0011] ELANE encodes neutrophil elastase (NE), which is involved in the function of neutrophil extracellular traps, a network of fibers that bind pathogens. Several studies suggest that mutated ELANE products disrupt neutrophil production in the bone marrow, causing neutropenia. These studies show that mutations in NE trigger the unfolded protein response (UPR), leading to cell loss during the neutrophil formation process in the bone marrow (Makaryan et al., 2017).

[0012] Current treatment Granulocyte colony-stimulating factor (G-CSF) is considered the first-line treatment for SCN (Connelly, Choi, and Levine 2012). G-CSF stimulates the production of more neutrophils and delays apoptosis (Schaffer and Klein 2007). Although 10% of SCN patients still die from severe bacterial infections or sepsis, overall survival is now estimated to be over 80%, including patients who develop malignancies (Skokowa et al. 2017). Although G-CSF therapy has been successful in preventing deaths from sepsis, long-term treatment has been found to be associated with an increased risk of developing myelodysplastic syndromes (MDS) or leukemia in SCN patients. The most common leukemia in the SCN is AML, but acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), and biphenotypic leukemia have also been reported in the literature (Connelly, Choi, and Levine 2012). It has previously been reported that patients with strong G-CSF response (dose <8 μg / kg / day) had a cumulative incidence of 15% for the development of MDS / leukemia 15 years after the time of G-CSF, whereas patients with poor response to G-CSF despite high doses had an incidence of 34% (Rosenberg et al. 2010).

[0013] Hematopoietic stem cell transplantation (HSCT) is an alternative curative treatment for patients who do not respond to G-CSF therapy or who develop AML / MDS. However, chronic neutropenic patients undergoing HCT are at high risk of developing infectious complications, such as fungal and graft-versus-host disease (Skokowa et al., 2017). Furthermore, HCT requires a matched related donor to improve survival, but most patients do not have a matched donor available (Connelly, Choi, and Levine 2012). Summary of the Invention

[0014] Disclosed are techniques for knocking out expression of dominant mutant alleles by either destroying the dominant mutant allele or degrading the resulting mRNA.

[0015] The present disclosure provides methods that utilize at least one naturally occurring heterozygous nucleotide difference or genetic polymorphism (such as a single nucleotide polymorphism (SNP)) to distinguish / distinguish between two alleles of a gene, where one allele carries a mutation such that the mutation encodes a mutant protein that results in a disease phenotype (the "mutant allele") and the other allele encodes a functional protein (the "functional allele").

[0016] The present invention provides a method of utilizing at least one heterozygous SNP of a gene that expresses a dominant mutant allele in a certain cell or subject. In the present invention, the SNP utilized may or may not be associated with a disease phenotype. In the present invention, the RNA molecule comprising a guide sequence targets the mutant allele of a gene by targeting the nucleotide base that is present at the heterozygous SNP in the mutant allele of the gene and thus has a different nucleotide base in the functional allele of the gene.

[0017] In some embodiments, the method further comprises the step of knocking out expression of the mutant protein and allowing expression of a functional protein.

[0018] The present invention relates to a method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) gene in a cell, the mutant allele having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN), the cell comprising: rs10424470, rs78302854, and the method comprises the steps of: To the cells, A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and introducing a composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides; A method is provided in which a complex of a CRISPR nuclease and a first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene.

[0019] The invention provides modified cells obtained by the methods of the invention.

[0020] The present invention provides modified cells that lack at least a portion of one allele of the ELANE gene.

[0021] The present invention provides a composition comprising the modified cells and a pharma- ceutically acceptable carrier.

[0022] The invention provides an in vitro or ex vivo method for preparing a composition comprising mixing the cells of the invention with a pharma- ceutically acceptable carrier.

[0023] The present invention provides a method for preparing a composition comprising modified cells in vitro or ex vivo, the method comprising: a) isolating HSPCs from cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject being selected from the group consisting of rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7l335276, rs580 rs10424470, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470, and rs78302854; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double stranded break in the ELANE gene in the one or more cells; Thereby obtaining, introducing, and optionally and c) culturing the modified cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, are capable of giving rise to progeny cells.

[0024] The present invention relates to a) isolating HSPCs from cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject being selected from the group consisting of rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7l3352 rs10424470, rs78302854, and rs58082l77, rs3826946, rs104l3889, rs76148l944, rs376l008, rs10409474, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470, and rs78302854; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene of the one or more cells; Thereby obtaining, introducing, and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, of giving rise to progeny cells; and d) administering to a subject the cells of step (b) or step (c) for treating SCN or CyN in the subject.

[0025] The invention provides a method for treating a subject suffering from SCN or CyN comprising administering a therapeutically effective amount of a modified cell, composition or composition prepared by the method of the invention.

[0026] The present invention relates to a method for treating SCN or CyN in a subject in need of such treatment having an ELANE gene mutation associated with SCN or CyN, the subject having one of the following mutations: rs104l4837, rs376l005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs2859l229, rs7l335276, rs7l335276, rs7l335277, rs7l335278, rs7l335279 ... and heterozygous at one or more polymorphic sites selected from the group consisting of rs58082177, rs3826946, rs104l3889, rs76l481944, rs376l008, rs10409474, rs3761007, rs172l6649, rs10469327, rs8l07095, rs10424470 and rs78302854, and the method comprises: a) isolating HSPCs from cells obtained from a subject; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene of the one or more cells; Thereby obtaining, introducing, and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, of giving rise to progeny cells; and d) administering to the subject the cells of step (b) or step (c), thereby treating SCN or CyN in the subject.

[0027] The present invention relates to a method for treating SCN or CyN in a subject in need of such treatment having an ELANE gene mutation associated with SCN or CyN, the subject having one of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs71335276, ... and the individual is heterozygous at one or more polymorphic sites selected from the group consisting of rs58082177, rs3826946, rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10424470, and rs78302854, and the method comprises: administering to the subject an autologous modified cell or a progeny of the autologous modified cell, wherein the autologous modified cell is modified to cause a double strand break in a mutant allele of the ELANE gene; the double-stranded break results from introducing into the cell a CRISPR nuclease or a composition comprising a sequence encoding a CRISPR nuclease and a first RNA molecule, wherein the complex of the CRISPR nuclease and the first RNA molecule affects a double-stranded break in the mutant allele of the ELANE gene so as to inactivate the mutant allele of the ELANE gene in the cell; Thereby, methods are provided for treating SCN or CyN in a subject.

[0028] The present invention relates to a method for selecting a subject for treatment from a pool of subjects diagnosed with SCN or CyN, the method comprising: a) obtaining a cell from each subject in a pool of subjects; b) screening the cells of each subject for an ELANE gene mutation associated with SCN or CyN and selecting only those subjects with an ELANE gene mutation associated with SCN or CyN; c) screening for heterozygosity at one or more polymorphic sites selected from the group consisting of rs104l4837, rs376l005, rs1683564 by sequencing cells of the subjects selected in step (b); and d) selecting only those subjects having cells that are heterozygous at one or more polymorphic sites for treatment.

[0029] An embodiment of the present invention comprises: e) obtaining hematopoietic stem and progenitor cells (HSPCs) from the subject's bone marrow, either by peripheral blood aspiration or by mobilization and apheresis; f) HSPC cells from step (e) One or more CRISPR nucleases or sequences encoding one or more CRISPR nucleases A first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1 to 1192, which targets a nucleotide base of a heterozygous allele of one or more polymorphic sites present in a mutant allele of the ELANE gene; and Introducing a second RNA molecule comprising a guide sequence portion that targets a sequence in intron 3, intron 4 or 3'UTR of the ELANE gene, introducing a complex of a first RNA molecule and a CRISPR nuclease to affect a first double stranded break in a mutant allele of the ELANE gene in the one or more HSPC cells, and a complex of a second RNA molecule and a CRISPR nuclease to affect a second double stranded break in intron 3, intron 4 or the 3'UTR of both alleles of the ELANE gene in the one or more HSPC cells where the complex of the first RNA molecule and the CRISPR nuclease affected the first double stranded break, thereby obtaining modified cells; g) administering to the subject the modified cells of step (f), thereby treating the SCN or CyN in the subject.

[0030] The present invention provides an RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1 to 1192.

[0031] The invention provides a method for inactivating a mutant ELANE allele in a cell, the method comprising delivering to the cell an RNA molecule or composition of the invention.

[0032] The invention provides the use of an RNA molecule of the invention, a composition of the invention or a composition prepared by a method of the invention to inactivate a mutant ELANE allele in a cell.

[0033] The present invention provides a drug comprising an RNA molecule of the present invention, a composition of the present invention, or a composition prepared by a method of the present invention, for use in inactivating a mutant ELANE allele in a cell, wherein the drug is administered by delivering the RNA molecule of the present invention, the composition of the present invention, or a composition prepared by a method of the present invention to the cell.

[0034] The invention provides use of a method of the invention, a modified cell of the invention, a composition of the invention or a composition prepared by a method of the invention, or an RNA molecule of the invention for treating, ameliorating or preventing SCN or CyN in a subject having or at risk of having SCN or CyN.

[0035] The present invention provides a medicament comprising an RNA molecule of the present invention, a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention for use in treating, ameliorating or preventing SCN or CyN, wherein the drug is administered by delivering an RNA molecule of the present invention, a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention to a subject having or at risk of having SCN or CyN.

[0036] The invention provides a kit for inactivating a mutant ELANE allele in a cell comprising an RNA molecule of the invention, a CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding a tracrRNA molecule, and instructions for delivering the RNA molecule, the CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or the tracrRNA or a sequence encoding a tracrRNA molecule to a cell to inactivate a mutant ELANE allele in the cell.

[0037] The present invention provides kits for treating SCN or CyN in a subject comprising an RNA molecule of the present invention, a CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding a tracrRNA molecule, and instructions for delivering the RNA molecule, the CRISPR nuclease and / or tracrRNA to a subject having or at risk of having SCN or CyN to treat SCN or CyN.

[0038] The invention provides a kit for inactivating a mutant ELANE allele in a cell comprising a composition of the invention, a composition prepared by a method of the invention, or a modified cell of the invention, and instructions for delivering the composition to the cell to inactivate the ELANE gene in the cell.

[0039] The invention provides kits for treating SCN or CyN in a subject comprising a composition of the invention, a composition prepared by a method of the invention, or a modified cell of the invention, and instructions for delivering a composition of the invention, a composition prepared by a method of the invention, or a modified cell of the invention to a subject having or at risk of having SCN or CyN to treat SCN or CyN. [Brief description of the drawings]

[0040] [Figure 1] Figure 1. Excision of the promoter region from upstream of the SNP position to intron 3, intron 4 or 3'UTR. In one embodiment, the first guide sequence targets a specific sequence at the heterozygous SNP position in the upstream region of the mutant allele (strategy la-rs104l4837, strategy lb-rs376l005) and the second guide sequence targets a sequence in intron 4 common to the two alleles of the gene. [Diagram 2] Figure 2. Excision of the promoter region from upstream of the SNP position to intron 3, intron 4 or 3'UTR. In one embodiment, the first guide sequence targets a specific sequence of the heterozygous SNP position in the upstream region of the mutant allele (strategy la-rs10414837, strategy lb-rs3761005), and the second guide sequence targets a sequence of intron 3 common to the two alleles of the gene. In another embodiment, the first guide sequence targets a specific sequence of the heterozygous SNP position in the upstream region of the mutant allele (strategy la-rs10414837, strategy lb-rs3761005), and the second guide sequence targets a sequence of the 3'UTR common to the two alleles of the gene. [Diagram 3]Figure 3. Excision of intron 3, intron 4 or the region from the 3'UTR to the downstream of the 3'UTR. In one embodiment, the first guide sequence targets a specific sequence at a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence in intron 4 that is common to the two alleles of the gene. In another embodiment, the first guide sequence targets a specific sequence at a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence in intron 3 that is common to the two alleles of the gene. In a further embodiment, the first guide sequence targets a specific sequence at a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence in the 3'UTR that is common to the two alleles of the gene. [Figure 4] Figure 4. Excision of intron 3, intron 4 or the 3'UTR to a region downstream of the 3'UTR. This strategy is designed to specifically knock out disease-causing alleles ("mutant alleles") while leaving healthy alleles intact. Allele-specific editing is achieved by using guides that target discriminating (heterozygous) SNP positions with relatively high heterozygous frequency in the population. [Diagram 5] Figure 5. Expected coverage in the population based on heterozygosity frequency and overlap / linkage between selected heterozygous SNPs. Designing alternative solutions for the three treatment strategies may allow inclusion of approximately 80% of the population. Approximately 80% of the population is assessed as carrying one or more heterozygous SNPs from the list above. Of these, 33% carry one heterozygous SNP, 31% carry two heterozygous SNPs from the list, 16% carry three heterozygous SNPs, while 20% do not carry any SNPs from the list above. [Figure 6]Figure 6. HeLa cells were seeded in 96-well plates (3K / well). After 24 hours, they were co-transfected with 65ng of WT-Cas9 or Dead-Cas9 and 20ng of gRNA plasmids, identified as g36-g66 and targeting different regions and SNPs of ELANE, using Turbofect reagent (Thermo Scientific). The percentage of editing was calculated according to the following formula: 100%-(unedited band intensity / total band intensity)X100. The average activity of each gRNA ±SD after subtracting the Dead-Cas9 background activity of three independent experiments is shown. [Figure 7] Figure 7. HSCs from healthy donors were nucleofected with spCas9-WT and the RNA components of gRNAs targeting either EMX1 (sgEMXl) or ELANE (g35:INT4; g58:rs376l005; g62:rs1683564). 72 hours after nucleofection, gDNA was extracted and editing levels were quantified by IDAA. The mean % editing ± SD of two independent experiments performed in duplicate is shown. [Figure 8] Figure 8. Specific knockout of a mutant allele of the ELANE gene is mediated by excising intron 4 and exon 5 of the mutant allele of the ELANE gene. This is accomplished by mediating a DSB in intron 4 and utilizing SNP rs1683564 to mediate an allele-specific DSB. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] An embodiment of the present invention is a method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) gene in a cell, the mutant allele having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN), the cell comprising: rs28591229, rs7l335276, rs58082l77, rs3826946, rs10413889, rs76l48l944, rs376l008, rs10409474, rs3761007, rs172l6649, rs10469327, rs8l07095, rs10424470 and rs78302854, and the method comprises: To the cells, A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and introducing a composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides; A method is provided in which a complex of a CRISPR nuclease and a first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene.

[0042] In an embodiment of the invention, a CRISPR nuclease and a first RNA molecule affect a double-stranded break in a mutant allele of the ELANE gene, and the mutant allele is targeted for double-stranded break based on one or more polymorphic sites.

[0043] In an embodiment of the invention, a CRISPR nuclease and a first RNA molecule affect a double-stranded break in a mutant allele of the ELANE gene, and the mutant allele is targeted for double-stranded break based on the sequence of the mutant allele at one or more polymorphic sites.

[0044] In an embodiment of the invention, the CRISPR nuclease and the first RNA molecule affect a double-stranded break in a mutant allele of the ELANE gene based on nucleotide bases at one or more polymorphic sites present in the mutant allele of the ELANE gene.

[0045] An embodiment of the invention further includes introducing a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene.

[0046] In an embodiment of the present invention, the composition may comprise one, two, three or more CRISPR nucleases or sequences encoding CRISPR nucleases. In an embodiment of the present invention, introducing a composition into a cell comprises introducing one, two, three or more compositions into the cell. In an embodiment of the present invention, each composition may comprise a different CRISPR nuclease or sequences encoding CRISPR nucleases or sequences encoding the same CRISPR nuclease or sequences encoding CRISPR nucleases. In an embodiment of the present invention comprising two RNA molecules, the second RNA molecule may form a complex with the same CRISPR nuclease as the first RNA molecule, or may form a complex with a different CRISPR nuclease.

[0047] In an embodiment of the invention, the second double stranded break is within a non-coding region of the ELANE gene. In an embodiment of the invention, the non-coding region of the ELANE gene is selected from an intron or an untranslated region (UTR). In an embodiment of the invention, the non-coding region is intron 3 or intron 4. In an embodiment of the invention, the UTR is the 3'UTR.

[0048] In an embodiment of the present invention, the guide sequence portion of the first RNA molecule comprises 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-1192.

[0049] According to an embodiment of the present invention, the guide sequence portion of the second RNA molecule comprises 17 to 20 nucleotides in a sequence of 17 to 20 adjacent nucleotides set forth in any one of SEQ ID NOs: 1 to 1192, or any one of SEQ ID NOs: 1193 to 2000.

[0050] In an embodiment of the invention, the second double-stranded break is within a non-coding region of the ELANE gene.

[0051] In an embodiment of the invention, the cell is heterozygous at rs104l4837 or rs376l005 and the complex of the second RNA molecule and the CRISPR nuclease affects a double stranded break in intron 4 of the ELANE gene.

[0052] In an embodiment of the invention, the cell is heterozygous at rs10414837 or rs376l005 and the complex of the second RNA molecule and the CRISPR nuclease affects a double stranded break in intron 3 of the ELANE gene.

[0053] In an embodiment of the invention, the cell is heterozygous at rs10414837 or rs376l005, and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene.

[0054] In an embodiment of the invention, the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene.

[0055] In an embodiment of the invention, the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 3 of the ELANE gene.

[0056] In an embodiment of the invention, the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene.

