[0001] The present invention is directed to a composition for the targeted knockout of a gene on double-stranded DNA in a biological cell, a method for the targeted knockout of a gene on double-stranded DNA in a biological cell, a preparation comprising a biological cell prepared in vitro, said biological cell comprises a gene on double- stranded DNA, which is knocked-out in a targeted manner, a kit for the targeted knockout of a gene on double-stranded DNA in a biological cell, a method of treating a subject af flicted with a disease associated with a mutated gene, nucleic acid molecules which can be a component of said composition and methods, and a method of treating a subject af flicted with a disease associated with a mutated gene. FIELD OF THE INVENTION

[0002] The present invention relates to the field of molecular medicine, more particularly to the field of genetic engineering applications, preferably to the targeted knockout of disease-associated genes.

BACKGROUND OF THE INVENTION

[0003] A genetic disorder is a health problem caused by one or more abnormal ities in the genome. It can be caused by a mutation in a single gene (monogenic) or multi ple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders are the most common, the term is mostly used when discussing disorders with a single ge netic cause, either in a gene or chromosome.

[0004] Two copies of the gene must be mutated for a person to be affected by an autosomal recessive disorder. An affected person usually has unaffected parents who each carry a single copy of the mutated gene and are referred to as genetic carriers. Each parent with a defective gene normally do not have symptoms. Two unaffected people who each carry one copy of the mutated gene have a 25% risk with each pregnancy of having a child affected by the disorder.

[0005] Examples of autosomal recessive disorders are albinism, medium-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis, sickle cell disease, Tay-Sachs dis ease, Niemann-Pick disease, spinal muscular atrophy, and Roberts syndrome.

[0006] Only one mutated copy of the gene will be necessary for a person to be affected by an autosomal dominant disorder. Each affected person usually has one af fected parent. The chance a child will inherit the mutated gene is 50%. Autosomal domi nant conditions sometimes have reduced penetrance, which means although only one mutated copy is needed, not all individuals who inherit that mutation go on to develop the disease. [0007] Examples of autosomal dominant disorders are Huntington's disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, hereditary nonpol yposis colorectal cancer, hereditary multiple exostoses (a highly penetrant autosomal dominant disorder), tuberous sclerosis, Von Willebrand disease, and acute intermittent porphyria. Birth defects are also called congenital anomalies.

[0008] Neutropenia is an abnormally low concentration of neutrophils in the blood. Neutrophils make up the majority of circulating white blood cells and serve as the primary defense against infections by destroying bacteria, bacterial fragments and immu noglobulin-bound viruses in the blood. People with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life- threatening. Neutropenia can be divided into congenital and acquired, with severe con genital neutropenia and cyclic neutropenia.

[0009] Severe congenital neutropenia (SCN or CN), also often known as Kost- mann syndrome or disease, is an inherited bone marrow failure syndrome. CN affects myelopoiesis, usually without other physical malformations.

[0010] Autosomal-dominant mutations in the so-called "elastase neutrophil ex pressed gene" ( ELANE) are the most common cause of CN (ELAA/E-CN). "Maturation ar rest", the failure of the bone marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELAA/E-CN. Mutated neutrophil elastase could not be properly pro cessed, accumulates in the cells, inducing unfolded protein response (UPR) and endo plasmic reticulum (ER) stress. This leads to the death of hematopoietic cells and defective granulopoiesis. ELAA/E-CN manifests in infancy with life-threatening bacterial infections.

[0011] Another congenital bone marrow failure syndrome which is caused by ELANE mutations is cyclic neutropenia (ELANE-CyN). In CyN patients the numbers of mature neutrophils show cycling from zero to almost normal levels (under G-CSF treat ment) in approximately 21 -days intervals with a high probability of severe infections during the neutropenia phase. [0012] Daily administration of granulocyte colony stimulating factor (G-CSF), mostly in recombinant form or the human variant (rhG-CSF; filgrastim), to patients suffer ing from EL4A/E-CN and EL4A/E-CyN in many cases clinically improves neutrophil counts and immune function. G-CSF stimulates the production of more neutrophils and delays their apoptosis. It often decreases the severity and frequency of infections and, therefore, is the mainstay of therapy. Overall survival is now estimated to exceed 80% although 10% of the patients still die from severe bacterial infections or sepsis.

[0013] This form of G-CSF therapy of EL4A/E-CN and EL4A/E-CyN may, how ever, increase the risk for long term side effects. Approximately 20 % of the patients de velop myelodysplasia or acute myeloid leukemia. The most common leukemia in CN and CyN is AML, but acute lymphoid leukemia (ALL), juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), and bi- phenotypic leukemia are also reported in the literature. It was previously demonstrated that patients who had a robust response to G-CSF (doses<8 pg/kg/day) had a cumulative incidence of 15% for develop ing MDS/leukemia after 15 years on G-CSF, while an incidence of 34% was reported in patients with poor response to G-CSF despite high doses. In addition, approximately 15 % of CN and CyN patients do not respond to G-CSF even at doses of up to 50 pg/kg/day.

[0014] Hematopoietic stem cell transplant (HSCT) is an alternative, curative therapy for patients who do not respond to G-CSF therapy or who develop AML/MDS. However, patients with chronic neutropenia who undergo HCT are at increased risk of de veloping infectious complications such as fungal and graft-versus-host disease. Moreover, HCT requires a matched related donor for successful survival but most patients will not have an available matched donor.

[0015] WO 2019/217294 discloses a method for inactivating a mutant allele of the ELANE gene associated with CN and CyN. The known method involves the introduc tion to a biological cell of a CRISPR nuclease or a sequence encoding the CRISPR nucle ase together with a guide RNA molecule. The complex of the CRISPR nuclease and the RNA molecule is described to affect a double strand break in the mutant allele of the ELANE gene. This method is complex and cumbersome and has to be adapted to each patient individually. It depends on the existence of single nucleotide polymorphisms (SNPs) which, however, cannot be found or used in all patients in the same manner.

[0016] Nasri et al. (2020), CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients, Haematologica 105(3), pp. 598-609, disclose a similar method. In primary hematopoietic stem and progenitor cells from ELAA/E-CN and ELAA/E-CyN patients the mutated ELANE gene is knocked-out by causing DNA double-strand breaks by introducing a CRISPR/Cas9-sgRNA ribonucleo- protein.

[0017] However, the approaches disclosed in WO 2019/27294 and Nasri et al. may result in the introduction of novel ELANE mutations and malignant transformations of the treated cells and, for this reason, may be inappropriate for an application in clinical practices.

[0018] Against this background it is an object underlying the invention to pro vide an improved method and composition for the targeted knockout of a gene on double- stranded DNA in a biological cell, by means of which the disadvantages of the methods known in the art can be avoided or at least reduced. In, particular a method and composi tion should be provided allowing the treatment of an autosomal-dominantly inherited ge netic disorder, such as congenital neutropenia (CN) or cyclic neutropenia (CyN), in partic ular ELAA/E-CN and ELAA/E-CyN, in a reliable and safe manner.

[0019] The present invention satisfies these and other needs.

SUMMARY OF THE INVENTION

[0020] The present invention provides a composition for the targeted knockout of a gene on double-stranded DNA in a biological cell, comprising: - a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease;

- a second CRISPR endonuclease or a nucleic acid molecule encoding said second CRISPR endonuclease; said first and said second CRISPR endonucleases are configured to cre ate single-strand DNA breaks,

- a first single guide RNA (sgRNA), and

- a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a ge netic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the antisense strand of said genetic element controlling the expression of said gene.