[0057] In some embodiments, the cell is heterozygous at the ELANE rs104l4837 polymorphic site, and a complex of a first RNA molecule comprising a guide sequence portion having 17-20 nucleotides and a CRISPR nuclease affects a double-stranded break in a mutant allele of the ELANE gene and does not affect a double-stranded break in a functional allele of the ELANE gene. In such an embodiment, the guide sequence portion of the first RNA molecule has 17 to 20 nucleotides and is selected from the group consisting of SEQ ID NOs: 82, 86, 93, 94, 115, 119, 135, 173, 180, 213, 215, 224, 225, 262, 263, 307, 308, 319, 323, 351, 352, 374, 461, 462, 466, 467, 474, 477, 478, 491, 504, 505, 533, 537, 538, 550, 556, 569, 570, 583, 584, 684, 695, 706, 707, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 85, 714, 745, 790, 791, 845, 846, 854, 857, 858, 861, 863, 864, 880, 881, 886, 890, 891, 901, 911, 912, 936, 937, 939, 940, 960, 961, 972, 978, 979, 983, 984, 1018, 1034, 1035, 1040, 1086, 1110, 1111, 1135, 1144 and 1145.

[0058] In some embodiments, the cell is heterozygous at the ELANE rs376l005 polymorphic site, and a complex of a first RNA molecule comprising a guide sequence portion having 17-20 nucleotides and a CRISPR nuclease affects a double-stranded break in a mutant allele of the ELANE gene and does not affect a double-stranded break in a functional allele of the ELANE gene. In such an embodiment, the guide sequence portion of the first RNA molecule has 17-20 nucleotides and is selected from the group consisting of SEQ ID NOs: 26, 57, 65, 66, 70, 187, 188, 191, 206, 220, 221, 243, 245, 261, 275, 356, 357, 392, 417, 418, 431, 441, 442, 447, 488, 513, 514, 545, 546, 548, 598, 599, 604, 607, 608, 612, 613, 639, 648, 658, 659, 660, 680, 681, 74 2, 743, 755, 756, 759, 762, 763, 767, 771, 772, 773, 786, 787, 815, 816, 818, 819, 820, 831, 836, 849, 850, 870, 871, 898, 899, 907, 908, 1009, 1010, 1013, 1023, 1029, 1030, 1082, 1083, 4093, 1099, 1100, 1101, 1107, 1108 and 1182.

[0059] In some embodiments, the cell is heterozygous at the ELANE rs1683564 polymorphic site, and a complex of a first RNA molecule comprising a guide sequence portion having 17-20 nucleotides and a CRISPR nuclease affects a double-stranded break in a mutant allele of the ELANE gene and does not affect a double-stranded break in a functional allele of the ELANE gene. In such an embodiment, the guide sequence portion of the first RNA molecule has 17-20 nucleotides and is selected from the group consisting of SEQ ID NOs: 52, 87, 122, 164, 175, 199, 214, 290, 326, 345, 346, 373, 404, 412, 436, 437, 451, 452, 483, 484, 517, 520, 525, 617, 618, 621, 641, 661, 676, 722, 736, 806, 855, 856, 87 8, 879, 888, 889, 896, 903, 905, 913, 914, 929, 933, 934, 935, 982, 998, 1021, 1022, 1026, 1046, 1047, 1053, 1097, 1121, 1122, 1124, 1126, 1127, 1131, 1134, 1175, 1176, 1183 and 1190.

[0060]

[0013] An embodiment of the present invention includes obtaining cells having an ELANE gene mutation associated with severe congenital neutropenia (SCN) or CyN from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject having one or more of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs19972 rs10424470, rs78302854, and / or rs28591229, rs71335276, rs58082177, rs3826946, rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10424470, and / or rs78302854.

[0061] An embodiment of the present invention includes first selecting a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject having one or more of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs71335276, rs580 The method includes determining whether a subject is heterozygous at one or more polymorphic sites selected from the group consisting of rs104177, rs3826946, rs10413889, rs761481944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8107095, rs10424470 and rs78302854, and obtaining cells from the subject.

[0062] Embodiments of the invention include obtaining cells from a subject by mobilization and / or by apheresis.

[0063] An embodiment of the invention involves obtaining cells from a subject by bone marrow aspirate.

[0064] In an embodiment of the invention, the cells are primed prior to introducing the composition into the cells.

[0065] An embodiment of the invention involves growing cells in culture to obtain a cell population.

[0066] In an embodiment of the invention, the cells are cultured with one or more of stem cell factor (SCF), IL-3 and GM-CSF.

[0067] In an embodiment of the invention, the cells are cultured with at least one cytokine.

[0068] In an embodiment of the invention, at least one cytokine is a recombinant human cytokine.

[0069] In an embodiment of the invention, the cell is one of a plurality of cells, and a composition comprising a first RNA molecule or both the first and second RNA molecules is introduced into at least the cell and other cells of the plurality of cells, and a mutant allele of the ELANE gene is inactivated in at least the cell and other cells of the plurality of cells, thereby obtaining a plurality of modified cells.

[0070] In an embodiment of the invention, introducing a composition comprising a first RNA molecule or introducing a second RNA molecule comprises electroporation of a cell or a plurality of cells.

[0071] An embodiment of the present invention provides modified cells obtained by the methods of the present invention.

[0072] In an embodiment of the invention, the modified cells are further propagated in culture.

[0073] In an embodiment of the invention, the modified cells are capable of engraftment.

[0074] In an embodiment of the invention, the modified cells are capable of long-term engraftment when infused into a patient, producing differentiated hematopoietic cells for at least 12 months, preferably at least 24 months, and even more preferably at least 30 months after infusion. In a further embodiment, the modified cells are capable of long-term engraftment when infused into an autologous subject. In a further embodiment, the modified cells are capable of long-term engraftment when infused into a subject without myeloablation. In an embodiment of the invention, the modified cells are delivered to a human subject in sufficient numbers to provide long-term engraftment when engrafted in the subject.

[0075] In an embodiment of the invention, the modified cell or modified cells are capable of giving rise to progeny cells.

[0076] In an embodiment of the invention, the modified cell or cells are capable of giving rise to progeny cells after engraftment.

[0077] In an embodiment of the invention, the modified cell or cells are capable of giving rise to progeny cells following autologous transplantation.

[0078] In embodiments of the invention, the modified cell or cells are capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment.

[0079] In one embodiment, the cell or cells are stem cells. In one embodiment, the cells are embryonic stem cells. In one embodiment, the stem cells are hematopoietic stem / progenitor cells (HSPCs).

[0080] In an embodiment of the invention, the modified cell or cells are CD34+ hematopoietic stem cells.

[0081] In an embodiment of the invention, the modified cells or cells are bone marrow cells or peripheral mononuclear cells (PMCs).

[0082] An embodiment of the present invention provides modified cells that lack at least a portion of one allele of the ELANE gene.

[0083] In an embodiment of the invention, the modified cell has been modified from a cell that is heterozygous at one or more polymorphic sites selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs2859l229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs76l48l944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8107095, rs10424470, and rs78302854.

[0084] An embodiment of the present invention provides a composition comprising modified cells and a pharma- ceutically acceptable carrier.

[0085] An embodiment of the invention provides an in vitro or ex vivo method for preparing a composition comprising mixing the cells of the invention with a pharma- ceutically acceptable carrier.

[0086] An embodiment of the invention is a method for preparing a composition comprising modified cells in vitro or ex vivo, the method comprising: a) isolating HSPCs from a plurality of cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject being selected from the group consisting of rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7l335276, rs5 isolating and obtaining cells from the subject that are heterozygous at one or more polymorphic sites selected from the group consisting of rs8l07095, rs10424470, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470, and rs78302854; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene in the one or more cells, thereby obtaining a modified cell. and c) culturing the modified cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, are capable of giving rise to progeny cells.

[0087] An embodiment of the present invention comprises: a) isolating HSPCs from cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, the subject being selected from the group consisting of rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7l3352 rs10424470, rs78302854, and rs58082l77, rs3826946, rs104l3889, rs76148l944, rs376l008, rs10409474, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470, and rs78302854; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double stranded break in the ELANE gene in the one or more cells; Thereby obtaining, introducing, and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, of giving rise to progeny cells; and d) administering to a subject the cells of step (b) or step (c) for treating SCN or CyN in the subject.

[0088] An embodiment of the invention provides a method of treating a subject suffering from SCN or CyN comprising administering a therapeutically effective amount of a modified cell, composition, or composition prepared by a method of the invention.

[0089] An embodiment of the present invention is a method of treating SCN or CyN in a subject in need of such treatment having an ELANE gene mutation associated with SCN or CyN, the subject having any of the following mutations: rs104l4837, rs376l005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs2859l229, rs7l33527 6, rs58082177, rs3826946, rs104l3889, rs76l481944, rs376l008, rs10409474, rs3761007, rs172l6649, rs10469327, rs8l07095, rs10424470 and rs78302854, and the method comprises: a) isolating HSPCs from cells obtained from a subject; b) inactivating the mutant allele of the ELANE gene in one or more cells of the cell of step (a), A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in a mutant allele of the ELANE gene in the one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease affects a second double stranded break in the ELANE gene in the one or more cells; Thereby obtaining, introducing, and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, after engraftment, of giving rise to progeny cells; and d) administering to the subject the cells of step (b) or step (c), thereby treating SCN or CyN in the subject.

[0090] An embodiment of the present invention is a method of treating SCN or CyN in a subject in need of such treatment having an ELANE gene mutation associated with SCN or CyN, the subject having any of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7133527 6, rs58082177, rs3826946, rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854, and the method comprises: administering to the subject an autologous modified cell or a progeny of the autologous modified cell, wherein the autologous modified cell is modified to cause a double strand break in a mutant allele of the ELANE gene; administering to the cell, the double stranded break resulting from introducing into the cell a CRISPR nuclease or a composition comprising a sequence encoding a CRISPR nuclease and a first RNA molecule, wherein the complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene so as to inactivate the mutant allele of the ELANE gene in the cell; Thereby, methods are provided for treating SCN or CyN in a subject.

[0091] An embodiment of the invention is a method for selecting a subject for treatment from a pool of subjects diagnosed with SCN or CyN, the method comprising: a) obtaining a cell from each subject in a pool of subjects; b) screening the cells of each subject for an ELANE gene mutation associated with SCN or CyN and selecting only those subjects with an ELANE gene mutation associated with SCN or CyN; c) screening for heterozygosity at one or more polymorphic sites selected from the group consisting of rs104l4837, rs376l005, rs1683564 by sequencing cells of the subjects selected in step (b); and d) selecting only those subjects having cells that are heterozygous at one or more polymorphic sites for treatment.

[0092] An embodiment of the present invention comprises: e) obtaining HSPC cells from the subject's bone marrow, either by peripheral blood aspiration or by mobilization and apheresis; f) HSPC cells from step (e) One or more CRISPR nucleases or sequences encoding one or more CRISPR nucleases A first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1 to 1192, which targets a nucleotide base of a heterozygous allele of one or more polymorphic sites present in a mutant allele of the ELANE gene; and Introducing a second RNA molecule comprising a guide sequence portion that targets a sequence in intron 3, intron 4 or 3'UTR of the ELANE gene, introducing a complex of a first RNA molecule and a CRISPR nuclease to affect a first double stranded break in a mutant allele of the ELANE gene in the one or more HSPC cells, and a complex of a second RNA molecule and a CRISPR nuclease to affect a second double stranded break in intron 3, intron 4 or the 3'UTR of both alleles of the ELANE gene in the one or more HSPC cells where the complex of the first RNA molecule and the CRISPR nuclease affected the first double stranded break, thereby obtaining modified cells; g) treating SCN or CyN in the selected subject, comprising administering to the subject the modified cells of step (f), thereby treating SCN or CyN in the subject.

[0093] An embodiment of the present invention provides an RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1 to 1192.

[0094] Embodiments of the invention further include a second RNA molecule that includes a guide sequence portion.

[0095] In an embodiment of the invention, the second RNA molecule targets a non-coding region of the ELANE gene.

[0096] In an embodiment of the invention, the nucleotide sequence of the guide sequence portion of the second RNA molecule is a different nucleotide sequence than the sequence of the guide sequence portion of the first RNA molecule.

[0097] In an embodiment of the invention, the first RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In an embodiment of the invention, the second RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease.

[0098] In an embodiment of the invention, the sequence that binds to the CRISPR nuclease is a tracrRNA sequence.

[0099] In an embodiment of the invention, the first RNA molecule further comprises a portion having a tracr mate sequence.In an embodiment of the invention, the second RNA molecule further comprises a portion having a tracr mate sequence.

[0100] In an embodiment of the invention, the first RNA molecule further comprises one or more linker moieties.In an embodiment of the invention, the second RNA molecule further comprises one or more linker moieties.

[0101] In an embodiment of the invention, the first RNA molecule is up to 300 nucleotides in length. In an embodiment of the invention, the second RNA molecule is up to 300 nucleotides in length.

[0102] In an embodiment of the invention, the composition further comprises one or more CRISPR nucleases or sequences encoding one or more CRISPR nucleases.In an embodiment of the invention, the composition further comprises one or more tracrRNA molecules or sequences encoding one or more tracrRNA molecules.

[0103] An embodiment of the present invention provides a method for inactivating a mutant ELANE allele in a cell, the method comprising delivering to the cell an RNA molecule or composition of the present invention.

[0104] In embodiments of the invention, one or more CRISPR nucleases or a sequence encoding one or more CRISPR nucleases and an RNA molecule or multiple RNA molecules are delivered to a subject and / or cell at substantially the same time or at different times.

[0105] In an embodiment of the invention, the tracrRNA molecule or a sequence encoding one or more tracrRNA molecules and the RNA molecule or RNA molecules are delivered to a subject and / or cell at substantially the same time or at different times.

[0106] In an embodiment of the invention, the method comprises the steps of: The method includes removing an exon containing a disease-causing mutation from a mutant allele, wherein the first RNA molecule or the first and second RNA molecules target regions adjacent to the entire exon or part of the exon.

[0107] In an embodiment of the invention, the method involves removing an entire open reading frame of a gene, multiple exons, or removing the entire gene.

[0108] In an embodiment of the invention, the first RNA molecule or the first and second RNA molecules target an alternative splicing signal sequence between an exon and an intron of a mutant allele.

[0109] In an embodiment of the invention, the second RNA molecule targets a sequence that is present in both the mutant allele and the functional allele.

[0110] In an embodiment of the invention, the second RNA molecule targets an intron.

[0111] In an embodiment of the invention, the method results in a mutant allele undergoing an insertion or deletion by an error-prone non-homologous end joining (NHEJ) mechanism, resulting in a frameshift in the sequence of the mutant allele.

[0112] In an embodiment of the invention, the frameshift results in the inactivation or knockout of the mutant allele.

[0113] In an embodiment of the invention, the frameshift creates a premature stop codon in the mutant allele or the frameshift leads to nonsense-mediated mRNA decay of the transcript of the mutant allele.

[0114] In an embodiment of the invention, inactivation or treatment results in a truncated protein encoded by the mutant allele and a functional protein encoded by the functional allele.

[0115] In an embodiment of the invention, the cell or subject is heterozygous at rs104l4837 or rs376l005, and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene.

[0116] In an embodiment of the invention, the cell or subject is heterozygous at rs104l4837 or rs376l005, and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 3 of the ELANE gene.

[0117] In an embodiment of the invention, the cell or subject is heterozygous at rs104l4837 or rs376l005, and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene.

[0118] In an embodiment of the invention, the cell or subject is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene.

[0119] In an embodiment of the invention, the cell or subject is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in intron 3 of the ELANE gene.

[0120] In an embodiment of the invention, the cell or subject is heterozygous at rs1683564 and the complex of the second RNA molecule and the CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene.

[0121] An embodiment of the present invention provides the use of an RNA molecule of the present invention, a composition of the present invention, or a composition prepared by a method of the present invention, to inactivate a mutant ELANE allele in a cell.

[0122] An embodiment of the present invention provides a drug comprising an RNA molecule of the present invention, a composition of the present invention, or a composition prepared by a method of the present invention, for use in inactivating a mutant ELANE allele in a cell, wherein the drug is administered by delivering the RNA molecule of the present invention, the composition of the present invention, or a composition prepared by a method of the present invention to the cell.

[0123] Embodiments of the invention provide use of a method of the invention, a modified cell of the invention, a composition of the invention or a composition prepared by a method of the invention, or an RNA molecule of the invention for treating, ameliorating or preventing SCN or CyN in a subject having or at risk of having SCN or CyN.

[0124] An embodiment of the present invention provides a medicament comprising an RNA molecule of the present invention, a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention for use in treating, ameliorating or preventing SCN or CyN, wherein the medicament is administered by delivering the RNA molecule of the present invention, the composition of the present invention, the composition prepared by a method of the present invention, or the modified cell of the present invention to a subject having or at risk of having SCN or CyN.

[0125] An embodiment of the present invention provides a kit for inactivating a mutant ELANE allele in a cell comprising an RNA molecule of the present invention, a CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding a tracrRNA molecule, and instructions for delivering the RNA molecule, the CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or the tracrRNA or a sequence encoding a tracrRNA molecule to a cell to inactivate a mutant ELANE allele in the cell.

[0126] An embodiment of the present invention provides a kit for treating SCN or CyN in a subject comprising an RNA molecule of the present invention, a CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding a tracrRNA molecule, and instructions for delivering the RNA molecule, the CRISPR nuclease or a sequence encoding a CRISPR nuclease, and / or the tracrRNA or a sequence encoding a tracrRNA molecule to a subject having or at risk of having SCN or CyN to treat SCN or CyN.

[0127] An embodiment of the present invention provides a kit for inactivating a mutant ELANE allele in a cell comprising a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention, and instructions for delivering the composition to the cell to inactivate the ELANE gene in the cell.

[0128] An embodiment of the present invention provides a kit for treating SCN or CyN in a subject comprising a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention, and instructions for delivering a composition of the present invention, a composition prepared by a method of the present invention, or a modified cell of the present invention to a subject having or at risk of having SCN or CyN to treat SCN or CyN.

[0129] definition Unless otherwise specified, technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0130] The terms "a" and "an," as used above and elsewhere herein, should be understood to refer to "one or more" of the listed components. One of ordinary skill in the art would recognize that the use of the singular also includes the plural unless specifically stated otherwise. Thus, the terms "a," "an," and "at least one" are used interchangeably in this application.