[0021] According to the invention, a "targeted knockout" or "gene knockout" re fers to a specific functional elimination of the expression of a gene on double stranded DNA by a genetic technique, however without destructing and/or affecting the coding re gion of said gene.

[0022] According to the invention "double-stranded DNA" (dsDNA) refers to a molecule of DNA consisting of two parallel polynucleotide chains joined by hydrogen bonds between complementary purines and pyrimidines.

[0023] According to the invention a "biological cell" includes both prokaryotic and eukaryotic cells, such as animal, plant or bacterial cells comprising DNA, in particular dsDNA. Human cells comprising dsDNA are of particular preference. [0024] According to the invention, a "CRIPR endonuclease" refers to an en zyme which cleaves the phosphodiester bond within a polynucleotide chain and which is a component of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) complex. The CRISPR endonuclease is, therefore, capable to specifically bind to a guide or sgRNA which directs the endonuclease to a target nucleic acid. A CRISPR endonucle ase is also referred to as 'CRISPR associated protein' (Cas) or CRISPR associated endo nuclease, respectively.

[0025] According to the invention, in a first embodiment the first and second CRISPR endonucleases are identical, and in a second embodiment the first and second CRISPR endonucleases differ from each other.

[0026] According to the invention "a nucleic acid molecule encoding the CRISPR endonuclease" refers to a DNA, or cDNA or mRNA, which comprise or consist of the coding information for the synthesis of the CRISPR endonuclease.

[0027] According to the invention the first and second CRISPR endonucleases are configured to create single-strand DNA breaks on the target nucleic acid, but no dou ble-strand DNA breaks, as typically carried out by naturally occurring CRISPR endonucle ases, such as the types I, II, and Ilia. Such a configuration of the CRISPR endonucleases to be used in the invention can be achieved by a targeted modification of wild type CRISPR endonucleases. The modification typically addresses the catalytic center of the enzyme, thereby converting the nuclease into a nicking enzyme or "nickase", respectively. As a consequence the modified CRISPR endonuclease according to the invention cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequences known as a restriction site but is no longer capable to introduce DNA double-strand breaks. It hydrolyses (cuts) only one strand of the DNA duplex, to produce DNA molecules that are "nicked", rather than cleaved.

[0028] According to the invention a "single guide RNA" (sgRNA) or guide RNA is a component of the CRISPR complex which serves to guide the CRISPR endonuclease to its target. The sgRNA is a non-coding short RNA sequence which binds to the comple mentary target DNA sequence. The sgRNA first binds to the CRISPR endonuclease en zyme and the sgRNA sequence guides the complex via pairing to a specific location on the DNA, where the modified CRISPR endonuclease performs its nickase activity by cut ting the target single DNA strand.

[0029] According to the invention a "genetic element controlling the expression of said gene" refers to one or several regions located on the DNA bearing the target gene to be knocked-out, said region(s) control(s) the initiation of transcription of the gene. It is typically located near the transcription start of the gene, mostly upstream of the coding se quence. This region can be short (only a few nucleotides in length) or quite long (hun dreds of nucleotides long). A typical genetic element controlling the expression of said gene according to the invention is a promoter.

[0030] According to the invention the first sgRNA comprises a nucleotide se quence which is essentially complementary to a sequence on the sense strand of the DNA bearing the coding sequence of said gene to be knocked-out. Said complementary sequence is located in a genetic element controlling the expression of said gene, typically upstream of the gene.

[0031] According to the invention the second sgRNA comprises a nucleotide sequence which is essentially complementary to a sequence on the antisense strand of the DNA bearing the coding sequence of said gene to be knocked-out. Said complemen tary sequence is located in a genetic element controlling the expression of said gene.

[0032] The essential complementary sequences of the first and second sgRNA allow the specific hybridization of the complex consisting of modified CRISPR endonucle ase and sgRNA to the sense and antisense strand of the expression controlling element and the performance of a single-strand break thereof.

[0033] The inventors provide a genetic tool by means of which the targeted knockout of any gene on double-stranded DNA can be achieved in a reliable and safely manner. The safety profile of this approach is very high because the disruption of the con trolling element, e.g. the promoter, occurs via the use of two CRISPR nickases with two sgRNAs. The nickase may be a mutated variant of a conventional CRISPR endonuclease, such as Cas9, that can only generated single-strand DNA breaks (SSB).

[0034] By using two guide RNAs or sgRNAs, respectively, as paired nickases, designed for the sense (plus) and antisense (minus) DNA strands, it is finally possible to create a double-strand break (DSB) by generating two single-strand breaks at the control ling element, e.g. the promoter upstream of the transcription start site of the gene to be knocked-out. The DSB would be created only if the two CRISPR endonucleases will cut simultaneously at the target sites. If any of the sgRNA will target any unwanted site in the genome and introduce a SSB non-specifically, there will be no adverse effects because the SSB will be repaired efficiently by the cellular repair machinery. Therefore, the compo sition according to the invention comprises one sgRNA that introduces a nick at the plus or sense strand of the controlling element upstream of the transcription start site (TSS) and one sgRNA that introduce the complementary nick at the minus or antisense strand upstream of the TSS. Simultaneous activity of both CRISPR endonulcease/sgRNA com plexes lead to small genomic deletions in the controlling element, that permanently re press the gene transcription initiation complex.

[0035] The invention does not affect the coding sequence of the gene to be knocked-out in contrast to what is taught in the art, e.g. in Nasri et al. (2019, l.c.) or WO 2019/217294. The disruption of the coding sequence may result in malignant transfor mations. By using the invention the risk of malignant transformation is excluded or at least significantly reduced. Thus the composition of the invention is characterized by a very high safety profile which qualifies it for an administration in clinical routine applications.

[0036] The problem underlying the invention is herewith completely solved.

[0037] In an embodiment of the invention said genetic element controlling the expression of said gene is a promoter. [0038] This measure has the advantage that such a controlling element is tar geted which reliably and safely ensures the permanent knockout of the gene, however without affecting the coding region of the gene. According to the invention the promoter is a sequence of DNA to which proteins bind that initiate transcription of a single RNA from the DNA downstream of it. This transcribed RNA encodes the gene to be knocked-out.

The promoter is typically located near the transcription start site of the gene, upstream on the DNA, i.e. towards the 5' region of the sense strand. Promoters can be about 100-1000 base pairs long.

[0039] In another embodiment of the invention said first and/or second CRISPR endonuclease is a variant of the CRISPR associated protein 9 (Cas9).

[0040] This measure has the advantage that such CRISPR endonuclease is used for a conversion into a nickase, which is well established and characterized in the art. Cas9, formerly also called Cas5, Csn1, or Csx12, is a 160 kilodalton protein. It origi nates from the Gram-positive, aerotolerant bacterium Streptococcus pyogenes where it plays a vital role in the immunological defense against DNA viruses and plasmids. Strep tococcus pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to in duce site-directed double-strand breaks in DNA. Cas9 can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA: DNA complementarity and not modifications to the protein itself, engineering Cas9 to target new DNA is straightforward. Native Cas9 requires a guide RNA composed of two disparate RNAs that associate - the CRISPR RNA (crRNA), and the trans-activat ing crRNA (tracrRNA).Cas9 targeting has been simplified through the engineering of a chi meric single guide RNA (chiRNA or sgRNA). [0041] In another embodiment of the invention said variant of Cas9 comprises mutation(s) in the nuclease domain(s) RuvC and/or HNH, said mutation(s) conferring DNA nickase activity.

[0042] This measure has the advantage that the nickase conversion of Cas9 is effectively and reliably achieved by specifically addressing those catalytic domains in Cas9 which are responsible for the DNA cleavage activity. The mutations in the RuvC and/or HNH domains eliminate the activity of Cas9 to carry out DNA double strand breaks but establish the activity to effect DNA single-strand breaks.

[0043] In another embodiment of the invention said variant of Cas 9 is Cas9 D10A nickase and/or Cas9 H840A nickase.

[0044] This measure has the advantage that CRISPR endonucleases are em ployed, which are well established and characterized by their nicking enzyme activities which makes them predestined for a use in the invention. By the D10A amino acid ex change the RuvC catalytic center is disrupted, and by the H840A amino acid exchange the HNH catalytic domain is disrupted, both converting the enzyme into a nicking enzyme.

[0045] In another embodiment of the invention said gene is a mutated form of a wild type gene (mutated gene), preferably said mutated gene is subject of a gain-of-func- tion mutation.

[0046] This measure has the advantage that the composition according to the invention is adapted to eradicate the phenotypical changes of a gene mutation, especially such a mutation which may play a causative gain-of-function role in hereditary diseases.

[0047] In view of the aforesaid, in another embodiment said mutated gene is a disease-associated gene.

[0048] An example of such a gain-of-function and disease-associated mutation is a mutation in the ELANE gene, such as the p.A57V CN mutation. [0049] In a further embodiment said wild-type gene is a non-essential gene.

[0050] This measure has the advantage that the targeted knockout of the gene according to the invention can, on the one hand, avoid the consequences of the expres sion of a potential disease-associated gene, and, on the other hand, does not result in the loss of a vital function in the affected organism.

[0051] In another embodiment of the invention the mutated gene is a mutated form of the gene encoding neutrophil elastase or elastase neutrophil expressed gene (ELANE).

[0052] This measure has the advantage that a targeted knockout of such a gene is achieved which, if mutated, is considered of being responsible for most cases of severe congenital neutropenia (CN), and cyclic neutropenia (CyN). The inventors there fore provide a genetic tool for the effective treatment of disorders of the neutrophil produc tion, which so far cannot be cured in a satisfying manner. An example is the p.A57V CN mutation in the ELANE gene.

[0053] In another embodiment of the composition of the invention said first and/or second sgRNA comprises a nucleotide sequence which is selected from the group consisting of SEQ ID NOS: 1 to 252.

[0054] This measure has the advantage that sgRNA sequences are used which demonstrably guide the CRISPR endonucleases to the ELANE promoter, allowing the lat ter to be simultaneously cut in the sense and the antisense DNA strands. Such simultane ous DNA single-strand breaks result in a DNA double strand break and a functional dis ruption of the promotor’s function. This concerted action finally results in the inhibition of the expression of the ELANE gene.

[0055] Another subject-matter of the invention relates to the composition ac cording to the invention for use as a medicament, preferably for use in the treatment of an autosomal-recessive disorder, alternatively an autosomal-dominantly inherited genetic dis order, further preferably for use in the treatment of congenital neutropenia, highly prefera bly afore-said disorders with gain-of-function mutations.

[0056] The features, characteristics, and embodiments of the composition ac cording to the invention apply to the composition for use as a medicament and for use in said treatment according to the invention in a corresponding manner, even if not specifi cally indicated.

[0057] Another subject-matter of the invention relates to a method for the tar geted knockout of a gene on double-stranded DNA in a biological cell, comprising the fol lowing steps:

1) providing a biological cell comprising a gene on double-stranded DNA;

2) introducing into said biological cell a composition comprising:

- a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease;

- a second CRISPR endonuclease or a nucleic acid molecule encod ing said second CRISPR endonuclease; said first and said second CRISPR endonucleases are con figured to create single-strand DNA breaks,

- a first single guide RNA (sgRNA), and

- a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a genetic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the anti- sense strand of said genetic element controlling the expres sion of said gene, and 3) incubating said cell and said composition, thereby allowing the creation of a single-strand DNA break on the sense strand of a genetic element controlling the expression of said gene and a single-strand DNA break on the antisense strand of the genetic element controlling the expression of said gene.

[0058] The features, characteristics, and embodiments of the composition ac cording to the invention apply to this method according to the invention in a corresponding manner.

[0059] Therefore, in an embodiment of this method said composition is the composition according to the invention.

[0060] In another embodiment of this method said biological cell is a primary cell, preferably a hematopoietic stem and progenitor cell (HSPC).

[0061] By this measure therapeutic cells are generated which can be used in an autologous stem cell transplantation or in vivo administration, respectively. The obtained HSPCs can be a component of a hematopoietic stem cell transplant (HSCT).

[0062] In another embodiment of the method according to the invention said bi ological cell originates from a subject with a mutated gene, further preferably said mutated gene is subject of a gain-of-function mutation, further preferably said mutated gene is a disease-associated gene, further preferably the wild-type counterpart gene of said mu tated gene is a non-essential gene, and highly preferably said mutated gene is a mutated form of the gene encoding neutrophil elastase or the elastase neutrophil expressed gene (ELANE).

[0063] Another subject-matter relates to a preparation comprising a biological cell prepared in vitro by a method comprising the following steps:

1) providing a biological cell comprising a gene on double-stranded DNA; 2) introducing into said biological cell a composition comprising:

- a first CRISPR endonuclease or a nucleic acid molecule encoding the said CRISPR endonuclease;

- a second CRISPR endonuclease or a nucleic acid molecule encod ing said second CRISPR endonuclease; said first and said second CRISPR endonucleases are con figured to create single-strand DNA breaks,

- a first single guide RNA (sgRNA), and

- a second sgRNA, said first sgRNA is configured to hybridize to the sense strand of a genetic element controlling the expression of said gene, and said second sgRNA is configured to hybridize to the anti- sense strand of said genetic element controlling the expres sion of said gene;

3) incubating said cell and said composition, thereby allowing the creation of a single-strand DNA break on the sense strand of a genetic element controlling the expression of said gene and a single-strand DNA break on the antisense strand of the genetic element controlling the expression of said gene, and

4) recovering said cell.

[0064] The features, characteristics, and embodiments of the composition ac cording to the invention and the method for the targeted knockout according to the inven tion apply to the preparation according to the invention in a corresponding manner. Again, the obtained cell, such as a hematopoietic stem and progenitor cell (HSPC), can be a component of a hematopoietic stem cell transplant (HSCT).

[0065] Therefore, in an embodiment said method used for the production of the preparation is the method according to the invention. [0066] Another subject-matter of the invention is a kit for the targeted knockout of a gene on double-stranded DNA in a biological cell, comprising the composition accord ing to the invention and instructions for delivering the composition to said biological cell so as to knockout the gene.

[0067] A "kit" is a combination of individual elements useful for carrying out the methods of the invention, wherein the elements are optimized for use together in the methods. The kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the methods according to the invention. Such kits unify all essential elements required to work the methods according to the invention, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the methods according to the invention.

[0068] The features, characteristics, advantages and embodiments specified herein apply to the kit according to the invention, even if not specifically indicated.