[0131] For the purpose of providing a better understanding of the present teachings, and not in any way limiting the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or ratios, and other numerical values ​​used in the specification and claims should be understood as being modified in all instances by the term "translation." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0132] Unless otherwise expressly stated, adjectives such as "substantially" and "approximately" modifying a condition or relationship specific to a feature or group of features of an embodiment of the invention are understood to mean that the condition or characteristic is defined within an acceptable range for the process of the embodiment for the application for which it is intended. Unless otherwise indicated, the word "or" in the specification and claims is deemed to be an inclusive "or" rather than an exclusive or, indicating at least one or any combination of the items it conjoins.

[0133] In the description and claims of this application, the verbs "comprise," "include," and "have" and their conjugations are used to indicate that the object or group of objects of the verb is not necessarily an exhaustive list of components, elements, or parts of the object or group of objects of the verb. Other terms used herein are defined according to their known meaning in the art.

[0134] As used herein, the term "heterozygous single nucleotide polymorphism" or "SNP" refers to a single nucleotide position in a genome that differs between paired chromosomes in a population. As used herein, the most common or most prevalent nucleotide base at this position is referred to as the reference (REF), wild type (WT), common or predominant form. The less common nucleotide base at this position is referred to as the alternative (ALT), minor, rare or variant form.

[0135] A "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize to a specific target DNA sequence, for example, the guide sequence portion has a nucleotide sequence that is fully complementary to the targeted DNA sequence over the length of the guide sequence portion. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length, or approximately 17-24, 18-22, 19-22, 18-20 or 17-20 nucleotides in length. The entire length of the guide sequence portion is fully complementary to the targeted DNA sequence over the length of the guide sequence portion. The guide sequence portion may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, where the guide sequence portion serves as the DNA targeting portion of the CRISPR complex. When a DNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, the RNA molecule can target the CRISPR nuclease to a specific target DNA sequence. Each possibility represents a separate embodiment. The RNA molecule can be custom designed to target any desired sequence.

[0136] The term "targeting" as used herein refers to the guide sequence portion that RNA molecule preferentially hybridizes to the nucleic acid having the nucleotide sequence targeted.The term "targeting" is understood to include variable hybridization efficiency, such that the nucleic acid having the nucleotide sequence targeted preferentially hybridizes, but unintended off-target hybridization may occur in addition to hybridization on target.When RNA molecule targets a sequence, it is understood that the complex of RNA molecule and CRISPR nuclease molecule targets this sequence for nuclease activity.

[0137] In the context of targeting a DNA sequence present in a plurality of cells, this targeting is understood to include the hybridization of the guide sequence portion of the RNA molecule to a sequence in one or more cells, and the hybridization of the RNA molecule to the target sequence in fewer than all of the plurality of cells. Thus, when an RNA molecule targets a sequence in a plurality of cells, it is understood that the complex of the RNA molecule and CRISPR nuclease hybridizes to the target sequence in one or more cells, and may hybridize to the target sequence in fewer than all of the cells. Thus, the complex of the RNA molecule and CRISPR nuclease may introduce double-strand breaks in association with hybridization to the target sequence in one or more cells, and may introduce double-strand breaks in association with hybridization to the target sequence in fewer than all of the cells. As used herein, the term "modified cell" refers to a cell in which a double-strand break is caused by the complex of the RNA molecule and CRISPR nuclease as a result of hybridization to the target sequence, i.e., hybridization on the target.

[0138] In embodiments of the invention, an RNA guide molecule may target a mutant allele based on the nucleotide base present at the polymorphic site of the mutant allele.

[0139] In an embodiment of the invention, the RNA molecule is set forth in any one of SEQ ID NOs: 1 to 1192, or in the following group of SEQ ID NOs: 6, 75, 76, 252, 253, 287, 295, 296, 311, 536, 592, 595, 596, 597, 695, 729, 737, 812, 839, 915, 947, 1048, 1049, 1070, 1071, 1169, or in the following group of SEQ ID NOs: 6, 75, 76, 93, 94, 97, 98, 148, 149, 171, 173, 180, 182, 183, 184, 187, 188, 21 3, 232, 234, 249, 252, 253, 264, 272, 287, 291, 295, 296, 305, 306, 307, 308, 311, 326, 333, 334, 337, 338, 339, 340, 351, 352, 358, 359, 378, 379, 385, 388 , 399, 408, 410, 419, 420, 426, 427, 428, 429, 430, 436, 437, 449, 450, 465, 468, 476, 477, 478, 480, 495, 497, 499, 500, 508, 511, 521, 522, 523, 524, 529, 530, 532, 536, 542, 545, 546, 564, 565, 566, 573, 574, 583, 584, 591, 592, 595, 596, 597, 598, 599, 601, 602, 604, 612, 613, 616, 622, 623, 634, 644, 645, 6 58, 659, 661, 670, 671, 678, 680, 681, 684, 685, 688, 689, 694, 695, 714, 715, 716, 722, 723, 724, 729, 736, 737, 739, 740, 745, 755, 756, 760, 761, 769, 77 0, 771, 772, 775, 776, 786, 787, 806, 809, 812, 818, 819, 821, 822, 826, 829, 830, 833, 834, 839, 845, 846, 861, 862, 874, 875, 876, 877, 884, 888, 889, 890 , 891, 893, 894, 911, 912, 913, 914, 915, 925, 928, 930, 931, 939, 940, 942, 946, 947, 948, 949, 950, 957, 972, 974, 982, 994, 998, 1006, 1007, 1008, 1021,1022, 1026, 1027, 1028, 1031, 1032, 1034, 1035, 1039, 1046, 1047, 1048, 1049, 1057, 1070, 1071, 1072, 1074, 1075, 1076, 1079, 1084, 1090, 1091, 1093, 1094, 1095, 1112, 1113, 1116, 1117, 1118, 1119, 1121, 1122, 1124, 1140, 1168, 1169, 1170, 1171, 1179, or the following group SEQ ID NOs: 6, 10, 13, 14, 19, 21, 22, 23, 24, 25, 26, 29, 30, 34, 35, 36, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 57, 58, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 82 , 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 107, 108, 109. 115, 119, 122, 123. 124. 125. 126, 127, 128, 130, 133, 134, 135, 136, 1 37, 138, 139, 140, 141, 142, 143, 144, 148, 149, 150, 152, 153, 155, 156, 158, 159, 160, 161, 162, 163, 164, 167, 168, 169, 170, 171, 172, 173, 175, 176, 1 77, 178, 179, 180, 181, 182, 183, 184, 187, 188, 189, 190, 191, 192, 195, 196, 198, 199, 201, 203, 206, 209, 211, 212, 213, 214, 215, 216, 217, 219, 220, 22 1, 223, 224, 225, 226, 227, 232, 233, 234, 236, 237, 238, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 261, 262, 263 , 264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 277, 281, 282, 285, 286, 287, 290, 291, 292, 293, 294, 295, 296, 302, 303, 305, 306, 307, 308,310、311、314、319、323、326、327、328、329、331、332、333、334、335、336、337、338、339、340、341、343、344、345、346、349、350、351、352、353、354、356、357、358、359、360、363、364、366、370、371、372、373、374、375、376、377、378、379、380、381、382、385、386、387、388、389、390、391、392、393、394、395、396、397、399、400、404、405、406、407、408、410、411、412、415、417、418、419、420、422、423、424、425、426、427、428、429、430、431、432、433、435、436、437、440、441、442、443、444、445、446、447、449、450、451、452、454、455、456、457、460、461、462、464、465、466、467、468、469、470、471、474、475、476、477、478、479、480、483、484、485、486、488、489、491、492、493、494、495、496、497、498、499、500、501、502、504、505、506、508、509、510、511、513、514、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、573、574、577、579、580、582、583、584、586、587、588、590、591、592、593、595、596、597、598、599、600、601、602、604、605、606、607、608、609、610、612、613、614、615、616、617、618、619、620、621、622、623、624、625、628、629、630、633、634、635、636、637、638、639、640、641、644、645、648、650、651、652、653、654、655、658、659、660、661、663、664、665、667、670、671、672、673、675、676、678、680、681、683、684、685、686、688、689、690、691、692、694、695、698、700、701、702、703、704、705、706、707、708、709、712、713、714、715、716、718、719、720、722、723、724、727、728、729、730、731、732、733、734、735、736、737、738、739、740、742、743、744、745、746、747、748、749、753、754、755、756、757、758、759、760、761、762、763、764、767、769、770、771、772、773、775、776、778、779、780、781、786、787、788、789、790、791、792、794、795、798、800、801、805、806、807、808、809、811、812、813、814、815、816、818、819、820、821、822、823、826、829、830、831、832、833、834、835、836、837、838、839、843、844、845、846、849、850、852、853、854、855、856、857、858、861、862、863、864、865、866、867、868、870、871、872、874、875、876、877、878、879、880、881、882、883、884、886、887、888、889、890、891、893、894、895、896、897、898、899、901、902、903、905、906、907、908、909、910、911、912、913、914、915、916、917、920、921、925、926、927、928、929、930、931、932、933、934、935、936、937、939、940、942、943、945、946、947、948、949、950、951、952、953, 954, 955, 956, 957, 958, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 972, 974, 975, 976, 978, 979, 982, 983, 984, 988, 989, 990, 991, 992, 993, 994, 996, 997, 998, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1061, 1062, 1063, 1064, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1 078, 1079, 1080, 1081, 1082, 1083, 1084, 1086, 1090, 1091, 1093, 1094, 1095, 1097, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1107, 1108, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1126, 1127, 1128, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 11 38, 1139, 1140, 1142, 1143, 1144, 1145, 1147, 1148, 1149, 1151, 1152, 1153, 1154, 1155, 1156, 1158, 1159, 1160, 1162, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1175, 1176, 1179, 1180, 1181, 1182, 1183. In any embodiment of the invention, the guide sequence portion of the RNA molecule comprisesIt is understood that any single sequence of SEQ ID NOs: 1 to 1192, or any single sequence from the above group of sequences, may contain 17 to 20 contiguous nucleotides as described.

[0140] As used herein, "contiguous nucleotides" set forth in a SEQ ID NO: refer to nucleotides that are in the sequence of nucleotides in the order set forth in the SEQ ID NO: without any intervening nucleotides.

[0141] In an embodiment of the invention, the guide sequence portion may be 20 nucleotides in length and is composed of 20 nucleotides in a sequence of 20 contiguous nucleotides as set forth in any one of SEQ ID NOs: 1-1192. In an embodiment of the invention, the guide sequence portion may be shorter than 20 nucleotides in length. For example, in an embodiment of the invention, the guide sequence portion may be 17, 18 or 19 nucleotides in length. In such an embodiment, the guide sequence portion may be composed of 17, 18 or 19 nucleotides in a sequence of 17 to 20 contiguous nucleotides as set forth in any one of SEQ ID NOs: 1-1192. For example, a 17 nucleotide guide sequence portion in a sequence of 17 contiguous nucleotides as set forth in SEQ ID NO: 1 may be composed of any one of the following nucleotide sequences (nucleotides excluded from the contiguous sequence are crossed out): TIFF0007611702000001.tif44162

[0142] In embodiments of the invention, the guide sequence portion may be greater than 20 nucleotides in length. For example, in embodiments of the invention, the guide sequence portion may be 21, 22, 23 or 24 nucleotides in length. In such embodiments, the guide sequence portion comprises 20 nucleotides that are a sequence of 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-1192 and additional nucleotides that are perfectly complementary to the sequence of nucleotides or nucleotides adjacent to the 3' end of the target sequence, the 5' end of the target sequence, or both.

[0143] In an embodiment of the present invention, a CRISPR nuclease and an RNA molecule comprising a guide sequence form a CRISPR complex that binds to a target DNA sequence and causes cleavage of the target DNA sequence. The CRISPR nuclease, such as Cpfl, may form a CRISPR complex comprising a CRISPR nuclease and an RNA molecule without a further tracrRNA molecule. Alternatively, the CRISPR nuclease, such as Cas9, may form a CRISPR complex between the CRISPR nuclease, an RNA molecule and a tracrRNA molecule.

[0144] In embodiments of the invention, the RNA molecule may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of a guide portion of the RNA molecule and a transactivating crRNA (tracrRNA). (See Jinek (2012) Science). Embodiments of the invention may also utilize separate tracrRNA molecules and separate RNA molecules that include a guide sequence portion to form a CRISPR complex. In such embodiments, the tracrRNA molecule may form a hybrid with the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described herein.

[0145] The term "tracr mate sequence" refers to a sequence that is sufficiently complementary to the tracrRNA molecule to hybridize with the tracrRNA through base pairing and promote the formation of a CRISPR complex. (See US8906616). In an embodiment of the present invention, the RNA molecule may further comprise a portion having a tracr mate sequence.

[0146] According to embodiments of the invention, the RNA molecule may be up to 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110 or 100 nucleotides in length. Each possibility represents a separate embodiment. In embodiments of the invention, the RNA molecule may be 17-300 nucleotides in length, 100-300 nucleotides in length, 150-300 nucleotides in length, 200-300 nucleotides in length, 100-200 nucleotides in length or 150-250 nucleotides in length. Each possibility represents a separate embodiment.

[0147] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product and all DNA regions that regulate the synthesis of the gene product, and such regulatory sequences may or may not be contiguous with the coding and / or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border regions, origins of replication, matrix attachment regions, and locus control regions.

[0148] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

[0149] As used herein, the term HSPC refers to both hematopoietic stem cells and hematopoietic stem progenitor cells. Non-limiting examples of stem cells include bone marrow cells, myeloid progenitor cells, multipotent progenitor cells, and lineage-restricted progenitor cells.

[0150] As used herein, "progenitor cells" refer to lineage cells that are derived from stem cells and retain mitotic capacity and multipotency (e.g., capable of differentiating or developing into more than one, but not all, types of mature lineage cells). As used herein, "hematopoiesis" or "erythropoiesis" refers to the various types of blood cells (e.g., red blood cells, megakaryocytes, myeloid cells (e.g., monocytes, macrophages, and neutrophils), and lymphocytes) and other elements formed in the body (e.g., in the bone marrow).

[0151] The term "nuclease" as used herein refers to an enzyme that can cleave the phosphodiester bond between the nucleotide subunits of nucleic acid. Nucleases can be isolated or derived from natural sources. Natural sources can be any living organism. Alternatively, nucleases can be modified or synthetic proteins that retain the ability to cleave phosphodiester bonds. Nucleases, for example, CRISPR nucleases, can be used to achieve genetic modification.

[0152] In an embodiment of the invention, an RNA molecule is designed to target a heterozygous polymorphic site present in a mutant allele of the ELANE gene, and the RNA molecule targets a nucleotide base, REF or ALT, of the heterozygous polymorphic site present in a mutant allele of the ELANE gene.

[0153] The present disclosure provides methods that utilize at least one naturally occurring nucleotide difference or genetic polymorphism (such as a single nucleotide polymorphism (SNP)) to distinguish / distinguish between two alleles of a gene, one allele carrying a mutation such that the mutation encodes a mutant protein that results in a disease phenotype (the "mutated allele") and the other allele encodes a functional protein (the "functional allele"). The method further comprises knocking out expression of the mutant protein and allowing expression of the functional protein. In some embodiments, the method is for treating, ameliorating, or preventing a dominant negative genetic disorder.

[0154] An embodiment of the present invention provides a method of utilizing at least one heterozygous SNP of a gene that expresses a dominant mutant allele in a certain cell or subject. In an embodiment of the present invention, the SNP utilized may or may not be associated with a disease phenotype. In an embodiment of the present invention, an RNA molecule comprising a guide sequence targets a mutant allele of a gene by targeting a nucleotide that is present at a heterozygous SNP in the mutant allele of the gene and thus has a different nucleotide base in the functional allele of the gene.

[0155] According to an embodiment of the present invention, a first RNA molecule targets a first heterozygous SNP present in an exon or promoter of the ELANE gene, the first RNA molecule targets a nucleotide base, REF or ALT, of the first SNP present in a mutant allele of the ELANE gene, the second RNA molecule targets a second heterozygous SNP present in the same or different exon or intron of the ELANE gene, the second RNA molecule targets a nucleotide base, REF or ALT, of the second SNP present in a mutant allele of the ELANE gene, or the second RNA molecule targets a sequence of a non-coding region present in both the mutant allele and the functional allele.

[0156] According to an embodiment of the present invention, the first RNA molecule or the first and second RNA molecules target a heterozygous SNP present in the promoter region, start codon or untranslated region (UTR) of the ELANE gene, and the RNA molecule targets the nucleotide base, REF or ALT, of the SNP present in a mutant allele of the ELANE gene.

[0157] According to an embodiment of the invention, the first RNA molecule or the first and second RNA molecules target at least a part of the promoter and / or the start codon and / or a part of the UTR of a mutant allele of the ELANE gene.

[0158] According to an embodiment of the present invention, a first RNA molecule targets a portion of a promoter, a first heterozygous SNP present in the promoter of the ELANE gene, or a heterozygous SNP present upstream of the promoter of the ELANE gene, and a second RNA molecule targets a second heterozygous SNP, which is present in the ELANE gene downstream of the first heterozygous SNP, and which is in the promoter, UTR or intron or exon of the ELANE gene, and the first RNA molecule targets the nucleotide base, REF or ALT, of the first SNP present in a mutant allele of the ELANE gene, and the second RNA molecule targets the nucleotide base, REF or ALT, of the second SNP present in a mutant allele of the ELANE gene.

[0159] According to an embodiment of the present invention, a first RNA molecule is designed to target a heterozygous SNP present in the promoter, upstream of the promoter, or UTR of the ELANE gene, an RNA molecule targets the nucleotide base, REF or ALT, of the SNP present in a mutated allele of the ELANE gene, and a second RNA molecule is designed to target a sequence present in an intron of both the mutated and functional alleles of the ELANE gene.