[0069] Another subject-matter of the invention relates to a method of treating a subject afflicted with a disease associated with a mutated gene, comprising administration of a therapeutically effective amount of the composition according to the invention.

[0070] The features, characteristics, and embodiments of the composition ac cording to the invention apply to this method according to the invention in a corresponding manner, even if not specifically indicated.

[0071] Another subject-matter of the invention relate to a nucleic acid molecule, preferably an RNA molecule, comprising a nucleotide sequence which is selected from the group consisting of SEQ ID NOS: 1 to 252.

[0072] Still another subject-matter of the invention relates to a method of treat ing a subject afflicted with a disease associated with a mutated gene, preferably a domi nant-autosomal disease, comprising a step of inhibiting in said subject a genetic element controlling the expression of said gene, preferably knocking-out said genetic element con trolling the expression of said gene.

[0073] The features, characteristics, and embodiments of the composition ac cording to the invention apply to this method according to the invention in a corresponding manner, even if not specifically indicated.

[0074] It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respec tive case, but also in other combinations or in an isolated manner without departing from the scope of the invention.

[0075] The invention is now further explained by means of embodiments result ing in additional features, characteristics and advantages of the invention. The embodi ments are of pure illustrative nature and do not limit the scope or range of the invention. The features mentioned in the specific embodiments are general features of the invention which are not only applicable in the specific embodiment but also in an isolated manner and in the context of any embodiment of the invention.

[0076] The invention is now described and explained in further detail by refer ring to the following non-limiting examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 : Safe and efficient strategy to disrupt the ELANE gene expression by pro moter knockout using CRISPR/Cas9 D10A nickase restores granulocytic differentiation of congenital neutropenia patient’s HSPCs with p.A57V CN mutation. A, Scheme of the experiment. B, FACS gating strategy for cells generating during in vitro granulocytic differentiation C, Repre sentative FACS density plots for granulocytic CD45 ⁺ CD11b ⁺ CD15 ⁺ , CD45 ⁺ CD15 ⁺ CD16 ⁺ and CD45 ⁺ CD16b ⁺ C066b ⁺ cell subsets of differenti ated primary HSPCs of congenital neutropenia patient with p.A57V mu tation after CRISPR/Cas9 D10A double nickases editing.

Fig. 2: ELANE promoter knockout restored granulocytic differentiation of con genital neutropenia patient’s HSPCs with p.A57V CN mutation. A-D, Granulocytic differentiation capacity of ELANE promoter knockout HSPCs of congenital neutropenia patient's with p.A57V mutation was as sessed by liquid culture differentiation after 13 days by investigating neu trophilic surface marker expression. Percentage (A), absolute cell counts (B), morphologic analysis of cytospin slides (C) and representative im ages of Wright-Giemsa stained cytopsins (D) are shown. E, Colony forming unit assay of the ELANE promoter knockout cells showed higher numbers of CFU-G compared to control cells. Data represent means ± standard deviation (SD) from triplicates. *P<0.05, **P<0.01,

****p<0.0001.

Fig. 3: ELANE promoter knockout significantly impaired the ELANE gene ex pression and improved the functionality of differentiated neutrophils. A, ROS production was significantly elevated in promoter knockout cells treated with fMLP, as compared to non-target Cas9 cells. B, ELANE mRNA expression in in vitro generated CN neutrophils was 5-fold re duced compared to unedited healthy donor neutrophils. C, Indel's preva lence was assessed by inference of CRISPR edits (ICE) at day 13 post differentiation and was 87 %.

Fig. 4: Independent in silico on- and off-target profiling of sgRNA confirms the safety of the selected sgRNAs. Independent in silico on- and off-target profiling of each sgRNA was done using Doench 2016 algorithm using CRISPRitz tool for up to 4 mismatches using reference genome GRCh37 (hg19) for sgRNA 2 (A) and sgRNA 30 (B) shows low off-target profile for each sgRNA. Fig. 5: Analyzing ELANE promoter knockout genome editing outcome by Next generation sequencing. A, Quantification of editing frequency deter mined by the percentage and number of sequence reads showing un modified and modified alleles. B, Nucleotide distribution across am- plicon. At each base in the reference amplicon, the percentage of each base as observed in sequencing reads is shown (A = green; C = orange; G = yellow; T = purple). Black bars show the percentage of reads for which that base was deleted. Brown bars between bases show the per centage of reads having an insertion at that position. Sequence is listed as SEQ ID NO: 259. C, Combined frequency of any modification across the amplicon. Modifications outside of the quantification window are also shown.

Fig. 6: ELANE promoter knockout did NOT impair granulocytic differentiation of healthy control’s HSPCs. Granulocytic differentiation capacity of ELANE promoter knockout HSPCs of healthy controls was assessed by liquid cul ture differentiation after 14 days by investigating neutrophilic surface marker expression and ELANE, PRTN3 and AZU1 mRNA expression in in vitro generated neutrophils from (A), healthy control 98 and (B), healthy control 99. Data represent means ± standard deviation (SD) from tripli cates. ^* P<0.05, ^** P<0.01, ^**** P<0.0001.

Fig. 7: Unbiased genome-wide profiling of off-target cleavage by CRISPR-Cas9 double nickase ELANE promoter knockout using GUIDE-seq. Profiling of off-target cleavage upon double nickase ELANE promoter knockout treat ment using GUIDEseq showed only 2 potential off-target sites, LINC00992 - Long Intergenic Non-Protein Coding RNA 992- and chr9:93703739 - a non-coding intergenic region-. The ELANE promoter target sequence is shown in the top line with cleaved sites shown under neath and with mismatches to the on-target site shown and highlighted in color. GUIDE-seq sequencing read counts are shown to the right of each site. The on-target site is marked with a green circle and off-target sites with red circle in (A), sgRNA 30 (upper sequence "Reads" is listed as SEQ ID NO: 260) and (B), sgRNA 2 (upper sequence "Reads" is listed as SEQ ID NO: 261). All the genomic coordinates are based on reference genome GRCh37 (hg19).

EXAMPLES

1. Material and methods

Patients

[0077] One severe congenital neutropenia patient harboring an ELANE muta tion (EL4A/E-CN) was used in the study. Healthy individuals served as control group.

Bone marrow and peripheral blood samples were collected from the patient in association with an annual follow-up recommended by the Severe Chronic Neutropenia International Registry. Study approval was obtained from the Ethical Review Board of the Medical Fac ulty, University of Tubingen. Informed written consent was obtained from the participant of this study.

Cell culture

[0078] Human CD34 ⁺ HSPC were isolated from bone marrow mononuclear cells by magnetic bead separation using human CD34 progenitor cell isolation kit. (Mil- tenyi Biotech, #130-046-703). CD34 ⁺ cells were cultured in a density of 2 x 10 ⁵ cells/ml in Stemline II hematopoietic stem cell expansion medium (Sigma Aldrich, #50192) supple mented with 10 % FBS. 1 % penicillin/streptomycin, 1 % L-glutamine and a cytokine cock tail consisting of 20 ng/ml IL-3, 20 ng/ml IL-6. 20 ng/ml TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Systems).

Design of the sgRNAs

[0079] Specific D10A nickase-guide-RNAs (sgRNAs) to target promoter region of ELANE gene (nick sites: chr19 [GGGCTATAAGAGGAGCCGGG (SEQ ID NO: 1; sgRNA 2), GAGGCCGTTGCATTGCCCCA (SEQ ID NO: 2; sgRNA 30)] were designed by using the Desktop genetics website. In total 252 sgRNAs specific for the ELANE promoter were developed by the inventors. They are listed in the following table along with their ge nomic positions according to the Genome Reference Consortium Human Build 37 (GRCh37), the specificity for the sense (1) or antisense strand (-1) and the protospacer adjacent motif ("PAM").

SEQ-ID Genomic position

NO. (hg 19) Strand Sequence PAM

3 851023 -1 CTCACTTTCACCCAGGCTCA CGG

4 851030 -1 CGGGGAACTCACTTTCACCC AGG

5 851045 1 GGTGAAAGTGAGTTCCCCGT TGG

6 851048 1 GAAAGTGAGTTCCCCGTTGG AGG

7 851048 -1 CTCGTCTGTTGCCTCCAACG GGG

8 851049 -1 CCTCGTCTGTTGCCTCCAAC GGG

9 851050 -1 TCCTCGTCTGTTGCCTCCAA CGG

10 851060 1 CCCGTTGGAGGCAACAGACG AGG 11 851065 1 TGGAGGCAACAGACGAGGAG AGG 12 851069 1 GGCAACAGACGAGGAGAGGA TGG

13 851073 1 ACAGACGAGGAGAGGATGGA AGG

14 851078 1 CGAGGAGAGGATGGAAGGCC TGG

15 851085 -1 AGGGCTCATTCTTGGGGGCC AGG

16 851090 -1 ACCTCAGGGCTCATTCTTGG GGG

17 851091 -1 AACCTCAGGGCTCATTCTTG GGG

18 851092 -1 GAACCTCAGGGCTCATTCTT GGG

19 851093 -1 TGAACCTCAGGGCTCATTCT TGG

20 851100 1 GCCCCCAAGAATGAGCCCTG AGG 21 851104 -1 CAGCCGCTCCCTGAACCTCA GGG 22 851105 -1 CCAGCCGCTCCCTGAACCTC AGG

23 851106 1 AAGAATGAGCCCTGAGGTTC AGG

24 851107 1 AGAATGAGCCCTGAGGTTCA GGG

25 851112 1 GAGCCCTGAGGTTCAGGGAG CGG

26 851116 1 CCTGAGGTTCAGGGAGCGGC TGG

27 851126 1 AGGGAGCGGCTGGAGTGAGC CGG

28 851134 -1 GCTGGACGGAGATCTGGGGC CGG 29 851138 -1 CGCAGCTGGACGGAGATCTG GGG 851139 -1 CCGCAGCTGGACGGAGATCT GGG

851140 -1 CCCGCAGCTGGACGGAGATC TGG

851148 -1 CTCTGGGACCCGCAGCTGGA CGG

851150 1 CCCAGATCTCCGTCCAGCTG CGG

851151 1 CCAGATCTCCGTCCAGCTGC GGG

851152 -1 AGGCCTCTGGGACCCGCAGC TGG

851160 1 CGTCCAGCTGCGGGTCCCAG AGG

851164 -1 CGAGTGTAACCCAGGCCTCT GGG

851165 1 AGCTGCGGGTCCCAGAGGCC TGG

851165 -1 GCGAGTGTAACCCAGGCCTC TGG

851166 1 GCTGCGGGTCCCAGAGGCCT GGG

851172 -1 AGGAGCTGCGAGTGTAACCC AGG

851185 1 TGGGTTACACTCGCAGCTCC TGG

851186 1 GGGTTACACTCGCAGCTCCT GGG

851187 1 GGTTACACTCGCAGCTCCTG GGG

851188 1 GTTACACTCGCAGCTCCTGG GGG

851191 1 ACACTCGCAGCTCCTGGGGG AGG

851192 -1 GCACGTCAAGGGCCTCCCCC AGG

851203 -1 TGGGAACTGAGGCACGTCAA GGG

851204 -1 TTGGGAACTGAGGCACGTCA AGG

851214 -1 GGGTTCCTGTTTGGGAACTG AGG

851220 1 ACGTGCCTCAGTTCCCAAAC AGG

851222 -1 CCTTCCCAGGGTTCCTGTTT GGG

851223 -1 TCCTTCCCAGGGTTCCTGTT TGG

851228 1 CAGTTCCCAAACAGGAACCC TGG

851229 1 AGTTCCCAAACAGGAACCCT GGG

851233 1 CCCAAACAGGAACCCTGGGA AGG

851234 -1 CACTTCTCTGGTCCTTCCCA GGG

851235 -1 GCACTTCTCTGGTCCTTCCC AGG

851246 -1 CTGCGCAATAGGCACTTCTC TGG

851257 -1 TCGGGCACTCACTGCGCAAT AGG

851275 -1 CGGCCACATGCAGCTGTGTC GGG

851276 -1 CCGGCCACATGCAGCTGTGT CGG

851283 1 GTGCCCGACACAGCTGCATG TGG

851287 1 CCGACACAGCTGCATGTGGC CGG

851295 -1 TACCCAGGGCCCTGTGATAC CGG

851296 1 CTGCATGTGGCCGGTATCAC AGG

851297 1 TGCATGTGGCCGGTATCACA GGG

851303 1 TGGCCGGTATCACAGGGCCC TGG

851304 1 GGCCGGTATCACAGGGCCCT GGG 851309 -1 CGCCTGCCTCAGTTTACCCA GGG

851310 -1 TCGCCTGCCTCAGTTTACCC AGG

851314 1 ACAGGGCCCTGGGTAAACTG AGG

851318 1 GGCCCTGGGTAAACTGAGGC AGG

851336 1 GCAGGCGACACAGCTGCATG TGG

851340 1 GCGACACAGCTGCATGTGGC CGG

851348 -1 TACCCAGGGCCCTGTGATAC CGG

851349 1 CTGCATGTGGCCGGTATCAC AGG

851350 1 TGCATGTGGCCGGTATCACA GGG

851356 1 TGGCCGGTATCACAGGGCCC TGG

851357 1 GGCCGGTATCACAGGGCCCT GGG

851362 -1 CGCCTGCCTCAGTTTACCCA GGG

851363 -1 TCGCCTGCCTCAGTTTACCC AGG

851367 1 ACAGGGCCCTGGGTAAACTG AGG

851371 1 GGCCCTGGGTAAACTGAGGC AGG

851389 1 GCAGGCGACACAGCTGCATG TGG

851393 1 GCGACACAGCTGCATGTGGC CGG

851401 -1 TACCCAGGGCCCTGTGATAC CGG

851402 1 CTGCATGTGGCCGGTATCAC AGG

851403 1 TGCATGTGGCCGGTATCACA GGG

851409 1 TGGCCGGTATCACAGGGCCC TGG

851410 1 GGCCGGTATCACAGGGCCCT GGG

851415 -1 CGCCTGCCTCAGTTTACCCA GGG

851416 -1 TCGCCTGCCTCAGTTTACCC AGG

851420 1 ACAGGGCCCTGGGTAAACTG AGG

851424 1 GGCCCTGGGTAAACTGAGGC AGG

851442 1 GCAGGCGACACAGCTGCATG TGG

851454 1 GCTGCATGTGGCCGTATCAC AGG

851454 -1 TTACCCAGGGCCCTGTGATA CGG

851455 1 CTGCATGTGGCCGTATCACA GGG

851461 1 GTGGCCGTATCACAGGGCCC TGG

851462 1 TGGCCGTATCACAGGGCCCT GGG

851467 -1 CACCTGCCTCAGTTTACCCA GGG

851468 -1 TCACCTGCCTCAGTTTACCC AGG

851472 1 ACAGGGCCCTGGGTAAACTG AGG

851476 1 GGCCCTGGGTAAACTGAGGC AGG

851494 1 GCAGGTGACACAGCTGCATG TGG

851498 1 GTGACACAGCTGCATGTGGC CGG

851506 1 GCTGCATGTGGCCGGTATCA CGG

851506 -1 TATCCAGGGCCCCGTGATAC CGG 851507 1 CTGCATGTGGCCGGTATCAC GGG

851508 1 TGCATGTGGCCGGTATCACG GGG

851514 1 TGGCCGGTATCACGGGGCCC TGG

851520 -1 CGCCTGCCTCTGTTTATCCA GGG

851521 -1 TCGCCTGCCTCTGTTTATCC AGG

851525 1 ACGGGGCCCTGGATAAACAG AGG

851529 1 GGCCCTGGATAAACAGAGGC AGG

851547 1 GCAGGCGACACAGCTGCATG TGG