[0160] According to an embodiment of the present invention, a first RNA molecule targets a sequence upstream of a promoter present in both a mutant allele and a functional allele of the ELANE gene, and a second RNA molecule targets a heterozygous SNP present at any position in the ELANE gene, and the second RNA molecule targets a nucleotide base, REF or ALT, of the SNP present in the mutant allele of the ELANE gene.

[0161] According to an embodiment of the invention, there is provided a method comprising removing an exon containing a disease-causing mutation from a mutant allele, wherein a first RNA molecule, or first and second RNA molecules, target regions flanking all or part of the exon.

[0162] According to embodiments of the invention, methods are provided that include removing an entire open reading frame of a gene, multiple exons, or removing the entire gene.

[0163] According to an embodiment of the present invention, a first RNA molecule targets a first heterozygous SNP present in an exon or promoter of the ELANE gene, a second RNA molecule targets a second heterozygous SNP present in the same or different exon or intron of the ELANE gene, the second RNA molecule targets a nucleotide base, REF or ALT, of a second SNP present in a mutant allele of the ELANE gene, or the second RNA molecule targets an intron sequence present in both the mutant allele and the functional allele of the ELANE gene.

[0164] According to an embodiment of the invention, the first RNA molecule or the first and second RNA molecules target an alternative splicing signal sequence between an exon and an intron of the mutant allele.

[0165] According to an embodiment of the invention, the second RNA molecule targets a sequence present in both the mutant and functional alleles of the ELANE gene.

[0166] According to an embodiment of the invention, the second RNA molecule targets an intron.

[0167] According to an embodiment of the invention, a method is provided which comprises subjecting a mutant allele to an insertion or deletion by an error-prone non-homologous end joining (NHEJ) mechanism, resulting in a frameshift in the sequence of the mutant allele.

[0168] According to an embodiment of the invention, the frameshift results in the inactivation or knockout of the mutant allele.

[0169] According to an embodiment of the invention, a frameshift creates a premature stop codon in the mutant allele.

[0170] According to an embodiment of the invention, the frameshift causes the transcript of the mutant allele to be subjected to nonsense-mediated mRNA decay.

[0171] According to embodiments of the invention, inactivation or treatment results in a truncated protein encoded by the mutant allele and a functional protein encoded by the functional allele.

[0172] The compositions and methods of the disclosure may be utilized to treat, prevent, ameliorate, or slow the progression of SCN or CyN.

[0173] In some embodiments, the mutant allele is inactivated by delivering to a cell an RNA molecule that targets a heterozygous SNP present in the promoter region, start codon or untranslated region (UTR) of the ELANE gene, wherein the RNA molecule targets the nucleotide base, REF or ALT, of the SNP present in the mutant allele of the ELANE gene.

[0174] In some embodiments, the mutant allele is inactivated by removing at least a portion of the promoter and / or removing the start codon and / or a portion of the UTR. In some embodiments, the method of inactivating the mutant allele comprises removing at least a portion of the promoter. In such embodiments, one RNA molecule is designed to target a first heterozygous SNP present in the promoter or upstream of the promoter of the ELANE gene, and another RNA molecule targets a second heterozygous SNP, which is downstream of the first SNP and present in the promoter, UTR or intron or exon of the ELANE gene. Alternatively, one RNA molecule may be designed to target a heterozygous SNP present in the promoter or upstream of the promoter or UTR of the ELANE gene, and another RNA molecule is designed to target a sequence present in an intron of both the mutant allele and the functional allele of the ELANE gene. Alternatively, one RNA molecule may be designed to target a sequence upstream of the promoter present in both the mutant allele and the functional allele, and the other guide is designed to target a heterozygous SNP present anywhere in the ELANE gene, such as in an exon, intron, UTR or downstream of the promoter of the ELANE gene, with the RNA molecule targeting the nucleotide base, REF or ALT of the SNP present in the mutant allele of the ELANE gene.

[0175] In some embodiments, the method of inactivating a mutant allele comprises an exon skipping step, which comprises removing an exon containing a disease-causing mutation from the mutant allele. Removal of an exon containing a disease-causing mutation from a mutant allele requires two RNA molecules that target regions flanking the entire or partial exon of the exon. Removal of an exon containing a disease-causing mutation may be designed to remove the disease-causing activity of the protein while allowing expression of the remaining protein product that retains some or all of its wild-type activity. As an alternative to single exon skipping, multiple exons, entire open reading frames or entire genes can be excised using two RNA molecules flanking the region desired to be excised.

[0176] In some embodiments, a method of inactivating a mutant allele comprises delivering two RNA molecules to a cell, one RNA molecule targeting a first heterozygous SNP present in an exon or promoter of the ELANE gene, an RNA molecule targeting the nucleotide base, REF or ALT, of the first SNP present in the mutant allele of the ELANE gene, and the other RNA molecule targeting a second heterozygous SNP present in the same or a different exon or intron of the ELANE gene, an RNA molecule targeting the nucleotide base, REF or ALT, of the second SNP present in the mutant allele of the ELANE gene, or the second RNA molecule targeting an intron sequence present in both the mutant allele or the functional allele.

[0177] In some embodiments, an RNA molecule is used to target a CRISPR nuclease to an alternative splicing signal sequence between an exon and an intron of a mutant allele, thereby destroying the alternative splicing signal sequence of the mutant allele.

[0178] Any one or combination of the above mentioned strategies for inactivating mutant alleles may be used in the context of the present invention.

[0179] Additional strategies may be used to inactivate mutant alleles. For example, in an embodiment of the present invention, an RNA molecule is used to target CRISPR nuclease to exons or splice sites of mutant alleles to generate double-strand breaks (DSBs), insert or delete nucleotides by error-prone non-homologous end joining (NHEJ) mechanism, and form frameshift mutations of mutant alleles. Frameshift mutations may (1) inactivate or knock out mutant alleles by creating premature stop codons in mutant alleles, creating truncated proteins, or (2) cause nonsense-mediated mRNA decay in mutant allele transcripts. In a further embodiment, an RNA molecule is used to target CRISPR nuclease to promoters of mutant alleles.

[0180] In some embodiments, the method of inactivating a mutant allele further comprises enhancing the activity of the functional protein, such as by providing a small molecule, such as a protein / peptide, a nucleic acid encoding the protein / peptide, or a chemical, that can activate / enhance the activity of the functional protein.

[0181] According to some embodiments, the present disclosure provides RNA molecules that bind / associate with and / or target an RNA-guided DNA nuclease, such as a CRISPR nuclease, to a sequence that includes at least one nucleotide (such as a heterozygous SNP) that differs between a mutant allele and a functional allele of a gene of interest (such as a sequence of the mutant allele that is not present in the functional allele).

[0182] In some embodiments, the methods include contacting a mutant allele of a gene of interest with a CRISPR nuclease, such as an allele-specific RNA molecule and a Cas9 protein, which binds to a nucleotide sequence of the mutant allele of the gene of interest that differs from the nucleotide sequence of a functional allele of the gene of interest by at least one nucleotide, thereby modifying or knocking out the mutant allele.

[0183] In some embodiments, the allele-specific RNA molecule and the CRISPR nuclease are introduced into a cell encoding a gene of interest. In some embodiments, the cell encoding the gene of interest is in a mammalian subject. In some embodiments, the cell encoding the gene of interest is in a plant.

[0184] In some embodiments, the cleaved mutant allele undergoes insertion or deletion (indel) by an error-prone non-homologous end joining (NHEJ) mechanism, generating a frameshift in the sequence of the mutant allele. In some embodiments, the generated frameshift results in the inactivation or knockout of the mutant allele. In some embodiments, the generated frameshift creates a premature stop codon in the mutant allele, generating a truncated protein. In such embodiments, the method results in a truncated protein encoded by the mutant allele and a functional protein encoded by the functional allele. In some embodiments, the frameshift generated in the mutant allele using the methods of the invention results in the transcript of the mutant allele undergoing nonsense-mediated mRNA decay.

[0185] In some embodiments, the mutant allele is an allele of the "neutrophil elastase" gene (ELANE gene). In some embodiments, the RNA molecule targets a heterozygous SNP in the ELANE gene that coexists / is associated with a mutant sequence associated with an SCN or CyN genetic disorder. In some embodiments, the RNA molecule targets a heterozygous SNP in the ELANE gene, where heterozygosity for said SNP is highly prevalent in the population. In an embodiment of the invention, the REF nucleotide is prevalent in the mutant allele but not in the functional allele of the individual subject to be treated. In an embodiment of the invention, the ALT nucleotide is prevalent in the mutant allele but not in the functional allele of the individual subject to be treated. In some embodiments, a disease-causing mutation in a mutant ELANE gene is targeted.

[0186] In an embodiment of the invention, the heterozygous SNP may or may not be associated with an ELANE-associated disease phenotype. In an embodiment of the invention, the heterozygous SNP is associated with an ELANE-associated disease phenotype. In an embodiment of the invention, the SNP is not associated with an ELANE-associated disease phenotype.

[0187] In some embodiments, the heterozygous SNP is within an exon of a gene of interest. In such embodiments, the guide sequence portion of the RNA molecule may be designed to target the exon of the gene of interest.

[0188] In some embodiments, the heterozygous SNP is within an intron or exon of the gene of interest. In some embodiments, the heterozygous SNP is at a splice site between an intron and an exon. In some embodiments, the heterozygous SNP is at a PAM site of the gene of interest.

[0189] Those skilled in the art will understand that in each embodiment of the present invention, individually, each of the RNA molecules of the present invention can be complexed with a nuclease, such as a CRISPR nuclease, associated with a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM). The nuclease then mediates cleavage of the target DNA to create a double-stranded break within the protospacer. Thus, in embodiments of the present invention, the guide sequence and RNA molecule of the present invention can target a position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides upstream or downstream from the PAM site.

[0190] Thus, in an embodiment of the invention, an RNA molecule of the invention complexed with a nuclease, such as a CRISPR nuclease, may affect a double strand break at an allele 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 genes upstream or downstream from a target site. One skilled in the art will appreciate that if a heterozygous polymorphic site is present and used to define the target, the RNA molecule may be designed to target only the REF or ALT nucleotide base of the heterozygous polymorphic site to affect a double strand break.

[0191] It is understood that if the heterozygous polymorphic site is within the PAM site, the RNA molecule may be designed to target a sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides upstream or downstream from the PAM site, and the complex of the RNA molecule and nuclease is designed to target only one of the REF or ALT nucleotide bases of the heterozygous polymorphic site of the PAM site to affect cleavage at the PAM site, e.g., tracrRNA is designed to target one of the REF or ALT nucleotide bases of the heterozygous polymorphic site.

[0192] In an embodiment of the invention, the RNA molecule is designed to target a heterozygous polymorphic site present in the ELANE gene and the RNA molecule and / or a complex of the RNA molecule and a CRISPR nuclease is designed to target a nucleotide base, REF or ALT, of the heterozygous polymorphic site present in a mutant allele of the ELANE gene.

[0193] In an embodiment of the present invention, the RNA molecule, composition, method, cell, kit or drug is used to treat a subject with a disease phenotype caused by a heterozygous ELANE gene. In an embodiment of the present invention, the disease is SCN or CyN. In such an embodiment, the method improves, ameliorate or prevents the disease phenotype.

[0194] In an embodiment of the invention, the RNA molecule, composition, method, cell, kit or drug of the invention is used in combination with a second treatment for SCN or CyN to treat a subject. In an embodiment of the invention, the RNA molecule, composition, method, cell, kit or drug of the invention is administered prior to, during and / or after administration of the second treatment.

[0195] In an embodiment of the invention, the RNA molecule, composition, method, cell, kit or medicament of the invention is administered in combination with granulocyte colony stimulating factor (G-CSF) treatment.

[0196] The above embodiments refer to the CRISPR nuclease, the RNA molecule(s) and the tracrRNA molecule being effective simultaneously in the subject or cell. The CRISPR, RNA molecule(s) and the tracrRNA molecule can be delivered substantially simultaneously, or can be delivered at different times but be effective simultaneously. For example, this includes delivering the CRISPR nuclease to the subject or cell before the RNA molecule and / or the tracrRNA molecule are substantially present in the subject or cell.

[0197] According to an embodiment of the invention, there is provided a method for inactivating a mutant allele of the ELANE gene in a cell, the method comprising: a) selecting cells which have an ELANE gene mutation associated with SCN or CyN and are heterozygous at one or more polymorphic sites in the ELANE gene selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs201048029, rs199720952, rs2859l229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs761481944, rs376l008, rs10409474, rs376l007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854; b) In cells A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, introducing a complex of a CRISPR nuclease and a first RNA molecule that affects a double strand break in a mutant allele of the ELANE gene of the cell, but does not affect a double strand break in a functional allele of the ELANE gene of the cell; Thereby, a method is provided for inactivating a mutant allele of the ELANE gene in a cell.

[0198] According to an embodiment of the invention, there is provided a method for inactivating a mutant allele of the ELANE gene in a cell, the method comprising: a) selecting cells which have an ELANE gene mutation associated with SCN or CyN and are heterozygous at one or more polymorphic sites in the ELANE gene selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs201048029, rs199720952, rs2859l229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs761481944, rs376l008, rs10409474, rs376l007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854; b) In cells A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A step of introducing a composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affecting a double stranded break in a mutant allele of the ELANE gene of the cell but not affecting a double stranded break in a functional allele of the ELANE gene; The method further includes introducing a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex between the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene; Thereby, a method is provided for inactivating a mutant allele of the ELANE gene in a cell.

[0199] According to an embodiment of the present invention, there is provided a method for inactivating a mutant allele of the ELANE gene in a cell having a mutation associated with SCN or CyN, the method comprising the steps of: inactivating a mutant allele of the ELANE gene in a cell having a mutation associated with SCN or CyN, the cell being selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs201048029, rs199720952, rs2859l229, rs7l335276, rs and the individual is heterozygous at one or more polymorphic sites in the ELANE gene selected from the group consisting of rs10409474, rs376l007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854, and the method comprises: To cells A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affecting and introducing a double stranded break in a mutant allele of the ELANE gene; Thereby, a method is provided for inactivating a mutant allele of the ELANE gene in a cell.

[0200] According to an embodiment of the present invention, a method for the treatment of a cell comprising administering to a patient having an ELANE gene mutation associated with SCN or CyN, comprising administering to said patient a therapeutic agent selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs201048029, rs199720952, rs2859l229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs 1. A method for inactivating a mutant allele of the ELANE gene that is heterozygous at one or more polymorphic sites in the ELANE gene selected from the group consisting of s761481944, rs376l008, rs10409474, rs376l007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854, the method comprising: To cells A CRISPR nuclease or a sequence encoding a CRISPR nuclease, and a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides; a complex of the CRISPR nuclease and the first RNA molecule affects a double-stranded break in a mutant allele of the ELANE gene; The method further includes introducing a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex between the second RNA molecule and the CRISPR nuclease affects a second double-stranded break in the ELANE gene; Thereby, a method is provided for inactivating a mutant allele of the ELANE gene in a cell.

[0201] In an embodiment of the invention, a complex of a CRISPR nuclease and a first RNA molecule affects a double-stranded break in a mutant allele of the ELANE gene of the cell, but does not affect a double-stranded break in a functional allele of the ELANE gene of the cell.

[0202] In an embodiment of the invention, the cells are also heterozygous at at least one further polymorphic site in the ELANE gene selected from the group consisting of rs104l4837, rs376l005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs28591229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs76l48l944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8l07095, rs10424470 and rs78302854.

[0203] In an embodiment of the invention, the cells having an ELANE gene mutation associated with SCN or CyN may be derived from a subject having an ELANE gene mutation and / or afflicted with SCN or CyN. Thus, selecting a cell having an ELANE gene mutation may comprise selecting a subject having an ELANE gene mutation. In a further embodiment of the invention, selecting a cell may comprise selecting a cell from a subject having an ELANE gene mutation. In an embodiment of the invention, introducing a composition of the subject invention into a cell may comprise introducing a composition of the invention into a cell of a subject afflicted with an ELANE gene mutation.

[0204] Thus, in an embodiment of the invention, a method is provided for inactivating a mutant allele of the ELANE gene in a subject having an ELANE gene mutation that causes SCN or CyN, the method comprising the steps of: Methods are provided that include selecting a subject who is heterozygous at one or more polymorphic sites in the ELANE gene selected from the group consisting of rs1041095, rs77, rs3826946, rs10413889, rs76l48l944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8107095, rs10424470 and rs78302854n.

[0205] Thus, embodiments of the present invention include screening subjects or cells for the ELANE gene. Those skilled in the art will readily understand how to screen for mutations in the ELANE gene in the prior art, including, but not limited to, sequencing-by-synthesis, Sanger sequencing, karyotyping, fluorescent in situ hybridization, and / or microarray testing. In embodiments of the present invention, mutations in the ELANE gene are screened by exon sequencing.

[0206] In an embodiment of the present invention, a subject is diagnosed with or has been diagnosed with SCN or CyN by measuring absolute neutrophil count (ANC) in peripheral blood. In an embodiment of the present invention, SCN is diagnosed or has been diagnosed before the subject reaches 6 months of age. In an embodiment of the present invention, CyN is diagnosed or has been diagnosed between 12 and 24 months of age or after 24 months of age. In an embodiment of the present invention, SCN or CyN is diagnosed by one or more recurrent acute oral medical diseases. In an embodiment of the present invention, SCN or CyN is diagnosed by bone marrow examination, preferably bone marrow examination is a cytogenetic bone marrow survey. In an embodiment of the present invention, SCN or CyN is diagnosed by one or more of antineutrophil antibody assay, immunoglobulin assay (Ig GAM), lymphocyte immunophenotyping, pancreatic markers (serum trypsinogen and fecal elastase) and fat-soluble vitamin levels (vitamins A, E and D). It is understood that all diagnostic methods may be used together with any other diagnostic methods.

[0207] In an embodiment of the invention, subjects diagnosed with SCN or CyN are screened by exon sequencing to identify ELANE pathogenic mutations in the ELANE gene. In a further embodiment, subjects are screened by Sanger sequencing to confirm heterozygosity of at least one SNP in Table 1. In an embodiment of the invention, the SNP is one of rs1683564, rsI0414837 and rs3761005. In an embodiment of the invention, the nucleotide of the heterozygous SNP of the mutant allele of the ELANE gene is determined using BAC bio. In an embodiment of the invention, a suitable guide is selected according to Table 2. In an embodiment of the invention, the selected guide is introduced into cells, such as PBMCs, obtained from the subject, and the reduction of pathogenic ELANE mutations in the cells is measured, such as by next-generation sequencing technology.

[0208] Using the CR1SPR / Cas9 gene editing system, nucleases can be targeted to target sites in a sequence-specific manner to combat disease-causing mutations. Hematopoietic stem and progenitor cells (HSPCs) have therapeutic potential because they are capable of both self-renewal and differentiation (Yu, Natanson, and Dunbar 2016). Thus, an embodiment of the present invention applies genome editing to HSPCs.

[0209] In an embodiment of the invention, the home-grown therapy utilizes autologous CRISPR / Cas9 edited CD34+ hematopoietic stem cells from patients diagnosed with SCN or CyN. In an embodiment of the invention, the CD34+ cells are isolated from bone marrow or peripheral blood mononuclear cells (PBMCs) following apheresis in the patient.

[0210] In cases where there are dominant negative (or compound heterozygous) indications, such as SCN or CyN, the strategy is to edit the mutant allele and avoid cleavage of the non-mutated allele or other off-targets by targeting the heterozygous SNP sequence.

[0211] An embodiment of the present invention includes the following steps. Selection of patients diagnosed with SCN or CyN identified as heterozygous for at least one of the SNPs in Table 1 herein below. In an embodiment of the invention, the subject is heterozygous for rs104l4837, rs376l005 or rs1683564, Selection of treatment strategies based on identified heterozygous SNP locations in candidate patients. Obtaining HSPC cells from the subject's bone marrow, either by peripheral blood withdrawal or by mobilization and apheresis; optionally processing the HSPC cells (enriching, stimulating, both, etc.) To HSPC cells (by ex vivo electroporation, etc.) A sequence (e.g., mRNA) encoding a CRISPR nuclease or the like; A unique RNA molecule that targets a specific sequence (REF / ALT sequence) of the identified heterozygous SNP of the mutant allele and introducing a composition comprising a non-characteristic RNA molecule that targets a sequence in intron 3, intron 4, or the 3'UTR that is common to both the mutant allele and the other allele; thereby editing HSPC cells to knock out the expression of the mutant allele of ELANE; and The edited HSPCs are introduced into candidate patients.

[0212] In an embodiment of the invention, CD34+ cells may be isolated from bone marrow or peripheral blood mononuclear cells (PBMCs) after patient apheresis. Bone marrow or PBMCs may be collected from patients by apheresis after HSPC mobilization. In an embodiment of the invention, the apheresis product may be washed to remove platelets and the CD34+ cell population may be enriched by purification using, for example, the CliniMACS system (Miltenyi Biotec). In an embodiment of the invention, selected cells may be pre-stimulated ex vivo with, for example, a mixture of recombinant human cytokines. In an embodiment of the invention, cells may be electroporated. In an embodiment of the invention, stimulated cells (such as CD34+ cells), CRISPR nuclease, mRNA and gRNA may be pre-incubated under defined conditions prior to electroporation. In an embodiment of the invention, cells are electroporated ex vivo with CRISPR nuclease, mRNA / gRNA mixture or with pre-assembled RNPs (ribonuclease protein, which is the CRISPR nuclease protein, and gRNA), followed by cell washing. In an embodiment of the invention, cells are suspended into the final formulation. In an embodiment of the invention, the cells may be resuspended. In an embodiment of the invention, the resuspended cells may be loaded into an infusion bag. In an embodiment of the invention, the bag may be frozen using a freeze-down step in a controlled rate freezer and / or stored in vapor phase liquid nitrogen. In an embodiment of the invention, the product may be administered to a patient via intravenous (IV) administration.

[0213] Dominant genetic disorders Those skilled in the art will understand that any subject with any type of heterozygous genetic disorder (such as a dominant genetic disorder) may undergo the methods described herein. In one embodiment, the present invention may be used to target genes involved in, associated with, or responsible for a dominant genetic disorder, such as SCN or CyN. In some embodiments, the dominant genetic disorder is SCN or CyN. In some embodiments, the target gene is the ELANE gene (Entrez Gene, gene ID No: 335).

[0214] CRISPR nucleases and PAM recognition In some embodiments, the sequence-specific nuclease is selected from a CRISPR nuclease or a functional variant thereof. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, an RNA sequence (such as Cpfl) that guides the RNA-guided DNA nuclease binds to and / or directs the RNA-guided DNA nuclease to a sequence that contains at least one nucleotide (such as a SNP) that differs between a mutant allele and its functional counterpart. In some embodiments, the CRISPR complex does not further comprise a tracrRNA. In a non-limiting example, the RNA-guided DNA nuclease is a CRISPR nuclease, but the at least one nucleotide that differs between the dominant mutant allele and the functional allele may be within the PAM site and / or proximal to the PAM site within the region to which the RNA molecule is designed to hybridize. One skilled in the art will understand that RNA molecules can be engineered to bind to a selected target in a genome by methods well known in the art.

[0215] In an embodiment of the present invention, the Type II CRISPR system utilizes mature crRNA, i.e., as an additional target recognition requirement, the tracrRNA complex targets a CRISPR nuclease, such as Cas9, to the target DNA via Watson-Crick base pairing between the crRNA and the protospacer of the target DNA adjacent to the protospacer adjacent to the protospacer adjacent motif (PAM). The CRISPR nuclease then mediates cleavage of the target DNA to create a double-stranded break within the protospacer. One of skill in the art will appreciate that each of the engineered RNA molecules of the present invention is further designed to associate with a target genomic DNA sequence of interest bordered by a protospacer adjacent motif (PAM), e.g., the PAM is further designed to match a sequence associated with the type of CRISPR nuclease utilized, such as, by way of non-limiting example, NGG or NAG for Streptococcus pyogenes Cas9 WT (SpCAS9), where "N" is any nucleobase, NNGRRT for Staphylococcus aureus (SaCas9), NNNVRYM for Jejuni Cas9 WT, NGAN or NGNG for SpCas9-VQR variants, NGCG for SpCas9-VRER variants, NGAG for SpCas9-EQR variants, NNNNGATT for Neisseria meningitidis (NmCas9), or TTTV for Cpfl. The RNA molecules of the present invention are each designed to form a complex in combination with one or more different CRISPR nucleases and are designed to target a polynucleotide sequence of interest using one or more different PAM sequences for each CRISPR nuclease utilized.

[0216] In some embodiments, RNA-guided DNA nucleases, such as CRISPR nucleases, can be used to cut DNA at desired positions in the genome of a cell.Although the most commonly used RNA-guided DNA nucleases are derived from the CRISPR system, other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein.See, for example, U.S. Patent Application Publication No. 2015-0211023, the contents of which are incorporated herein by reference.

[0217] There are many different CRISPR systems that can be used in the practice of the present invention. The CRISPR system can be a type I, type II, type III or type V system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl2a, Casl2b, Casl2c, Casl2d, Casl2d, Casl Od, CasF, CasG, Casli, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966 (see, e.g., Koonin 2017).

[0218] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease derived from a type II CRISPR system (such as Cas9). CRISPR nucleases have been shown to be effective in the detection and characterization of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces roseum, and other pathogenic bacteria. roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.), Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplankti, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Francisella cf. novicida Fxl, Alicyclobacillus acidoterrestris, Oleiphilus sp., Bacteria CC09 39 24, Deltaproteobacteria bacteria, or any species that encodes a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria may also be used in the context of the present invention (see Burstein et al. Nature, 2017). Variants of CRISPR proteins with known PAM sequences, such as spCas9 D1135E variant, spCas9 VQR variant, spCas9 EQR variant, or spCas9 VRER variant, may also be used in the context of the present invention.

[0219] Thus, RNA-guided DNA nucleases of one CRISPR system, such as Cas9 protein, modified Cas9, homologs or orthologs of Cas9, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpfl and its homologs and orthologs, may be used in the compositions of the invention.

[0220] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that has a qualitative biological characteristic in common with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of native sequences and derivatives of native sequence polypeptides and fragments thereof, provided that they have a biological activity in common with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate to fragments. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent modifications thereof, and fusions thereof. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, covalent modifications, or fragments thereof of Cas proteins. Cas proteins and derivatives of Cas proteins or fragments thereof, including Cas proteins or fragments thereof, may be available from cells, may be chemically synthesized, or may be available by a combination of these two processes. The cell may be a cell that naturally produces a Cas protein, or a cell that naturally produces a Cas protein and has been genetically modified to produce an endogenous Cas protein at high expression levels or to produce a Cas protein from an exogenously introduced nucleic acid encoding a Cas that is the same or different from the endogenous Cas. In some cases, the cell does not naturally produce a Cas protein and is genetically modified to produce a Cas protein.

[0221] In some embodiments, the CRISPR nuclease is Cpfl. Cpfl is a single RNA-guided endonuclease that utilizes T-rich protospacer adjacent motifs. Cpfl cleaves DNA by staggered DNA double-strand breaks. Two Cpfl enzymes from the genera Acidaminococcus and Lachnospiraceae have been shown to perform efficient genome editing activity in human cells (see Zetsche et al., (2015) Cell.).

[0222] Thus, RNA-guided DNA nucleases of type II CRISPR systems, such as Cas9 protein or modified Cas9 or homologs, orthologs or variants of Cas9, or other RNA-guided DNA nucleases belonging to other types of CRISPR systems, such as Cpfl and its homologs, orthologs or variants, may be used herein.

[0223] In some embodiments, the guide molecule comprises one or more chemical modifications that impart new or improved properties (e.g., improved stability from degradation, improved hybridization energetics, or improved binding properties with RNA-guided DNA nucleases, etc.) Suitable chemical modifications include, but are not limited to, modified bases, modified sugar moieties, or modified internucleoside linkages.Non-limiting examples of suitable chemical modifications include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-0-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-0-methylpseudouridine, β,D-galactosylqueosine, 2'-0-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpropane ... pseudouridine, l-methylguanosine, 1-methylinosine, "2,2-dimethylguanosine", 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, "β,D-mannosylqueuosine", 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-Methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxosine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine uridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-0-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, "3-(3-amino-3-carboxy-propyl)uridine, (acp3)u", 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine or 1-methylpseudouridine. Each possibility represents a separate embodiment of the invention.

[0224] Further non-limiting examples of suitable chemical modifications include m 1 A(1-methyladenosine), m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms2 m 6 A(2-methylthio-N 6 -Methyladenosine);i 6 A(N 6 -Isopentenyladenosine);ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine;io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -Glycinylcarbamoyladenosine;t 6 A(N 6 -Threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -Threonylcarbamoyl adenosine);m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyl adenosine);hn 6 A(N 6 -Hydroxynorvalylcarbamoyl adenosine);ms 2 hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; Ar(p)(2'-O-ribosyladenosine (phosphate)); I(inosine); m 1 I(l-methylinosine);m 1 Im(l,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine);ac 4 C(N 4 -Acetylcytidine);f 5 C(5-formylcytidine);m 5 Cm(5,2'-O-dimethylcytidine);ac 4 Cm(N4 -acetyl-2'-O-methylcytidine);k 2 C(lycidin);m 1 G(l-methylguanosine);m 2 G(N 2 -Methylguanosine);m 7 G(7-methylguanosine);Gm(2'-O-methylguanosine);m 2 2 G(N 2 ,N 2 -Dimethylguanosine);m 2 Gm(N 2、2’ -O-dimethylguanosine);m 2 2 Gm(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine(phosphate));yW(Wybutosine); 02 yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (unmodified hydroxywybutosine); imG (uybutosine); mimG (methyluybutosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosylqueuosine); manQ (mannosyl-queuosine); preQ o (7-cyano-7-deazaguanosine);preQ 1 (7-aminomethyl-7-deazaguanosine);G + (Archaeosine);D(Dihydrouridine);m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine);m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine); 5 U(5-methoxyuridine); cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-oxyacetic acid methyl ester);chm 5U(5-(carboxyhydroxymethyl)uridine));mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm 5 U(5-Methoxycarbonylmethyluridine);mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine); nm 5 S 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5U(5-carbamoylmethyluridine);ncm5Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cmnm5Um(5-carboxymethylaminomethyl-2'-O-methyluridine);cmmm5s2U(5-carboxymethylaminomethyl-2-thiouridine);dimethyladenosine;Im(2'-O-methylinosine);m4C(N4-methylcytidine);m4Gm(N4,2'-O-dimethylcytidine);hm5C(5-hydroxymethylcytidine);m3U(3-methyluridine);cm5U(5-carboxymethyluridine);m6Am(N6,2'-O-dimethyladenosine) sine); m62Am (N6,N6,0-2'-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2,2,7G (N2,N2,7-trimethylguanosine); m3Um (3,2'-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0-methylcytidine); mlGm (l,2'-0-dimethylguanosine); m'Am (l,2'-0-dimethyladenosine); xm5U (5-taurinomethyluridine); xmVU (5-taurinomethyl-2-thiouridine); imG-l4 (4-demethyluiosine); imG2 (isouiosine); or ac6A (N6-acetyladenosine). Each possibility represents a separate embodiment of the present invention. (See U.S. Patent No. 9,750,824).

[0225] Guide sequences that specifically target mutant alleles A certain gene may contain thousands of SNPs. Using a target window of 24 base pairs to target each SNP of a gene would require hundreds of thousands of guide sequences. Any given guide sequence may degrade, have limited activity, lose activity, or have off-target effects when used to target SNPs. Therefore, a suitable guide sequence is required to target a certain gene. The present invention has identified a novel set of guide sequences to knock out the expression of mutant proteins, inactivate mutant alleles of ELANE gene, and treat SCN or CyN.

[0226] The present disclosure provides guide sequences that can specifically target mutant alleles to inactivate while functional alleles remain unmodified.The guide sequences of the present invention are designed to specifically distinguish between mutant alleles and functional alleles, and are most likely to do so.Of all possible guide sequences that target the mutant alleles that one wishes to inactivate, the specific guide sequences disclosed in the present invention are particularly effective in functioning in the disclosed embodiments.

[0227] Briefly, a guide sequence may have the following properties: (1) target a heterozygous SNP / insertion / deletion / indel that has a prevalence greater than 1% in a population, a particular ethnic group, or a patient population and where the SNP / insertion / deletion / indel heterozygosity rate in the same population is greater than 1%; (2) target the location of the SNP / insertion / deletion / indel proximal to a portion of a gene, e.g., within 5k bases of any portion of the gene, such as the promoter, UTR, exon, or intron; and (3) target mutant alleles using an RNA molecule that targets founder or common pathogenic mutations for the disease / gene. In some embodiments, the prevalence of the SNP / insertion / deletion / indel in the general population, a particular ethnic group, or a patient population is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, and the SNP / insertion / deletion / indel heterozygosity rate in the same population is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment and may be combined at will.

[0228] For each gene, according to the SNP / insertion / deletion / indel, any one of the following strategies may be used to inactivate the mutant allele: (1) a knockout strategy using one RNA molecule - one RNA molecule is used to target CRISPR nuclease to the mutant allele to create a double strand break (DSB) and form a frameshift mutation in an exon or splice site region of the mutant allele; (2) a knockout strategy using two RNA molecules - one RNA molecule is used, where one RNA molecule targets a region in the promoter of the mutant allele or an upstream region of the mutant allele, and another RNA molecule is used to target downstream of the first RNA molecule in the promoter, exon or intron of the mutant allele; (3) an exon(s) skipping strategy - one RNA molecule is used to target CRISPR nuclease to a splice site region either at the 5' end of the intron (donor sequence) or at the 3' end of the intron (acceptor sequence) to destroy the splice site. Alternatively, two RNA molecules may be utilized, with the first targeting the upstream region of the exon and the second targeting the downstream region of the first, thereby excising the exon(s). Based on the location of the SNPs / insertions / deletions / indels identified for each mutant allele, any one or combination of the above methods for inactivating mutant alleles may be utilized.

[0229] When only one RNA molecule is used, the location of the SNP is within an exon or very close (such as within 20 base pairs) to a splice site between an intron and an exon. When two RNA molecules are used, the guide sequence may target two SNPs such that the first SNP is upstream of exon 1, such as in the 5' untranslated region, in the promoter, or in the first 2 kilobases 5' of the transcription start point, and the second SNP is downstream of the first SNP, such as in the first 2 kilobases 5' of the transcription start point, in introns 1, 2 or 3, or in exons 1, 2 or 3.

[0230] The guide sequence of the present invention may target a SNP upstream of the target gene, preferably upstream of the last exon of the target gene. The guide sequence may target a SNP upstream of exon 1, for example, within the 5' untranslated region, within the promoter, or within the first 4-5 kilobases 5' of the transcription start site.

[0231] Guide sequences of the present invention may also target SNPs in close proximity (such as within 50 base pairs, more preferably within 20 base pairs) of known protospacer adjacent motif (PAM) sites.

[0232] The guide sequences of the present invention may also target (1) heterozygous SNPs in the targeted gene, (2) heterozygous SNPs upstream and downstream of the gene, (3) SNPs with a prevalence of SNP / insertion / deletion / indel greater than 1% in the general population, a particular ethnic group, or a patient population, (4) a guanidine-cytosine content greater than 30% and less than 85%, (5) no thymine / uracil repeats of 4 or more or no guanine, cytosine, or adenosine repeats of 8 or more, (6) no off-targets identified by off-target analysis, and (7) preferably targeting exons rather than introns or upstream of the SNP rather than downstream of the SNP.

[0233] In an embodiment of the invention, the SNP may be upstream or downstream of the gene. In an embodiment of the invention, the SNP is within 4,000 base pairs upstream or downstream of the gene.