851551 1 GCGACACAGCTGCATGTGGC CGG

851559 1 GCTGCATGTGGCCGGTATCA CGG

851559 -1 TACCCAGGGCCCCGTGATAC CGG

851560 1 CTGCATGTGGCCGGTATCAC GGG

851561 1 TGCATGTGGCCGGTATCACG GGG

851567 1 TGGCCGGTATCACGGGGCCC TGG

851568 1 GGCCGGTATCACGGGGCCCT GGG

851573 -1 CGCCTGCCTCAGTTTACCCA GGG

851574 -1 TCGCCTGCCTCAGTTTACCC AGG

851578 1 ACGGGGCCCTGGGTAAACTG AGG

851582 1 GGCCCTGGGTAAACTGAGGC AGG

851587 1 TGGGTAAACTGAGGCAGGCG AGG

851599 -1 CCTGAGGGACTTGATGGGGG TGG

851602 -1 AGACCTGAGGGACTTGATGG GGG

851603 -1 TAGACCTGAGGGACTTGATG GGG

851604 -1 CTAGACCTGAGGGACTTGAT GGG

851605 -1 CCTAGACCTGAGGGACTTGA TGG

851610 1 CCACCCCCATCAAGTCCCTC AGG

851614 -1 CCTGCCAAACCTAGACCTGA GGG

851615 -1 ACCTGCCAAACCTAGACCTG AGG

851616 1 CCATCAAGTCCCTCAGGTCT AGG

851621 1 AAGTCCCTCAGGTCTAGGTT TGG

851625 1 CCCTCAGGTCTAGGTTTGGC AGG

851630 1 AGGTCTAGGTTTGGCAGGTT TGG

851651 1 GGCAAAAACACAGCAACGCT CGG

851668 1 GCTCGGTTAAATCTGAATTT CGG

851669 1 CTCG GTT AAAT CT GAATTT C GGG

851683 1 AATTTCGGGTAAGTATATCC TGG

851684 1 ATTTCGGGTAAGTATATCCT GGG

851690 -1 GTCTCTTCCAAATGAGGCCC AGG

851694 1 AGTATATCCTGGGCCTCATT TGG

851696 -1 ATCT AAGT CT CTTCCAAAT G AGG 851735 1 AAAAAACGTCGAGACCAGCC CGG

851738 -1 TTTCACCGTGTTGGCCGGGC TGG

851742 -1 GGGGTTTCACCGTGTTGGCC GGG

851743 -1 CGGGGTTTCACCGTGTTGGC CGG

851744 1 CGAGACCAGCCCGGCCAACA CGG

851747 -1 GAGACGGGGTTTCACCGTGT TGG

851761 -1 TTGTATTTTTAGTAGAGACG GGG

851762 -1 TTT GT ATTTTT AGT AG AG AC GGG

851763 -1 TTTT GT ATTTTT AGT AG AG A CGG

851785 1 AAAAAT AC AAAAAATT AG C C AGG

851792 -1 CAGGCGTGAGCCACTGCGCC TGG

851793 1 AAAAAATT AGCCAGGCGCAG TGG

851811 -1 TCCCAGAGTGCTGGGATCAC AGG

851819 -1 CCTCAGCCTCCCAGAGTGCT GGG

851820 1 CGCCTGTGATCCCAGCACTC TGG

851820 -1 GCCTCAGCCTCCCAGAGTGC TGG

851821 1 GCCTGTGATCCCAGCACTCT GGG

851824 1 TGTGATCCCAGCACTCTGGG AGG

851830 1 CCCAGCACTCTGGGAGGCTG AGG

851834 1 GCACTCTGGGAGGCTGAGGC AGG

851837 1 CTCTGGGAGGCTGAGGCAGG CGG

851848 1 TGAGGCAGGCGGATCACCCG AGG

851853 -1 GGTCTTGAACATCTGACCTC GGG

851854 -1 TGGTCTTGAACATCTGACCT CGG

851871 1 TCAGATGTTCAAGACCAGCC TGG

851874 -1 TTTCGCCCTGTCGGCCAGGC TGG

851878 -1 AGTGTTTCGCCCTGTCGGCC AGG

851879 1 TCAAGACCAGCCTGGCCGAC AGG

851880 1 CAAGACCAGCCTGGCCGACA GGG

851883 -1 GAGACAGTGTTTCGCCCTGT CGG

851919 1 CTACAAATACAAAAATTAGC CGG

851920 1 T AC AAAT AC AAAAATT AG C C GGG

851925 1 AT AC AAAAATT AGCCGGGAG TGG

851927 -1 CAGGCACCTGCCACCACTCC CGG

851928 1 CAAAAATTAGCCGGGAGTGG TGG

851932 1 AATTAGCCGGGAGTGGTGGC AGG

851946 -1 TCCT GAAT AG CT GAG ATT AC AGG

851956 1 GCCTGTAATCTCAGCTATTC AGG

851959 1 TGTAATCTCAGCTATTCAGG AGG

851965 1 CTCAGCTATTCAGGAGGCTG AGG 851969 1 GCTATTCAGGAGGCTGAGGC AGG

851987 1 GCAGGAGAATCACTTGAACC TGG

851988 1 CAGGAGAATCACTTGAACCT GGG

851991 1 GAGAATCACTTGAACCTGGG AGG

851994 1 AATCACTTGAACCTGGGAGG CGG

851994 -1 CACGGCAACCTCCGCCTCCC AGG

851997 1 CACTTGAACCTGGGAGGCGG AGG

852011 1 AGGCGGAGGTTGCCGTGAGC CGG

852012 1 GGCGGAGGTTGCCGTGAGCC GGG

852012 -1 GGTGGCGTGATCCCGGCTCA CGG

852019 -1 GGAGTGCGGTGGCGTGATCC CGG

852030 -1 TCGCCCAGGCTGGAGTGCGG TGG

852033 -1 CTATCGCCCAGGCTGGAGTG CGG

852037 1 CACGCCACCGCACTCCAGCC TGG

852038 1 ACGCCACCGCACTCCAGCCT GGG

852040 -1 TCTTGCTCTATCGCCCAGGC TGG

852044 -1 AGAGTCTTGCTCTATCGCCC AGG

852071 -1 GGTTTTTTAATTTATTTTTT TGG

852092 -1 AAAT GT C AG AT AAT CAAT GT GGG

852093 -1 CAAAT GT C AG AT AAT CAAT G TGG

852131 -1 GGGGCCTCCAGACAAAATTC AGG

852135 1 TGTGCATCCTGAATTTTGTC TGG

852138 1 GCATCCTGAATTTTGTCTGG AGG

852150 -1 ACGCTGGATTGGCTCGGGTG GGG

852151 -1 GACGCTGGATTGGCTCGGGT GGG

852152 -1 AGACGCTGGATTGGCTCGGG TGG

852155 -1 ACAAGACGCTGGATTGGCTC GGG

852156 -1 GACAAGACGCTGGATTGGCT CGG

852161 -1 AGGGGGACAAGACGCTGGAT TGG

852166 -1 GGAGAAGGGGGACAAGACGC TGG

852178 -1 TG AT G AAAAG GGGGAGAAGG GGG

852179 -1 TT GAT G AAAAG GGGGAGAAG GGG

852180 -1 GTTG AT G AAAAG G G G G AG AA GGG

852181 -1 CGTTGATGAAAAGGGGGAGA AGG

852187 -1 ACAGGGCGTTGAT G AAAAG G GGG

852188 -1 CACAGGGCGTTGAT G AAAAG GGG

852189 -1 GCACAGGGCGTTGATGAAAA GGG

852190 -1 GGCACAGGGCGTTGATGAAA AGG

852204 1 TTTCATCAACGCCCTGTGCC AGG

852204 -1 ACTTCCTCTCCCCTGGCACA GGG 230 852205 1 TTCATCAACGCCCTGTGCCA GGG

231 852205 -1 CACTTCCTCTCCCCTGGCAC AGG

232 852206 1 TCATCAACGCCCTGTGCCAG GGG