[0234] At least one nucleotide that differs between the mutant allele and the functional allele may be upstream, downstream or inside the disease-causing mutation of the gene of interest. At least one nucleotide that differs between the mutant allele and the functional allele may be inside an exon or inside an intron of the gene of interest. In some embodiments, at least one nucleotide that differs between the mutant allele and the functional allele is inside an exon of the gene of interest. In some embodiments, at least one nucleotide that differs between the mutant allele and the functional allele is inside an exon or inside an intron of the gene of interest, i.e., very close to the splice site between the intron and the exon, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides upstream or downstream of the splice site.

[0235] In some embodiments, at least one nucleotide is a single nucleotide polymorphism (SNP). In some embodiments, each of the nucleotide variants of the SNP may be expressed in a mutant allele. In some embodiments, the SNP may be a founder or common pathogenic mutation.

[0236] The guide sequence may target a SNP that has both (1) a high population prevalence, such as greater than 1% in the population, and (2) a high population heterozygosity rate, such as greater than 1%. The guide sequence may target a SNP that is distributed worldwide. The SNP may be a founder or common pathogenic mutation. In some embodiments, the population prevalence is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment. In some embodiments, the population heterozygosity rate is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment.

[0237] In some embodiments, at least one nucleotide that differs between the mutant allele and the functional allele is associated with / coexists with a disease-causing mutation that is highly prevalent in the population. In such embodiments, "high prevalence" refers to at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Each possibility represents a separate embodiment of the invention. In one embodiment, at least one nucleotide that differs between the mutant allele and the functional allele is a disease-causing mutation. In some embodiments, the SNP is highly prevalent in the population. In such embodiments, "high prevalence" refers to at least 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, 60%, or 70% of the population. Each possibility represents a separate embodiment of the invention.

[0238] A guide sequence of the present invention may meet any one of the above criteria and is most likely to distinguish a mutant allele from a corresponding functional allele.

[0239] In some embodiments, the RNA molecule targets heterozygous SNPs present in the ELANE gene from SNPs as shown in Table 1 below. Details of the SNPs are shown in the first column from the left, including the SNP ID (based on NCBI's 2018 database of single nucleotide polymorphisms (dbSNP)). For variants without rs numbers, the characteristics of the variants are shown based on the gnomAD 2018 browser database. The second column shows the identifier assigned to each SNP. The third column shows the position of each SNP on the ELANE gene. [Table 1]

[0240] FIG. 5 discloses the heterogeneity of certain SNPs selected from Table 1 in human populations.

[0241] An embodiment of the invention may include excising the promoter region from upstream of the SNP position to intron 3, intron 4 or 3'UTR. In one embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP in the upstream region of the mutant allele (strategy 1a-rs104l4837, strategy 1b-rs376l005) and the second guide sequence targets a sequence of intron 4 common to the two alleles of the gene (Figure 1). In a further embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP in the upstream region of the mutant allele (strategy 1a-rs104l4837, strategy 1b-rs376l005) and the second guide sequence targets a sequence of intron 3 common to the two alleles of the gene. In another embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP in the upstream region of the mutant allele (Strategy 1a-rs104l4837, Strategy 1b-rs376l005) and the second guide sequence targets a sequence in the 3'UTR common to the two alleles of the gene (Figure 2).

[0242] An embodiment of the invention may include excising intron 3, intron 4 or a region downstream of the 3'UTR from the 3'UTR. In one embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence of intron 4 common to the two alleles of the gene. In a further embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence of intron 3 common to the two alleles of the gene. In a further embodiment, the first guide sequence targets a specific sequence of a heterozygous SNP position in the upstream region of the mutant allele (Strategy 2-rs1683564) and the second guide sequence targets a sequence of the 3'UTR common to the two alleles of the gene (Figure 3).

[0243] An embodiment of the present invention excises intron 3, intron 4, or the 3'UTR to a region downstream of the 3'UTR. This strategy is designed to specifically knock out disease-causing alleles ("mutated alleles") while leaving healthy alleles intact. Allele-specific editing is achieved by using guides that target discriminating (heterozygous) SNP positions with relatively high heterozygous frequency in the population (Figure 4).

[0244] Cellular delivery In the illustrated methods, it is understood that the RNA molecules and compositions described herein may be delivered to a target cell or subject by any suitable means. The following embodiments provide non-limiting examples of methods for delivering the RNA molecules and compositions of the present invention.

[0245] In some embodiments, the RNA molecules and compositions of the invention may be targeted to any cell that contains and / or expresses a dominant negative allele, including any mammalian or plant cell. For example, in one embodiment, the RNA molecule specifically targets a mutant ELANE allele and the target cell is a hepatocyte.

[0246] Any suitable viral vector system may be used to deliver the nucleic acid compositions, such as the RNA molecule compositions, of the subject invention. Conventional viral or non-viral based gene transfer methods can be used to introduce the nucleic acid and target tissue. In certain embodiments, the nucleic acid is administered in in vitro or ex vivo gene therapy applications. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle such as liposomes or poloxamers. For reviews of gene therapy methods, see Anderson (1992) Science 256:808-813, Nabel & Feigner (1993) TIBTECH 11:211-217, Mitani & Caskey (1993) TIBTECH 11:162-166, Dillon (1993) TIBTECH 11:167-175, Miller (1992) Nature 357:455-460, Van Brunt (1988) Biotechnology 6(10):1149-1154, Vigne (1995) Restorative Neurology and Neuroscience 8:35-36, Kremer & Perricaudet (1995) British Medical Bulletin 51(1):31-44, Haddada et al. (1995) in Current Topics in See Microbiology and Immunology Doerfler and Bohm (eds.), and Yu et al. (1994) Gene Therapy 1:13-26.

[0247] Non-viral delivery methods of nucleic acids and / or proteins include electroporation, lipofection, microinjection, gene guns, particle gun accelerators, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycation or lipid:nucleic acid complexes, artificial virions, and drug-enhanced uptake of nucleic acids, or can be delivered to plants by bacteria or viruses (such as Agrobacterium, Rhizobium, NGR234, Sinorhizobium meliloti, Mesorhizobium loti, Tobacco Mosaic Virus, Potato Virus X, Cauliflower Mosaic Virus, and Cassava Vein Mosaic Virus). (See, e.g., Chung et al. (2006) Trends Plant Sci. 1 l(l):l-4). Sonoporation, such as using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic-lipid mediated delivery of proteins and / or nucleic acids is also contemplated as an in vivo or in vitro delivery method. (See Zuris et al. (2015) Nat. Biotechnol. 33(l):73-80; see also Coelho et al. (2013) N. Engl. J. Med. 369, 819-829; Judge et al. (2006) Mol. Ther. 13, 494-505; and Basha et al. (2011) Mol. Ther. 19, 2186-2200.)

[0248] Further representative nucleic acid delivery systems include those provided by Amaxa.RTM Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787 and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam.TM., Lipofectin.TM. and Lipofectamine.TM. RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO91 / 17424, WO91 / 16024. Delivery can be to cells (ex vivo administration) or to target tissues (in vivo administration).

[0249] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; see U.S. Patent Nos. 186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787.

[0250] An additional delivery method involves the use of packaging the nucleic acid to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target cells using bispecific antibodies, where one arm of the antibody has specificity for the target cell and the other arm has specificity for the EDV. The antibody carries the EDV to the target cell surface, where it is then endocytosed into the cell. Once inside the cell, the contents are released (see MacDiarmid et al. (2009) Nature Biotechnology 27(7):643).

[0251] The use of RNA or DNA virus-based systems for virally mediated delivery of nucleic acids takes advantage of the highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or cells can be engineered in vitro and the modified cells administered to patients (ex vivo). Conventional virus-based systems for delivering nucleic acids include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors and herpes simplex vectors for gene transfer.

[0252] The tropism of retroviruses can be altered by incorporating heterologous envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and generally produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats that have the capacity to package heterologous sequences up to 6-10 kb. The minimal cis-acting LTRs are sufficient to replicate and package the vector, which is then used to integrate a therapeutic gene into the target cell to provide sustained transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchschacher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; PCT / US94 / 05700, etc.).

[0253] At least six viral vector approaches are currently available for clinical trials, utilizing approaches that involve complementation of a defective vector with a gene inserted into a helper cell line to produce the transducing agent.

[0254] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat Med. 1:1017-102; Malech et al. (1997) PNAS 94:2212133-12138). PA317 / pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al. (1995)). Transduction efficiencies of over 50% were observed for MFG-S packaged vectors (Ellem et al. (1997) Immunol Immunother. 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2).

[0255] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, AAV, and Psi-2 cells or PA317, which package retrovirus. Viral vectors used in gene therapy are usually made by a production cell line that packages a nucleic acid vector into a viral particle. This vector usually contains the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences being replaced by an expression cassette that codes for the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the inverted terminal repeats (ITRs) from the AAV genome, which are required for packaging and integration into the host genome. The viral DNA is packaged into a cell line that contains a helper plasmid that codes for the other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus facilitates replication of the AAV vector and expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in significant quantities because it lacks ITR sequences. Adenovirus contamination can be reduced by, for example, heat treatment, to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Pat. No. 7,479,554).

[0256] In many gene therapy applications, it is desirable for gene therapy vectors to be delivered with high specificity to a particular tissue type. Thus, viral vectors can be engineered to have specificity for certain cell types by expressing a ligand as a fusion protein on the outer surface of the virus by a viral coat protein. The ligand is selected to have affinity for a receptor known to be present in the cell type of interest. For example, Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia virus can be engineered to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells that express the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein that contains a ligand for the cell surface receptor. For example, filamentous phage can be engineered to exhibit antibody fragments (such as FAB or Fv) that have specific binding affinity for a receptor on virtually any selected cell. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that favor uptake by specific target cells.

[0257] Gene therapy vectors can be delivered to an individual patient in vivo, generally by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous or intracerebral injection) or local administration, as described above. Alternatively, vectors can be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, cell biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into the patient, typically after selection of cells that have incorporated the vector.

[0258] Ex vivo cell transfection for diagnostics, research, or gene therapy (e.g., via reinfusion of transfected cells into a host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject organism, transfected with a nucleic acid composition, and reinfused into the subject organism (e.g., a patient). A variety of cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al. (1994) Culture of Animal Cells, A Manual of Basic Technique, 3rd ed. and references cited therein for a discussion of methods for isolating and culturing cells from a patient).

[0259] Suitable cells include, but are not limited to, eukaryotic cells and / or cell lines. Non-limiting examples of such cells or cell lines made from such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV, etc.), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T, etc.), perC6 cells, any plant cell (differentiated or undifferentiated), and insect cells such as Spodoptera fugiperda (Sf) or fungal cells such as Saccharomyces, Pichia, and Schizosaccharomyces. In a particular embodiment, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo for reintroduction into the patient to be treated for subsequent treatment with guided nuclease systems (such as CRISPR / Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells and mesenchymal stem cells.

[0260] In one embodiment, stem cells are used in ex vivo methods for cell gene transfer and gene therapy. The advantage of using stem cells is that they can be differentiated in vitro into other cell types or can be introduced into a mammal (such as a cell donor) where they will engraft in the bone marrow. Methods are known for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ and TNF-α (see, for non-limiting examples, Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0261] Using known methods, stem cells are isolated for transduction and differentiation. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies that bind unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes) and lad (differentiated antibody presenting cells) (for a non-limiting example, see Inaba et al. (1992) J. Exp. Med. 176:1693-1702). In some embodiments, already modified stem cells may be used.

[0262] Generally, the cells are administered in a pharmaceutical composition comprising at least one pharma- ceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to a compound, substance, composition and / or dosage form that is suitable for use in contact with human and animal tissues, within the bounds of sound medical decency, without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharma- ceutical, composition or vehicle, such as a liquid, solid filler, diluent, excipient or solvent, that encapsulates a substance.

[0263] Any one of the RNA molecule compositions described herein is suitable for genome editing in any cell, whether postmitotic or not actively dividing, for example, arrested cell.The example of postmitotic cell that can be edited using the RNA molecule composition of the present invention includes, but is not limited to, hepatocyte.

[0264] Vectors (e.g., retroviruses, liposomes, etc.) containing therapeutic nucleic acid compositions can also be administered directly to an organism to transduce cells in vivo. Administration can be by any of the routes typically used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical administration (such as eye drops and creams), and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and effective response than another route. According to some embodiments, the composition is delivered by IV injection.

[0265] Suitable vectors for introducing transgenes into immune cells (such as T cells) include non-integrating lentiviral vectors, see US Patent Application Publication No. 2009-0117617.

[0266] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Thus, as described below, there are a wide variety of suitable formulations of pharmaceutical compositions available (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0267] According to some embodiments, an RNA molecule is provided that binds / associates with and / or targets an RNA-guided DNA nuclease to a sequence (such as a sequence in the mutator that is not present in the functional allele) that contains at least one nucleotide (such as a SNP) that differs between the mutator and functional allele of a gene of interest. The sequence may be within the disease-associated mutation. The sequence may be upstream or downstream of the disease-associated mutation. Any sequence differences between the mutant allele and the functional allele may be targeted to inactivate the mutant allele by the RNA molecule of the invention, while preserving the activity of the functional allele, thereby negating the otherwise dominant disease-causing effect of the mutant allele.

[0268] The disclosed compositions and methods may also be used in the manufacture of a medicament for treating a dominant genetic disorder in a patient.

[0269] Mechanism of Action for Some Embodiments Disclosed herein Mutations in ELANE demonstrated to cause SCN or CyN mediate translation from an alternatively framed ORF (open reading frame) generating truncated N-terminal proteins, resulting in ER and protein misfolding stress.

[0270] Without wishing to be bound by any theory or mechanism, the present invention is utilized to treat mutated pathogenic ELANE alleles but not functional ELANE alleles, such as by applying CRISPR nucleases to prevent expression of mutated pathogenic ELANE alleles, create truncated non-pathogenic peptides from mutated pathogenic alleles, or repair / correct mutated pathogenic ELANE, in order to prevent, ameliorate or treat SCN or CyN.

[0271] Several alternative editing strategies utilizing SNPs located upstream or downstream of the ORF may be applied, including removal of the entire gene, truncation of the gene to remove the C-terminus of the gene, and attenuation of expression of the gene.

[0272] In some embodiments, two guides (such as those disclosed in Table 2) may be utilized to remove the entire gene (i.e., exons 1, 2, 3, 4, and 5) to knock out the mutant protein. In some embodiments, a first guide RNA may be utilized to mediate an allele-specific DSB by targeting a SNP / WT sequence located upstream of the ORF of a mutant allele of the ELANE gene, and a second guide RNA may be utilized to mediate a DSB to a SNP / WT sequence located in exon 5 of the ELANE gene or downstream of the mutant allele, or a sequence located in intron 4, 3'UTR or downstream of an allele of the ELANE gene, or a SNP / WT sequence located in intron 4, 3'UTR or downstream of an allele of the ELANE gene.

[0273] There are records of healthy individuals harboring frameshift mutations that result in the acquisition of a stop codon located in exon 3. Thus, a potential strategy may be to truncate the mutant allele to include exons 1 to 3 at most. In some embodiments, two guides (such as those disclosed in Table 2) may be utilized to truncate the c-terminus of the mutant allele of the ELANE gene. In some embodiments, a first guide RNA may be utilized to mediate an allele-specific DSB by targeting exon 5 or downstream SNP / WT sequence of the mutant allele, and a second guide RNA may be utilized to mediate a DSB of a sequence or SNP / WT sequence located in intron 1, 2 or 3 of the ELANE gene. The peptide / protein encoded by the truncated mutant allele may not show a pathogenic effect. Alternatively, nonsense-mediated mRNA decay may be triggered to knock out expression of the mutant allele. The results may be verified by examining mRNA and protein expression.

[0274] In some embodiments, expression of mutant alleles may be attenuated by excising elements from the proximal promoter and enhancer regions using SNPs located upstream of the ORF. In a non-limiting example, a significant reduction may be achieved by excising a large portion of the enhancer region by targeting a SNP. An example of an RNA guide sequence that specifically targets a mutant allele of the ELANE gene

[0275] Although many guide sequences can be designed to target mutant alleles, the nucleotide sequences set forth in Table 2, identified by SEQ ID NOs: 1-1192 below, were specifically selected to effectively perform the methods described herein and to effectively distinguish between alleles.

[0276] Referring to columns 1-4, each of SEQ ID NOs: 1-1192 shown in column 1 corresponds to an engineered guide sequence. Details of the corresponding SNPs are shown in column 2. Details of the SNPs shown in the second column indicate the identifiers assigned to each SNP, which correspond to the SNP IDs shown in Table 1. Column 3 indicates whether each guide sequence targets a polymorphism or wild type sequence of the ELANE gene at the indicated site. Column 4 indicates the guanine-cytosine content of each guide sequence at the indicated site.