233 852211 1 AACGCCCTGTGCCAGGGGAG AGG

234 852211 -1 GCCCTCCACTTCCTCTCCCC TGG

235 852217 1 CTGTGCCAGGGGAGAGGAAG TGG

236 852220 1 TGCCAGGGGAGAGGAAGTGG AGG

237 852221 1 GCCAGGGGAGAGGAAGTGGA GGG

238 852227 1 GGAGAGGAAGTGGAGGGCGC TGG

239 852231 1 AGGAAGTGGAGGGCGCTGGC CGG

240 852237 1 TGGAGGGCGCTGGCCGGCCG TGG

241 852238 1 GGAGGGCGCTGGCCGGCCGT GGG

242 852239 1 GAGGGCGCTGGCCGGCCGTG GGG

243 852239 -1 CCGTTGCATTGCCCCACGGC CGG

244 852250 1 CCGGCCGTGGGGCAATGCAA CGG

245 852262 -1 TCTTATAGCCCTGTGCTGGG AGG

246 852264 1 ATGCAACGGCCTCCCAGCAC AGG

247 852265 1 TGCAACGGCCTCCCAGCACA GGG

248 852265 -1 TCCTCTTATAGCCCTGTGCT GGG

249 852266 -1 CTCCTCTTATAGCCCTGTGC TGG

250 852275 1 TCCCAGCACAGGGCTATAAG AGG

251 852281 1 CACAGGGCTATAAGAGGAGC CGG

252 852282 1 ACAGGGCTATAAGAGGAGCC GGG

Table 1 : sgRNAs specific for the human ELANE promoter

CRISPR/Cas9 D10A nickase-gRNA RNP mediated ELANE promoter KO in HSPC

[0080] Nucleofection was carried out using the Amaxa nucleofection system (P3 primary kit, #V4XP-3024) according to the manufacturer’s instructions. For 1 x 10 ⁶ hu man CD34 ⁺ HSPC, 300 pmol sgRNA and 300 pmol Hifi Cas9 protein (Integrated DNA Technologies) was used for nucleofection.

Assessing genome editing efficiency

[0081] Gene editing efficiency assessed either by Sanger sequencing of the promoter region qRT-PCR. Assessing gene editing efficiency by NGS

[0082] rhAmpSeq CRISPR analysis system approach was used to accurately estimate the on-target editing efficiency. The IDT’s rhAmpSeq design tool was used to de sign rhAMP PCR primers to amplify the target area in ELANE promoter. The PCR amplifi cation followed by rhAmpSeq library preparation was performed using rhAmpSeq CRISPR Library Kit (IDT, # 10007317) according to manufacturer's protocol. The rhAmpSeq librar ies was sequenced on an lllumina platform by Novogene. The NGS data was analysed by IDT’s rhAmpSeq CRISPR analysis pipeline followed by CRISPRESSO tool.

RNA isolation, cDNA synthesis and qRT-PCR

[0083] RNA was isolated using RNeasy Mini Kit (Qiagen) and cDNA was pre pared using Omniscript RT kit (Qiagen). qPCR was performed using SYBR Green qPCR master mix (Roche) and Light Cycler 480 (Roche). ELANE [primers: Fwd: GTGTCWTCCTCGCCTGTGTC (SEQ ID NO: 253), Rev: CCCACAATCTCCGAGGCCAG (SEQ ID NO: 254)] mRNA expression was normalized to GAPDH [primers: Fwd: CTGGGCTACACTGAGCACC (SEQ ID NO: 255), Rev: AAGTGGTCGTTGAGGGCAATG (SEQ ID NO: 256)] and for ACTB [primers: Fwd: CAT GT ACGTTGCT ATCCAGGC (SEQ ID NO: 257), Rev: CTCCTT AAT GTCACGCACG AT (SEQ ID NO: 258)] mRNA expression levels.

GUIDEseq off-target profiling

[0084] GUIDEseq was used to detect potential off-target cleavage sites after CRISPR/Cas9 editing. Primary human bone-marrow derived CD34+ cells were cultured in a density of 2 x 105 cells/mL in Stemline II hematopoietic stem cell expansion medium (Sigma Aldrich, #50192) supplemented with 10 % FBS, 1 % penicillin/streptomycin, 1 % L- glutamine and a cytokine cocktail consisting of 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Sys tems). CD34+ cells were electroporated with 8 pg Cas9 D10A Nickase V3 (IDT,

#1081063) assembled with 195 ng sgRNA 2 (IDT) and 195 ng sgRNA 30 (IDT). dsODN was added at a final concentration of 1 ,5 mM (5'- P-

G*T*TT AATT G AGTT GTCAT AT GTT AAT AACGGT*A*T -3' (SEQ ID 262), 5'-P- A*T*ACCGTT ATT AAC AT AT GACAACT C AATT AA*A*C -3' (SEQ ID 263) using the electro poration program CA-137 in 20 mI P3 buffer (Lonza, # V4XP-3032) on a Lonza 4-D nu- cleofector device according to the manufacturer’s instruction. 96 hours post-electro- poration, the genomic DNA was isolated using QIAamp DNA Blood Mini Kit (Qiagen, # 51104) according to manufacturer’s recommendations. Isolated DNA was sheared with Covaris S200 instrument to an average length of DNA pieces of 500bp. Sheared DNA was end-repaired, A-tailed and ligated to half-functional adapters, incorporating a 8-nt ran dom molecular index. Two rounds of nested anchored PCR with primers complementary to the oligo tag were used for target enrichment. The final library was sequenced on an II- lumina platform by Novogene. The NGS results were analyzed by GUIDEseq analysis pipeline.