[0277] Table 2 shows guide sequences designed for use as described in the above embodiments in association with different SNPs within mutant ELANE allele sequences. Each engineered guide molecule is further designed to be associated with a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM), such as a PAM matching the sequence NGG or NAG (where "N" is any nucleobase). The guide sequence is designed to function in combination with one or more different CRISPR nucleases, including, but not limited to, SpCas9WT (PAM SEQ:NGG), SpCas9.VQR.l (PAM SEQ:NGAN), SpCas9.VQR.2 (PAM SEQ:NGNG), SpCas9.EQR (PAM SEQ:NGAG), SpCas9.VRER (PAM SEQ:NGCG), SaCas9WT (PAM SEQ:NNGRRT), NmCas9WT (PAM SEQ:NNNNGATT), Cpfl (PAM SEQ:TTTV) or JeCas9WT (PAM SEQ:NNNVRYM). Each of the RNA molecules of the invention is designed to form a complex in combination with one or more different CRISPR nucleases and to target the polynucleotide sequence of interest using one or more different PAM sequences for each of the CRISPR nucleases utilized. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

[0278] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any manner. The content of any individual paragraph may be equally applicable to all paragraphs. Various embodiments of the present invention are presented below. 1. A method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) gene in a cell, the mutant allele having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN), the cell comprising: 91229, rs7l335276, rs58082l77, rs3826946, rs10413889, rs76l48l944, rs376l008, rs10409474, rs3761007, rs172l6649, rs10469327, rs8l07095, rs10424470 and rs78302854, wherein the method comprises: The cells, A CRISPR nuclease or a sequence encoding said CRISPR nuclease, and a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides; The complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene. 2. The method of claim 1, wherein the complex of the CRISPR nuclease and the first RNA molecule affects a double-stranded break in the mutant allele of the ELANE gene, and the mutant allele is targeted for the double-stranded break based on the one or more polymorphic sites. 3. The method of claim 1 or 2, further comprising introducing a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and CRISPR nuclease affects a second double-stranded break in the ELANE gene. 4. The method according to any one of 1 to 3 above, wherein the guide sequence portion of the first RNA molecule comprises 17 to 20 adjacent nucleotides set forth in any one of SEQ ID NOs: 1 to 1192. 5. The method according to claim 3 or 4, wherein the second double-stranded break is within a non-coding region of the ELANE gene. 6. A method according to any one of 3 to 5 above, wherein the cell is heterozygous for rs104l4837 or rs376l005, and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene. 7. A method according to any one of claims 3 to 5, wherein the cell is heterozygous for rs10414837 or rs376l005, and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in intron 3 of the ELANE gene. 8. A method according to any one of 3 to 5 above, wherein the cell is heterozygous at rs10414837 or rs376l005, and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene. 9. A method according to any one of 3 to 5 above, wherein the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene. 10. A method according to any one of claims 3 to 5, wherein the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in intron 3 of the ELANE gene. 11. A method according to any one of claims 3 to 5, wherein the cell is heterozygous at rs1683564 and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene. 12. Obtaining the cells having an ELANE gene mutation associated with severe congenital neutropenia (SCN) or CyN from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, wherein the subject has one or more of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs 12. The method according to any one of 1 to 11 above, wherein the nucleotide sequence is heterozygous at one or more polymorphic sites selected from the group consisting of rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854. 13. First, selecting a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, wherein the subject has one or more of the following mutations: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs71335276, rs58082177, rs3 13. The method of claim 12, comprising: the subject being heterozygous at one or more polymorphic sites selected from the group consisting of rs1046946, rs10413889, rs761481944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8107095, rs10424470 and rs78302854; and obtaining the cells from the subject. 14. The method according to claim 13 or 14, comprising obtaining the cells from the subject by mobilization and / or by apheresis. 15. The method according to claim 14, comprising obtaining the cells from the subject by bone marrow aspiration. 16. The method according to any one of 1 to 15 above, wherein the cells are pre-stimulated before the composition is introduced into the cells. 17. The method according to any one of 12 to 16 above, further comprising culturing and growing the cells to obtain a cell population. 18. The method according to claim 17, wherein the cells are cultured with one or more of stem cell factor (SCF), IL-3 and GM-CSF. 19. The method according to claim 17 or 18, wherein the cells are cultured with at least one cytokine. 20. The method according to claim 19, wherein said at least one cytokine is a recombinant human cytokine. 21. A method according to any of 1 to 20 above, wherein the cell is one of a plurality of cells, and the composition comprising the first RNA molecule or both the first and second RNA molecules is introduced into at least the cell and other cells of the plurality of cells, and the mutant allele of the ELANE gene in at least the cell and the other cells of the plurality of cells is inactivated, thereby obtaining a plurality of modified cells. 22. A method according to any one of 1 to 21 above, wherein introducing the composition comprising the first RNA molecule or introducing the second RNA molecule comprises electroporation of the cell or a plurality of cells. 23. A modified cell obtained by the method according to 21 or 22 above. 24. A modified cell obtained by culturing and growing a cell according to 21 above. 25. A modified cell or cells according to 23 or 24 above, capable of engrafting. 26. A modified cell or a plurality of modified cells according to any one of 23 to 25 above, capable of giving rise to progeny cells. 27. The modified cell or cells according to claim 26, which are capable of giving rise to progeny cells after engraftment. 28. The modified cell or cells according to claim 27, which are capable of giving rise to progeny cells following autologous transplantation. 29. The modified cell or cells according to any of 27 or 28 above, capable of giving rise to progeny cells for at least 12 months or at least 24 months after engraftment. 30. The modified cell or cells according to any one of 23 to 29 above, wherein the modified cell or cells are hematopoietic stem and / or progenitor cells (HSPCs). 31. The modified cell or cells according to claim 30, wherein the modified cell or cells are CD34+ hematopoietic stem cells. 32. The modified cell or cells according to any one of 23 to 31 above, wherein the modified cell or cells are bone marrow cells or peripheral mononuclear cells (PMCs). 33. A modified cell lacking at least a portion of one allele of the ELANE gene. 34. The modified cell according to claim 33, wherein the modified cell is modified from a cell that is heterozygous at one or more polymorphic sites selected from the group consisting of rs104l4837, rs3761005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs2859l229, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs76l48l944, rs376l008, rs10409474, rs376l007, rs172l6649, rs10469327, rs8107095, rs10424470 and rs78302854. 35. A composition comprising the modified cell according to any one of 23 to 34 above and a pharma- ceutically acceptable carrier. 36. An in vitro or ex vivo method for preparing the composition according to claim 35, comprising mixing said cells with said pharma- ceutically acceptable carrier. 37. A method for preparing a composition comprising modified cells in vitro or ex vivo, the method comprising: a) isolating HSPCs from a plurality of cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or afflicted with SCN or CyN, said subject having one or more of the following mutations: rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs7l335276 ... heterozygous at one or more polymorphic sites selected from the group consisting of rs58082l77, rs3826946, rs104l3889, rs76148l944, rs376l008, rs10409474, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470 and rs78302854, and obtaining said cells from said subject; b) administering to the cells of step (a) a gene encoding the mutant allele of the ELANE gene in one or more cells, A CRISPR nuclease or a sequence encoding said CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene in one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and a CRISPR nuclease affects a second double stranded break in the ELANE gene of the one or more cells; thereby obtaining a modified cell; and optionally c) culturing and growing the modified cells of step (b); The method, wherein the modified cells are capable of engrafting and giving rise to progeny cells after engraftment. 38.a) isolating HSPCs from a plurality of cells obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, said subject having any of the following mutations: rs104l4837, rs376l005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs2859122 9, rs7l335276, rs58082l77, rs3826946, rs104l3889, rs76148l944, rs376l008, rs10409474, rs3761007, rs17216649, rs10469327, rs8l07095, rs10424470, and rs78302854; b) administering to the cells of step (a) a gene encoding the mutant allele of the ELANE gene in one or more cells, A CRISPR nuclease or a sequence encoding said CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene in one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and a CRISPR nuclease affects a second double stranded break in the ELANE gene of the one or more cells; thereby obtaining a modified cell; and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, following engraftment, are capable of giving rise to progeny cells; and d) use of a composition prepared in vitro by a method comprising administering to said subject the cells of step (b) or step (c) for treating said SCN or CyN in said subject. 39. A method for treating a subject suffering from SCN or CyN, comprising administering a therapeutically effective amount of a modified cell described in any of 21 to 34 above, a composition described in 35 above, or a composition prepared by the method described in 36 or 37 above. 40. A method for treating SCN or CyN in a subject in need of treatment having an ELANE gene mutation associated with SCN or CyN, the subject having any of the following mutations: rs104l4837, rs376l005, rs1683564, rs9749274, rs74002l, rs20l048029, rs199720952, rs2859l229, rs7l335276, rs7l335276, rs7l335277, rs7l335278, rs7l335279 ... rs10424470, and rs78302854, and the method further comprises: a) isolating HSPCs from a plurality of cells obtained from the subject; b) administering to the cells of step (a) a gene encoding the mutant allele of the ELANE gene in one or more cells, A CRISPR nuclease or a sequence encoding said CRISPR nuclease, and A composition comprising a first RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene in one or more cells; Optionally, introducing into the cell a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and a CRISPR nuclease affects a second double stranded break in the ELANE gene of the one or more cells; thereby obtaining a modified cell; and optionally c) culturing the cells of step (b), wherein the modified cells are capable of engraftment and, following engraftment, are capable of giving rise to progeny cells; and d) administering to the subject the cells of step (b) or step (c), thereby treating the SCN or CyN in the subject. 41. A method for treating SCN or CyN in a subject in need of treatment having an ELANE gene mutation associated with SCN or CyN, the subject having any of the following: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs71335276, rs58082177, rs3826946, rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs1 and heterozygous at one or more polymorphic sites selected from the group consisting of rs78302854 and rs78304470, said method comprising: administering to the subject an autologous modified cell or a progeny of an autologous modified cell, wherein the autologous modified cell is modified to have a double strand break in the mutant allele of the ELANE gene; The double-stranded break results from introducing into the cell a CRISPR nuclease or a composition comprising a sequence encoding the CRISPR nuclease and a first RNA molecule, and a complex of the CRISPR nuclease and the first RNA molecule affects a double-stranded break in the mutant allele of the ELANE gene so as to inactivate the mutant allele of the ELANE gene in the cell, thereby treating the SCN or CyN in the subject. 42. A method for selecting a subject for treatment from a pool of subjects diagnosed with SCN or CyN, comprising: a) obtaining a cell from each subject in said pool of subjects; b) screening the cells of each subject for ELANE gene mutations associated with SCN or CyN and selecting only subjects with ELANE gene mutations associated with SCN or CyN; c) screening for heterozygosity at one or more polymorphic sites selected from the group consisting of rs104l4837, rs376l005, rs1683564 by sequencing the cells of the subject selected in step (b); and d) selecting only those subjects having cells that are heterozygous at said one or more polymorphic sites for treatment. 43.e) obtaining hematopoietic stem and progenitor cells (HSPCs) from said subject's bone marrow either by aspiration or by mobilization and apheresis of peripheral blood; f) the HSPC cells of step (e). One or more CRISPR nucleases or sequences encoding one or more CRISPR nucleases A first RNA molecule including a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 adjacent nucleotides set forth in any one of SEQ ID NOs: 1 to 1192, which targets a nucleotide base of the heterozygous allele of the one or more polymorphic sites present in the mutant allele of the ELANE gene; and Introducing a second RNA molecule comprising a guide sequence portion targeting a sequence in intron 3, intron 4 or 3'UTR of the ELANE gene, a complex of the first RNA molecule and CRISPR nuclease affects a first double stranded break in the mutant allele of the ELANE gene of one or more of the HSPC cells, and a complex of the second RNA molecule and CRISPR nuclease affects a second double stranded break in intron 3, intron 4 or 3'UTR of both alleles of the ELANE gene of the one or more HSPC cells in which the complex of the first RNA molecule and the CRISPR nuclease affected the first double stranded break, thereby obtaining modified cells; g) administering to the subject the modified cells of step (f), thereby treating SCN or CyN in the subject; 43. The method of claim 42, further comprising treating SCN or CyN in the subject selected in step (d). 44. An RNA molecule comprising a guide sequence portion having 17 to 20 nucleotides in a sequence of 17 to 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1 to 1192. 45. A composition comprising the RNA molecule described in 44 above and a second RNA molecule comprising a guide sequence portion. 46. ​​The composition according to claim 45, wherein the second RNA molecule targets a non-coding region of the ELANE gene. 47. The composition described in 45 or 46 above, wherein the nucleotide sequence of the guide sequence portion of the second RNA molecule is a nucleotide sequence different from the sequence of the guide sequence portion of the first RNA molecule. 48. The composition according to any one of claims 45 to 47, wherein the first and / or second RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. 49. The composition according to claim 48, wherein the sequence that binds to the CRISPR nuclease is a tracrRNA sequence. 50. The composition according to any one of 45 to 48 above, wherein the first and / or second RNA molecule further comprises a portion having a tracr mate sequence. 51. The composition according to any one of 45 to 50 above, wherein the second RNA molecule further comprises one or more linker portions. 52. The composition according to any one of claims 42 to 51, wherein the first and / or second RNA molecule is up to 300 nucleotides in length. 53. The composition according to any one of claims 45 to 52, further comprising one or more CRISPR nucleases or sequences encoding said one or more CRISPR nucleases, and / or one or more tracrRNA molecules or sequences encoding said one or more tracrRNA molecules. 54. A method for inactivating a mutant ELANE allele in a cell, the method comprising delivering to the cell an RNA molecule described in 44 above or a composition described in any of 45 to 53 above. 55. A method for treating SCN or CyN, the method comprising delivering to a subject having SCN or CyN an RNA molecule as described in 44 above or a composition as described in any one of 45 to 53 above, or a cell modified by the RNA molecule as described in 44 above or the composition as described in any one of 45 to 53 above. 56. The method of claim 54 or 55, wherein the one or more CRISPR nucleases and / or the tracrRNA molecule and the RNA molecule or RNA molecules are delivered to the subject and / or cell at substantially the same time or at different times. 57. The method comprises: a) removing an exon containing a disease-causing mutation from a mutant allele, wherein the first RNA molecule or the first and second RNA molecules target an entire exon or a region adjacent to a portion of the exon; b) removing multiple exons of a gene, an entire open reading frame, or removing the entire gene; c) the first RNA molecule or the first and second RNA molecules target an alternative splicing signal sequence between an exon and an intron of a mutant allele; d) said second RNA molecule targets a sequence present in both the mutant allele and the functional allele; e) the second RNA molecule targets an intron; f) subjecting the mutant allele to an insertion or deletion by an error-prone non-homologous end joining (NHEJ) mechanism to generate a frameshift in the sequence of the mutant allele; Optionally, the frameshift results in an inactivation or knockout of the mutant allele; 57. The method according to any one of claims 54 to 56, wherein the frameshift creates a premature stop codon in the mutant allele or the frameshift induces nonsense-mediated mRNA decay in a transcript of the mutant allele. 58. A method according to any of claims 55 to 57, wherein said inactivation or treatment results in a truncated protein encoded by said mutant allele and a functional protein encoded by said functional allele. 59. a) the cell or the subject is heterozygous for rs104l4837 or rs376l005, and the complex of the second RNA molecule and a CRISPR nuclease affects a double-stranded break in intron 4 of the ELANE gene; b) the cell or the subject is heterozygous at rs104l4837 or rs376l005, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 3 of the ELANE gene; c) the cell or the subject is heterozygous at rs104l4837 or rs376l005, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in the 3'UTR region of the ELANE gene; d) the cell or the subject is heterozygous at rs1683564, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 4 of the ELANE gene; e) the cell or the subject is heterozygous at rs1683564, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 3 of the ELANE gene; f) A method according to any of claims 55 to 58, wherein the cell or the subject is heterozygous at rs1683564 and the complex of the second RNA molecule and CRISPR nuclease affects a double-stranded break in the 3'UTR region of the ELANE gene. 60. Use of an RNA molecule described in 44 above, a composition described in any of 35, 45 to 53 above, or a composition prepared by the method described in 36 or 37 above, for inactivating a mutant ELANE allele in a cell. 61. A drug for use in inactivating a mutant ELANE allele in a cell, comprising an RNA molecule described in 44 above, a composition described in any of 35 or 45 to 53 above, or a composition prepared by the method described in 36 or 37 above, wherein the drug is administered by delivering to the cell the RNA molecule described in 44 above, a composition described in any of 35 or 45 to 53 above, or a composition prepared by the method described in 36 or 37 above. 62. Use of a method according to any one of 1 to 22 above, a modified cell according to any one of 23 to 34 above, a composition according to any one of 35 or 45 to 53 above, or a composition prepared by a method according to 36 to 37 above, or an RNA molecule according to 44 above, for treating, ameliorating or preventing SCN or CyN in a subject having or at risk of having SCN or CyN. 63. A drug comprising an RNA molecule of 44 above, a composition of any one of 35 or 45-53 above, a composition prepared by the method described in 36 or 37 above, or a modified cell of any one of 23-34 above, for use in treating, ameliorating or preventing SCN or CyN, wherein the drug is administered to a subject having or at risk of having SCN or CyN by delivering the RNA molecule of 44 above, the composition of any one of 35 or 45-53 above, the composition prepared by the method described in 36 or 37 above, or the modified cell of any one of 23-34 above. 64. A kit for inactivating a mutant ELANE allele in a cell comprising an RNA molecule, a CRISPR nuclease or a sequence encoding the CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding the tracrRNA, as described in 44 above, and instructions for delivering the RNA molecule, the CRISPR nuclease or a sequence encoding the CRISPR nuclease, and / or the tracrRNA molecule or a sequence encoding the tracrRNA to the cell to inactivate the mutant ELANE allele in the cell. 65. A kit for treating SCN or CyN in a subject comprising an RNA molecule, a CRISPR nuclease or a sequence encoding said CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding said tracrRNA, as described in 44 above, and instructions for delivering said RNA molecule, a CRISPR nuclease or a sequence encoding said CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding said tracrRNA to a subject having or at risk of having said SCN or CyN to treat SCN or CyN. 66. A kit for inactivating a mutant ELANE allele in a cell, comprising a composition described in any of 35 or 45-53 above, a composition prepared by the method described in 36 or 37 above, or a modified cell described in any of 23-34 above, and instructions for delivering the composition to the cell so as to inactivate the ELANE gene in the cell. 67. A kit for treating SCN or CyN in a subject, comprising a composition described in any of 35 or 45 to 53 above, a composition prepared by the method described in 36 or 37 above, or a modified cell described in any of 23 to 34 above, and instructions for delivering a composition described in any of 35 or 45 to 53 above, a composition prepared by the method described in 36 or 37 above, or a modified cell described in any of 23 to 34 above to treat SCN or CyN to a subject having or at risk of having SCN or CyN.

[0279] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any manner. The content of any individual paragraph may be equally applicable to all paragraphs.