Liquid culture differentiation of CD34 ⁺ cells

[0085] CD34 ⁺ cells were seeded in a density of 2 x 10 ⁵ cells/ml. Cells were in cubated for 7 days in RPMI 1640 GlutaMAX supplemented with 10 % FBS. 1 % penicil lin/streptomycin, 5 ng/ml SCF, 5 ng/ml IL-3, 5 ng/ml GM-CSF and 10 ng/ml G-CSF. Me dium was exchanged every second day. On day 7, cells were plated in RPMI 1640 Gluta MAX supplemented with 10 % FBS, 1 % penicillin/streptomycin and 10 ng/ml G-CSF. On day 14, cells were analyzed by FACS using following mouse anti-human antibodies: CD34 (BB, #343811). CD33 (BioLegend, #303416), CD45 (BioLegend, #304036), CD11b (BD, #557754), CD15 (BE, #555402), CD16 (BD, #561248), CD66b (Biolegend, #305104). Morphology of the cells was investigated on cytospin slides by Wright-Giemsa staining.

2. Results

[0086] To perform ELANE transcriptional repression using CRISPR/Cas9 D10A nickase, HSPCs of an ELANE-C patient with a p.A57V mutation were nucleofected with CRISPR/Cas9 D10A RNP and two days later cells were processed for liquid culture differ entiation towards neutrophils (Figure 1A). The same approach was used with healthy vol unteers (data not shown).

[0087] Using the double nick strategy in ELANE-C HSPCs, the inventors ob served markedly elevated neutrophil differentiation, as assessed by the percentage of CD45 ⁺ CD11b ⁺ CD15 ⁺ , CD45 ⁺ CD15 ⁺ CD16 ⁺ and CD45 ⁺ CD16 ⁺ CD66b ⁺ cells (Figure 1B, C and 2A, B) compared to ELANE-CN control cells nucleofected with non-targeting CRISPR/Cas9 D10A RNP. No effects were observed with healthy volunteers (data not shown).

[0088] Again, comparing morphological analysis of ELANE-C with promoter KO to control cells (see above) the strongly improved number of mature granulocytes was confirmed (Figure 2C, D).

[0089] Similar results were achieved by colony forming unit assay (CFU) show ing significantly more granulocytic colony-forming units (CFU-G colonies) in the ELANE promoter KO cells compared to non-targeting CRISPR/Cas9 D10A RNP control (Figure 2E). Again, effects were observed with healthy volunteers (data not shown).

[0090] The inventors further tested the functionality of in vitro generated ELANE promoter KO neutrophils by testing the reactive oxygen species (ROS) production in re sponse to fMLP. Indeed, ELANE promoter KO cells produced significantly more ROS) in response to fMLP, as compared to control cells with non-targeting CRISPR/Cas9 D10A RNP (Figure 3A).

[0091] ELANE mRNA expression was reduced by around 5-fold in neutrophils from ELANE promoter KO cells compared to neutrophils from healthy donors (Figure 3B). Indel’s prevalence at targeted site was 87% as assessed by inference of CRISPR edits (ICE) from Sanger Sequencing Trace Data (Figure 3C), indicating high gene-editing efficiency. [0092] No effects were observed with healthy volunteers (data not shown).

[0093] The inventors also performed an independent in silico on- and off-target profiling of each sgRNA for up to 4 mismatches using Doench 2016 algorithm of CRIS- PRitz tool (Figure 4A, B). The inventors found low off-target effects for each single sgR- NAs, indicating very low off-target effects of the combination of both sgRNAs (Figure 4A, B).

[0094] To further obtain an NGS grade resolution on on-target profile of editing outcome, the inventors used rhAmpSeq CRISPR Analysis System. They found that the editing efficiency was 93%. In Figure 5A, B, the nucleotide composition across the edited region is depicted along with the frequency of any modification across the edited site.

[0095] To investigate if the inventors' proposed CRISPR/Cas9 strategy may af fect granulocytic differentiation, HSPCs of two healthy controls were nucleofected with CRISPR/Cas9 D10A RNP and two days later cells were processed for liquid culture differ entiation towards neutrophils. The inventors observed no impairment in neutrophil differ entiation of edited cells, as assessed by the percentage of CD45+CD11b+CD15+, CD45+CD15+CD16+ and CD45+CD16+CD66b+ neutrophilic cells that was comparable to healthy control cells nucleofected with non-targeting CRISPR/Cas9 D10A RNP (Figure 6A, B). While ELANE mRNA expression was reduced significantly in neutrophils from ELANE promoter KO cells, compared to neutrophils from healthy donors nucleofected with non-targeting CRISPR/Cas9 D10A RNP, the inventors observed no changes in mRNA expression levels of two NE related serine proteases, PRTN3 and AZU1 (Figure 6A, B).

[0096] Therapeutic use of CRISPR/Cas9 in clinic needs a comprehensive knowledge of potential off-target effects to minimize the risk of harmful consequences. Therefore, the inventors performed GUIDE-seq analysis, to be able to have an unbiased genome-wide profiling of off-target cleavage by CRISPR/Cas9 D10A double nickase tar geting the ELANE gene promoter. The inventors observed two potential off-target sites, LINC00992 -Long Intergenic Non-Protein Coding RNA 992- and Chr9:93703739 - a non- coding intergenic region- which may happen if the RNA guided nuclease had 6 or 7 mis matches with the genome. The GUIDE-seq results highlights the safety profile of ELANE promoter editing approach for clinic (Figure 7A, B).

3. Conclusion

[0097] Taken together, the inventors established a safe approach for reducing a gene's expression by CRISPR/Cas9 double nick strategy without affecting the gene's cod ing sequence. This approach was exemplified as feasible using the ELANE gene involved in the development of congenital neutropenia. As demonstrated by examples the ap proach according to the invention can rescue granulopoiesis in ELANE-C patients. In healthy individuals the knock-out of the ELANE promoter has no effect on granulopoiesis. Therefore, the invention offers a long-term treatment option replacing the daily G-CSF therapy and decreasing the risk of leukemia development.

[0098] Neutrophil elastase (NE) is a proteolytic enzyme of the neutrophil serine protease (NSP) family, including also cathepsin G (CG), proteinase 3 (PR3) and azuro- cidin (AZU1). NSPs are stored in cytoplasmic granules, can be secreted into the extra- and peri-cellular space upon cellular activation and considered to be crucially involved in bacterial defense. Elane ^A mice have normal neutrophil counts, but there are conflicting re sults regarding the effect of NE-deficiency on neutrophil extravasation to sites of inflam mation, phagocytosis, and neutrophil extracellular traps in mice. NE may or may not be essential for these processes. Papillon-Lefevre Syndrome (PLS) is a human disorder known to cause NE deficiency. This rare autosomal recessive disease is due to loss-of- function mutations in the DPPI gene locus with the loss of the lysosomal cysteine prote ase cathepsin C/dipeptidyl peptidase I (DPPI). The activation NSP, including NE, depends on the N-terminal processing activity of DPPI. Therefore, PLS patients exhibit a severe re duction in the activity and stability of all three NSP. Intriguingly, patients with PLS have no defects in their ability to kill bacteria e.g. Staphylococcus aureus or Escherichia coii, sug gesting that redundancies in the neutrophil’s bactericidal mechanisms negate the neces sity for serine proteases for killing common bacteria. Based on these observations, at this juncture, the inventors believe that CRISPR/Cas9 based knockout of ELANE in HSPC of CN patients may restore defective granulopoiesis in CN patients without seriously impair ing neutrophil functions.