[0280] In order to facilitate a more complete understanding of the present invention, the following examples are provided to illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only.

[0281] Furthermore, the following examples of the present invention disclose methods that include using SpCas9 and guide sequences suitable for directing SpCas9 to the disclosed SNP positions. The examples demonstrate the feasibility of the disclosed strategies. Those skilled in the art will understand that the same guide sequence can be used with different CRISPR nucleases to target the disclosed SNPs for each specific strategy application. Furthermore, different guide sequences can be used with other nucleases to direct other CRISPR nucleases to the same SNP for each specific strategy application. EXAMPLES

[0282] Experimental details Example 1: Selecting SNP-targeting guide sequences suitable for functioning in combination with SpCas9 and following the disclosed strategy HeLa cells were seeded in 96-well plates (3K / well). After 24 hours, cells were co-transfected with 65 ng of WT-Cas9 or Dead-Cas9 and 20 ng of gRNA plasmids, identified as g36-g66 and targeting different regions and SNPs of ELANE, using Turbofect reagent (Thermo Scientific). After 12 hours, fresh medium was added, and at 72 hours post-transfection, genomic DNA was extracted and the predicted regions targeted by Cas9 were amplified, and the size of the products was analyzed by capillary electrophoresis with a DNA ladder. The intensity of the bands was analyzed using Peak Scanner software vl.0. The percentage of editing was calculated according to the following formula: 100%-(unedited band intensity / total band intensity)X100. Figure 6 shows the average activity of each gRNA ± SD after subtracting the Dead-Cas9 background activity of three independent experiments.

[0283] [Table 3]

[0284] Example 2: Proving the feasibility of the resection strategy HeLa cells were co-transfected with spCas9-WT and RNA pairs, either sg35 (INT4) and g39 (rs104l4837), g58 (rs376l005) or g62 (rs1683564) for strategies la, lb and 2, respectively. At 72 hours post-transfection, gDNA was extracted and excision efficiency was assessed by measuring the reduction in copy number of exon 1 (strategy 1) or exon 5 (strategy 2) using droplet digital PCR (ddPCR) kits (10042958 and 10031228, Bio-Rad Laboratories). Furthermore, exon 1 was used to normalize the excision rate for strategy 2, whereas exon 5 was used to normalize the excision rate for strategy 1. The results disclosed in Table 4 show the mean % resection ± SD (standard deviation) of two independent experiments.

[0285] [Table 4]

[0286] Example 3: Distinguishing between editing by different sgRNAs to evaluate alleles Ribonucleoprotein complexes (RNPs) targeting the reference sequences were constructed from the relevant gRNAs according to the manufacturer's instructions, and WT-Cas9 (#1081058) or HiFiCas9 (#1081060) were purchased from Integrated DNA Technology (IDT). The RNPs were then nucleofected into iPSCs harboring the relevant SNPs using the 4D-Nucleofecor® system (Lonza). After 72 hours, gDNA was extracted, the SNP regions were amplified, and sent for NGS analysis. Allelic discrimination was assessed according to the editing percentages detected in the reference and alternative alleles. The indel frequency at each site was calculated using Cas-Analyzer software. The results are summarized in Table 5.

[0287] [Table 5]

[0288] Example 4: Editing efficiency in HSCs HSCs from healthy donors were nucleofected with spCas9-WT and the RNA components of gRNAs targeting either EMX1 (sgEMXl) or ELANE (g35:INT4; g58:rs376l005; g62:rs1683564). 72 hours after nucleofection, gDNA was extracted and editing levels were quantified by IDAA. Figure 7 shows the mean % editing ± SD of two independent experiments performed in duplicate.

[0289] Example 5: Functional Maturation Assay To prove the phenotypic rescue in the corrected cells, maturation assays starting from patient-derived induced pluripotent stem cells (iPSCs) were prepared from reprogrammed somatic cells. These cells provide a renewable source of patient-derived cells and have been shown to accurately recapitulate the disease phenotype. Briefly, patient PBMCs are transfected with episomal constructs expressing the reprogramming genes: Oct4, Sox2, Nanog, Lin28, L-Myc, Klf4 and SV40LT. Patient-derived and normal iPSCs harboring identical SNP genotypes are differentiated into hematopoietic progenitor cells using a commercial kit (STEMdiff™ Hematopoietic Kit, STEMCELL Technologies). After 12 days, differentiation efficiency is estimated by analyzing the cells for the expression of progenitor markers CD34 and CD45 by flow cytometry analysis. Normal and SCN-differentiated progenitor cells are gene-edited and grown for 5 days in conditioned medium containing stem cell factor (SCF), IL-3, and GM-CSF, which promotes differentiation into neutrophils. Normal unedited and edited cells, i.e., SCN-edited cells, differentiate into neutrophils, while unedited SCN-derived cells are arrested at an early stage of differentiation. The efficiency of neutrophil differentiation is measured by flow cytometry to detect neutrophil surface markers (e.g., increased expression of CD16, CD66b, and the pan-myeloid marker CD33).

[0290] Example 6: In vivo dose ranging and biodistribution pilot study in immunodeficient mice Dosing schedules, dose ranges and routes of administration are studied to measure the presence and number of HSCs with self-renewal and multilineage capacity in immunodeficient NSG mice after administration of G-CSF. Repopulation assays are performed to measure the presence and number of HSCs that can regenerate a functional immune system to establish long-term engraftment. For such validation, NSG cell lines that are highly supportive of human engraftment and hematopoietic repopulation are used. NSG mice are NOD SCID mice that lack mature T cells, B cells and natural killer (NK) cells, in addition to being deficient in multiple cytokine signaling pathways and having many defects in innate immunity. Engraftment is assessed 16 weeks after primary transplantation (analysis 12 weeks post-transplant).

[0291] A 16-week biodistribution pilot study in NSG mice will explore dose and maximum duration and will be performed at several cell dose levels. The study will include three dose levels and a group of mice receiving unedited SCN cells. The duration of the pilot study will be 16 weeks, with one of the two higher dose groups continuing for up to six months. Mice will survive for six months in this pilot study, with the duration of the core biodistribution study being six months. Persistence of expression by qPCR and immunohistochemistry will be studied during the study.

[0292] Example 7: Central biodistribution study in immunodeficient mice The pivotal biodistribution study will utilize NSG mice and will follow the pilot dose ranging findings of Example 6. This study will be performed in compliance with Good Laboratory Practice (GLP) and is a pivotal non-clinical pharmacokinetic, pharmacodynamic and toxicology study. Toxicity studies will be based on mortality, clinical findings, body and organ weights, and clinical and anatomic pathology following HSC infusion.

[0293] Transplantation of gene-edited CD34+ cells into NSG mice requires pretreatment to provide endogenous bone marrow depletion and allow engraftment of donor cells. Therefore, busulfan pretreatment is utilized to mimic the clinical situation. Three groups of 10 male and female mice per group (gene-edited cells, unedited cells, and busulfan vehicle-only control) are utilized.

[0294] Although the NSG mouse model does not fully resemble the human neutrophil-depleted situation, this study does not affect the validity of the model for use in biodistribution studies of gene-edited CD34+ cells, since a proportion of mice were treated with busulfan to deplete bone marrow function prior to in vivo injection of ex vivo gene-edited cells. Cre / Cre Mcl-l flox / flox (Mcl-l ΔMyelo ) myeloid cell leukemia (Mcl-1) anti-apoptotic protein shows that myeloid-specific deficiency of Mcl-1 leads to very severe neutropenia (Csepregi et al., 2018). ΔMyelo The mice were able to breed and their survival rates were close to normal under both specific pathogen-free and conventional housing conditions. However, in contrast to NSG mice, Mcl-1 ΔMyelo There is limited experience with mouse models.

[0295] Example 8: Modification Analysis Guide sequences comprising 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in SEQ ID NOs: 1-1192 are screened for high on-target activity, which is measured by DNA capillary electrophoresis analysis.

[0296] DNA capillary electrophoresis analysis indicates that a guide sequence comprising 17 to 20 nucleotides in the sequence of 17 to 20 adjacent nucleotides set forth in SEQ ID NOs: 1 to 1192 is suitable for correcting the ELANE gene.

[0297] Example 9: Efficacy of allele-specific knockout of ELANE Specific knockout of the mutant allele of the ELANE gene is mediated by excising intron 4 and exon 5 of the mutant allele of the ELANE gene. This is accomplished by mediating a DSB in intron 4 and utilizing SNPrs1683564 to mediate an allele-specific DSB as depicted in Figure 8. To demonstrate that HSCs can be efficiently differentiated into mature and functional neutrophils using this strategy, healthy donors are nucleofected with HSCs (Lonza) bearing RNPs containing g35 and g62 targeting the respective SNPs (see Table 1) in intron 4. Unedited cells are used as a positive control. Forty-eight (48) hours after nucleofection, HSCs are differentiated into neutrophils according to published protocols (Zhenwang Jie et al., PlosOne 2017). The differentiation efficiency of edited and unedited cells is measured by FACS after staining with neutrophil-specific markers CD66b and CD177.To assess HSC-mediated neutrophil function, the following assays are performed. 1. Phagocytosis is assayed using the EZCell™ Phagocytosis Assay Kit (BioVision), which utilizes pre-labeled zymosan particles as a tool for rapid and accurate detection and quantification of phagocytosis in vitro by flow cytometry. 2. Killing assays are performed by culturing HSC-derived neutrophils with E. coli and then plating the bacteria onto agar plates for colony formation. Untreated bacteria or bacteria cultured with undifferentiated HSCs are used as controls. Killing efficiency is calculated as follows: (colony number) Neutrophils / Colony count コントロール ) x 100. 3. Assay chemotaxis using the EZCell™ Cell Migration / Chemotaxis Assay Kit (Biovision).

[0298] Example 10: Patient Selection for Treatment Step 1: Four patients A-D diagnosed with SCN or CyN are selected by exon sequencing to identify ELANE pathogenic mutations in the ELANE gene. Step 2: Subjects with identified mutations are then selected by Sanger sequencing to confirm heterozygosity of at least one of rs1683564, rs104l4837 and rs376l005. Step 3. For each subject determined to be heterozygous at at least one of rs1683564, rs104l4837 and rs376l005, the nucleotide of the heterozygous SNP of the mutant allele in the ELANE gene is determined using BAC bio. Step 4. Select the appropriate guide according to Table 6. [Table 6]

[0299] Step 5. The selected guide is introduced into PBMCs obtained from each subject, and the reduction of pathogenic ELANE mutations in PBMCs is verified by next-generation sequencing technology. The methodology for patients A–D is described below. 1. Patient A is selected according to step 1 and is found to have a known pathogenic variant in ELANE that is consistent with the phenotype and clinical picture. Patient A is selected according to step 2 around the SNPs of interest and is found to be homozygous for all three SNPs, rs10414837, rs1683564 and rs376l005. Patient A is determined to be unsuitable for treatment. 2. Patient B is verified for a known pathogenic ELANE mutation. Patient B is selected according to step 2 and found to be homozygous at SNPrs1683564 and rs104l4837. Patient B is found to be heterozygous at rs376l005 in the promoter region. Patient B is determined to be suitable for treatment. The nucleotides present on the same allele as the pathogenic ELANE mutation (linkage determination) are determined according to step 3. Patient B is found to have a reference nucleotide base at the rs3761005 SNP position on the same allele as the ELANE pathogenic mutation. g58ref is selected with g35, which is perfectly complementary to the representation of this SNP and is directed to the non-coding region of intron 4. According to step 4, the selected guide composition comprises the pair of guides g58ref and g35. The successful excision of the mutated allele using this selected pair of guides is verified according to step 5 using NGS reads on the patient PMBC. 3. Patient C has suffered from severe neutropenia since early childhood. He was screened according to step A and no pathogenic mutation was found in the ELANE gene. Patient C is deemed unsuitable for treatment. 4. Patient D is verified to have a known pathogenic variant in the ELANE gene and upon SNP genotyping according to step 2, two of the three SNPs are found to be heterozygous, i.e., both rs104l4837 and rs1683564 are found to be heterozygous, while rs3761005 is found to be homozygous. Choose between the two potential SNPs to use for genetic manipulation. To make the selection, perform step 3 to determine the linkage between the SNP and the pathogenic variant, i.e., which nucleotides of the SNP (reference or alternative) are present on the same allele as the ELANE pathogenic variant (SNP presentation). Patient D is considered to have the reference presentation of rs10414837 and sg39ref is suitable for use. Referring to the rs1683564 SNP, the alternative presentation is found to be associated with the ELANE pathogenic variant and sg62alt is suitable for use as a guide. Each of these guides is used in conjunction with g35. Two pairs of potential guide compositions are identified according to step 4: sg39ref+g35 and g62alt+g35. To determine which of the guide pairs is preferred, a database of editing properties and characteristics of each of the guides and guide pairs is evaluated to determine off-target effects and editing efficiency. Guide pairs are selected based on the database evaluation and utilized according to step 5 to provide NGS reads to PMBC. TIFF0007611702000038.tif237166 TIFF0007611702000039.tif237166 TIFF0007611702000040.tif244167 TIFF0007611702000041.tif129166

Claims

1. For use in a method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) gene, the mutant allele having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN), in a cell; A CRISPR nuclease or a nucleic acid molecule comprising a sequence encoding said CRISPR nuclease, and A first RNA molecule comprising a guide sequence portion having a nucleotide sequence set forth in any one of SEQ ID NOs: 352 and 889; wherein the cells are heterozygous at one or more polymorphic sites selected from the group consisting of rs10414837 and rs1683564; The method includes introducing the composition into the cell, a complex of the CRISPR nuclease and the first RNA molecule affects a double stranded break in the mutant allele of the ELANE gene between 1 to 28 nucleotides upstream or downstream of a protospacer adjacent motif (PAM) site, the mutant allele being targeted for double stranded break by the first RNA molecule based on the one or more polymorphic sites; A composition designed to target one of the REF or ALT nucleotide bases of said one or more polymorphic sites.

2. 2. The composition of claim 1, further comprising a second RNA molecule comprising a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein in the method of inactivating a mutant ELANE allele in a cell, the complex of the second RNA molecule and CRISPR nuclease affects a second double-stranded break in the ELANE gene.

3. The composition of any one of claims 1 to 2, wherein the double-stranded break in the mutant allele of the ELANE gene is between 1 and 20 nucleotides upstream or downstream of a PAM site.

4. The composition of claim 2 , wherein the second double-stranded break is within a non-coding region of the ELANE gene.

5. The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs10414837, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 4 of the ELANE gene; The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs10414837, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 3 of the ELANE gene; The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs10414837, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in the 3'UTR region of the ELANE gene; The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs1683564, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 4 of the ELANE gene; The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs1683564, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in intron 3 of the ELANE gene; or The method of inactivating a mutant ELANE allele in a cell, wherein the cell is heterozygous at rs1683564, and the complex of the second RNA molecule and a CRISPR nuclease affects a double stranded break in the 3'UTR region of the ELANE gene. The composition of claim 2.

6. The composition of any one of claims 1 to 5, wherein in the method of inactivating a mutant ELANE allele in a cell, the cell having an ELANE gene mutation associated with severe congenital neutropenia (SCN) or CyN is obtained from a subject having an ELANE gene mutation associated with SCN or CyN and / or suffering from SCN or CyN, and the subject is heterozygous at one or more polymorphic sites selected from the group consisting of rs10414837 and rs1683564.

7. 7. The composition of claim 6, wherein in the method of inactivating a mutant ELANE allele in a cell, the cells are obtained from the subject by mobilization and / or by apheresis, or by bone marrow aspiration.

8. The method of inactivating a mutant ELANE allele in a cell, comprising pre-stimulating the cell prior to introducing the composition into the cell.

9. The composition of any one of claims 6 to 8, wherein the method of inactivating a mutant ELANE allele in a cell further comprises propagating the cell in culture to obtain a cell population.

10. Culturing the cells with one or more of stem cell factor (SCF), IL-3 and GM-CSF; and / or Culturing the cells with at least one cytokine; The composition of claim 9.

11. The composition of claim 10 , wherein the at least one cytokine is a recombinant human cytokine.

12. A modified cell comprising an inactivated allele of the ELANE gene obtained using a composition according to any one of claims 1 to 11.

13. A medicament comprising a composition according to any one of claims 1 to 11, or a modified cell according to claim 12, for use in treating, ameliorating or preventing SCN or CyN, wherein the medicament is administered by delivering the composition according to any one of claims 1 to 11, or the modified cell according to claim 12, to a subject having or at risk of having SCN or CyN.

14. 12. A kit for inactivating a mutant ELANE allele in a cell comprising the composition of any one of claims 1 to 11 and instructions for delivering to the cell the RNA molecule and a CRISPR nuclease or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease to inactivate the mutant ELANE allele in the cell, wherein the RNA molecule and the CRISPR nuclease are capable of forming a complex that affects a double-stranded break in the mutant ELANE allele.

15. The kit of claim 14, further comprising a tracrRNA molecule or a nucleic acid molecule comprising a sequence encoding the tracrRNA molecule, wherein the tracrRNA molecule comprises a sequence capable of hybridizing to an RNA molecule, and wherein the RNA molecule, the tracrRNA molecule, and the CRISPR nuclease are capable of forming a complex that affects a double-stranded break in the mutant ELANE allele.

16. 3. The composition of claim 1, wherein the PAM site is a nucleotide sequence selected from any of the group: NGG, NAG, NNGRRT, NNNVRYM, NGAN, NGNG, NGCG, NGAG, NNNNGATT, or TTTV.

17. 2. The composition of claim 1, wherein the first RNA molecule comprises a guide sequence portion consisting of the nucleotide sequence set forth in SEQ ID NO: 889, and the one or more polymorphic sites is rs1683564.

18. 2. The composition of claim 1, wherein the first RNA molecule comprises a guide sequence portion consisting of the nucleotide sequence set forth in SEQ ID NO: 352, and the one or more polymorphic sites is rs10414837.

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